Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M313268200 on December 29, 2003

J. Biol. Chem., Vol. 279, Issue 12, 10864-10871, March 19, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/12/10864    most recent
M313268200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Glowacki, R.
Right arrow Articles by Jakubowski, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Glowacki, R.
Right arrow Articles by Jakubowski, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cross-talk between Cys34 and Lysine Residues in Human Serum Albumin Revealed by N-Homocysteinylation*

Rafal Glowacki{ddagger}§ and Hieronim Jakubowski{ddagger}||

From the {ddagger}Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, International Center for Public Health, Newark, New Jersey 07103 and Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland

Received for publication, December 4, 2003 , and in revised form, December 19, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein N-homocysteinylation involves a post-translational modification by homocysteine (Hcy)-thiolactone. In humans, about 70% of circulating Hcy is N-linked to blood proteins, mostly to hemoglobin and albumin. It was unclear what protein site(s) were prone to Hcy attachment and how N-linked Hcy affected protein function. Here we show that Lys525 is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo. We also show that the reactivity of albumin lysine residues, including Lys525, is affected by the status of Cys34. The disulfide forms of circulating albumin, albumin-Cys34-S-S-Cys and albumin-Cys34-S-S-Hcy, are N-homocysteinylated faster than albumin-Cys34-SH. Although N-homocysteinylations of albumin-Cys34-SH and albumin-Cys34-S-S-Cys yield different primary products, subsequent thiol-disulfide exchange reactions result in the formation of a single product, N-(Hcy-S-S-Cys)-albumin-Cys34-SH. We also show that N-homocysteinylation affects the susceptibility of albumin to oxidation and proteolysis. The data suggest that a disulfide at Cys34 of albumin promotes conversion of N-(Hcy-SH)-albumin-Cys34-SH to a proteolytically sensitive form N-(Hcy-S-S-Cys)-albumin-Cys34-SH, which would facilitate clearance of the N-homocysteinylated form of mercaptoalbumin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Since the 1960s, it has been known that elevated levels of homocysteine (Hcy),1 resulting from mutations in genes encoding Hcy-metabolizing enzymes, are harmful to humans (1, 2). During the past decade it has been established that even a mild increase in Hcy level is a risk factor for cardiovascular disease and stroke in humans (3, 4) and predicts mortality independently of traditional risk factors in patients with coronary artery disease (5). Plasma Hcy is also a risk factor for neurodegenerative disorders, such as dementia and Alzheimer's disease (6). In tissue cultures, Hcy does not support growth and induces apoptotic death in human endothelial cells (7). Animal and cell culture studies have shown that Hcy induces cell death and potentiates amyloid {beta}-peptide toxicity in neurons (8).

In humans, Hcy, formed from dietary methionine as a byproduct of cellular methylation reactions, is detoxified by folic acid- and vitamin B12-dependent re-methylation to methionine (2) or vitamin B6-dependent trans-sulfuration to cysteine (1). Whereas Hcy is formed in all human organs, most of its detoxification occurs in the liver and kidneys. Detoxification of Hcy in human vascular tissues and skin occurs only by re-methylation; enzymes of the trans-sulfuration pathway are not expressed in these tissues (9).

Hcy is perhaps the most reactive amino acid in biological systems (1, 2). In addition to re-methylation to methionine or trans-sulfuration to cysteine (via cystathionine), Hcy is also metabolically converted to Hcy-thiolactone, S-nitroso-Hcy, AdoHcy, Hcy-containing disulfides, or homocysteic acid, each of which has been implicated in the pathology of hyperhomocystinemia (1, 10).

Because of its similarity to the protein amino acid methionine, Hcy can exert its biological effects by interfering with protein biosynthesis (10–18). For example, Hcy is metabolized to Hcy-thiolactone by methionyl-tRNA synthetase in a two-step reaction (19, 20). In the first step (Reaction 1), methionyl-tRNA synthetase catalyzes activation of Hcy with ATP, which yields methionyl-tRNA synthetase (MetRS)-bound homocysteinyl adenylate.

(REACTION 1)

The second step (Reaction 2), in which the side chain thiolate of Hcy reacts with the activated carboxyl group of Hcy, yields Hcy-thiolactone.



View larger version (8K):
[in this window]
[in a new window]
  		REACTION 2
 
The energy of the anhydride bond in Hcy~AMP is conserved in the thioester bond in Hcy-thiolactone. Because of this, Hcythiolactone reacts with proteins, forming Hcy-containing adducts, in which the carboxyl group of Hcy is linked by an amide bond with {epsilon}-amino group of a protein lysine residue (21–23).

Originally discovered in cultured cells (21–23), protein N-homocysteinylation is now known to occur in humans (10, 12–18). Both Hcy-thiolactone (13, 24, 25) and N-linked protein-Hcy (12, 16, 26, 27) have been demonstrated in human blood. About 70% of circulating Hcy is N-linked to blood proteins, mostly hemoglobin and albumin (26). A protective mechanism against protein N-homocysteinylation appears to exist in humans (28, 29).

Two major forms of albumin exist in circulation (30): albumin-Cys34-SH, also known as mercaptoalbumin, and albumin-Cys34-S-S-Cys (Fig. 1), accounting for about two-thirds and one-third, respectively, of total plasma albumin (31). Two minor forms, accounting for 1–2% of total albumin, also exist in circulation: albumin-Cys34-S-S-Hcy (Fig. 1) (31) and Hcy-N-albumin (26). However, these minor forms carry >80% of plasma Hcy. The ability of albumin to form a disulfide with Hcy has been examined in vitro (22, 28), and the mechanism for the formation of albumin-Cys34-S-S-Hcy has been proposed (32).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 1.
Structures of the different forms of albumin.

 
Molecular mechanism and functional consequences of albumin N-homocysteinylation were not known. In this study we show that Lys525 is a predominant site of N-homocysteinylation in human serum albumin. We also show that the status of Cys34 affects the reactivity of albumin lysine residues, including Lys525, and that N-homocysteinylation affects the susceptibility of albumin to proteolysis and oxidation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of [35S]Hcy-thiolactone—Carrier-free L-[35S]Met (5 mCi, Amersham Biosciences) was supplemented with unlabeled methionine (Sigma) to a specific activity of 20,000 Ci/mol, lyophilized on a Labconco CentriVap concentrator, dissolved in 0.15 ml of 57% hydriodic acid, and digested for 4 h at 128 °C (33). The conversion of L-[35S]Met to L-[35S]Hcy-thiolactone was complete as determined by analytical thin layer chromatography. Excess hydriodic acid was removed by lyophilization, and L-[35S]Hcy-thiolactone was dissolved in deionized water at 15 µCi/ml, aliquoted, and stored at –80 °C.

Preparation of Albumin-Cys34-SH and Albumin-Cys34-S-S-Cys—Human serum albumin (50 mg/ml) was converted to albumin-Cys34-SH by treatment with 2 mM DTT in 0.1 M potassium phosphate buffer, pH 7.4, 0.2 mM EDTA for 5 min at room temperature, diluted 10-fold with 0.01 M potassium phosphate buffer, pH 5.8, and purified by anion exchange HPLC. Fractions containing albumin-Cys34-SH were concentrated to 10 mg/ml in 0.1 M potassium phosphate buffer, pH 7.4, 0.2 mM EDTA, and treated with 2-fold molar excess of Cys overnight at 37 °C. Excess Cys was removed from albumin-Cys34-S-S-Cys by ultrafiltration through an Ultrafree-0.5 10-kDa cut-off membrane (Millipore) at 4 °C. The conversion of albumin-Cys34-SH to albumin-Cys34-S-S-Cys was monitored by anion exchange HPLC.

Preparation of Albumin-Cys34-S-IAA—Albumin-Cys34-SH (10 mg/ml) was incubated with 4-fold molar excess IAA in 0.1 M potassium phosphate buffer, pH 7.4, 0.2 mM EDTA for 1 h at 37 °C in the dark. Excess IAA was removed by ultrafiltration. The conversion of albumin-Cys34-SH to albumin-Cys34-S-IAA was complete as determined by anion exchange HPLC.

Preparation of N-Hcy-Proteins—Individual proteins (obtained from Sigma) were dissolved at 50 mg/ml in 0.1 M potassium phosphate, pH 7.4, 0.2 mM EDTA, and incubated for 16 h at 37 °C with 1–2 molar excess of unlabeled L-Hcy-thiolactone. Under these conditions the extent of modification was 0.6–0.75 mol of Hcy/mol of protein. The extent of modification has been determined by the formation of N-[35S]Hcy-protein, assayed either by precipitation with trichloroacetic acid (22), in parallel reactions with identical concentrations of [35S]Hcy-thiolactone or by monitoring the increase in protein thiol groups with Ellman's reagent (Sigma) (34). Both methods gave similar results.

Susceptibility of Hcy-thiolactone-modified Albumin to Oxidation— Unmodified albumin or N-Hcy-albumin (at 10 mg/ml) were incubated with or without 5-fold molar excess of H2O2 in 0.1 M potassium phosphate buffer, pH 7.4, for 15 min at 37 °C. The formation of albumin aggregates after oxidation was analyzed by anion exchange HPLC.

Anion Exchange HPLC—Albumin species (5 mg/ml) were analyzed by anion exchange HPLC on a Vydac 301VHP575 DEAE column (7.5 x 50 mm, 5 µm, 900 Å) equilibrated with 10 mM potassium phosphate, pH 5.8 (buffer A), and eluted with a linear gradient 0–250 mM NaCl (40 min, 1 ml/min) in buffer A. The elution conditions were somewhat modified for each experiment to achieve optimal separation of analyzed albumin species. In some experiments, a nonporous Tosohaas TSK-gel DNA-NPR column (4.6 x 75 mm, 2.5 µm) was used with similar results. The effluent was monitored at 280 nm using a diode array detector.

Susceptibility of Hcy-thiolactone-modified Albumin to Proteolysis— Albumin-Cys34-SH, N-(Hcy-SH)-albumin-Cys34-SH, albumin-Cys34-S-S-Cys, N-(Hcy-S-S-Cys)-albumin-Cys34-SH, and N-(Hcy-S-S-Cys)-albumin-Cys34-S-S-Cys (Fig. 1) (1–2 mg/ml) were digested with trypsin (L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated), chymotrypsin, elastase, or cathepsin D (all from Sigma) at substrate/enzyme ratios from 1:400 to 1:1 in 0.1 M potassium phosphate buffer, pH 7.4, for 2.5 h at 37 °C. Digests were mixed 1:1 with SDS-PAGE sample buffer containing 1% 2-mercaptoethanol, denatured for 5 min at 100 °C, and subjected to SDS-PAGE on 12% polyacrylamide gels. Protein bands were visualized by staining with Coomassie Brilliant Blue R-250 (J. T. Baker Inc.) and quantified by densitometry.

Purification of Native Albumin from Human Serum—Human serum (0.3 ml) was diluted with 1 volume of 0.1 M potassium phosphate, pH 6.8, and loaded onto a 1-ml High-Trap Blue column (Amersham Biosciences). After washing off unbound protein, albumin was eluted with 2 M NaCl and desalted by ultrafiltration.

Enrichment of N-Hcy-albumin with Thiopropyl-Sepharose—Crystal structure of albumin-Cys34-SH shows that Cys34 is located in a crevice about 10 Å deep (30), and because of this, access to Cys34 is somewhat limited. In contrast, N-linked Hcy in N-(Hcy-SH)-albumin-Cys34-SH is expected to be freely accessible. Indeed, in control experiments we found that N-(Hcy-SH)-albumin-Cys34-SH binds well, whereas albumin-Cys34-SH binds very poorly to thiopropyl-Sepharose. We utilized this property to enrich native albumin in N-(Hcy-SH)-albumin. Native albumin (50 mg/ml) was first converted into mercaptoalbumin by treatment with 2 mM DTT in 0.1 M potassium phosphate, pH 7.4, 0.2 mM EDTA. After removal of low molecular weight thiols by ultrafiltration on Ultrafree-05 30-kDa cut-off membrane (Millipore), mercaptoalbumin (50 mg/ml) was allowed to bind to washed thiopropyl-Sepharose (120 mg) in 0.4 ml of 0.1 M potassium phosphate, pH 5.0, for 20 min at 21 °C. The resin was washed with 0.1 M potassium phosphate, pH 5.0, 0.1 M NaCl to remove unbound protein. Bound albumin was eluted with 0.5 ml of 25 mM DTT in 0.1 M potassium phosphate, pH 7.4, desalted, and concentrated to 50 µl in 0.1 M potassium phosphate, pH 5.0. Resulting albumin preparation was subjected to Thiopropyl-Sepharose enrichment two more times. About 7% of native albumin was bound to and recovered from thiopropyl-Sepharose after three cycles of enrichment.

Preparation of Tryptic Peptides—N-Hcy-albumin or native albumin (10 mg/ml) was reduced with 25 mM DTT in 10 M urea, 0.2 M Tris-HCl buffer, pH 8.6, 1 mM EDTA (5 h at 21 °C), and the liberated thiols were blocked with 100 mM IAA for 1 h at 37 °C in the dark. IAA-modified albumin was desalted by ultrafiltration and digested with L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin (enzyme/substrate ratio 1:50) in 0.1 M ammonium bicarbonate overnight at 37 °C.

Reversed Phase C18 HPLC—HPLC analyses of tryptic peptides were carried out using a reversed phase C18 peptide-protein column (4.6 x 100 mm, 5 µ, 300 Å) from Vydac. The detection was by monitoring at 215 nm using a diode array detector. After sample application, the C18 HPLC column was eluted with a linear gradient from 0 to 40% acetonitrile (60 min) in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min.

Cation Exchange HPLC—N-Hcy-peptides from a C18 column were further purified by cation exchange on a PolySULFOETHYL AspartamideTM column (2.1 x 200 mm, 5 µm, 300 Å, PolyLC, Inc.). Solution A (50 mM potassium phosphate-HCl, pH 3.4) and solution B (500 mM NaCl in solution A) were used as solvents, and the flow rate was 0.6 ml/min. After sample application, the column was eluted with a linear gradient from 0 to 60% solution B for 20 min.

HPLC Instrumentation—HPLC instrumentation was Beckman-Coulter System Gold Noveau as described before (24, 26). It consisted of the following modules: advanced gradient solvent delivery module 126 and a high resolution diode array 168 detector module. Manual injector (7725i Rheodyne) with a 0.1-ml loop was used. Chromatograms were analyzed using Gold Noveau chromatography work station software for Windows.

Mass Spectroscopy—Peptide mass analyses were carried out by Dr. Hong Li on a MALDI-TOF Voyager-DETM PRO BiospectrometryTM work station (PerSeptive Biosystems) at the New Jersey Medical School Center for Advanced Proteomics Research facility (njms.umdnj.edu/biochemistry/proteomics/index.htm).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hcy-thiolactone Reacts Faster with Albumin-Cys34-S-S-Cys and Albumin-Cys34-S-S-Hcy than with Albumin-Cys34-SH—To determine whether the status of Cys34 in human serum albumin affects the susceptibility of that protein to N-homocysteinylation, albumin-Cys34-S-S-Cys, albumin-Cys34-S-S-Hcy, and albumin-Cys34-SH (Fig. 1), major forms of circulating albumin (30, 31), were prepared as described under "Materials and Methods" and incubated with [35S]Hcy-thiolactone. The formation of N-[35S]Hcy-albumin was monitored by precipitation with trichloroacetic acid. As shown in Fig. 2, albumin-Cys34-S-S-Cys and albumin-Cys34-S-S-Hcy were N-homocysteinylated at similar rates. However, both disulfide forms of albumin were N-homocysteinylated 47 ± 5% faster than mercaptoalbumin, albumin-Cys34-SH. Similar results were obtained when corresponding forms of albumin were prepared in situ by treatments with 5-fold molar excess Cys, Hcy, or DTT. These results suggest that the reactivity of albumin lysine residues depends on the status of Cys34.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 2.
Time courses of N-homocysteinylation of human serum albumins. Reactions were carried out at 37 °C in mixtures contained 50 mg/ml (0.74 mM) HPLC-purified indicated form of albumin (Alb), 7.5 µM L-[35S]Hcy-thiolactone (20,000 Ci/mol), 0.1 M potassium phosphate, pH 7.4, 0.2 mM EDTA. Incorporation of radiolabel into albumin-Cys34-S-S-Cys ({blacksquare}), albumin-Cys34-S-S-Hcy ({circ}), and albumin-Cys34-SH ({blacktriangleup}) was determined by trichloroacetic acid precipitation of DTT-reduced samples. Errors in the rate measurements were 5%.

 
Thiol-Disulfide Exchange in N-Homocysteinylated Albumin-Cys34-S-S-Cys Leads to Formation of Free Sulfhydryl at Cys34—Albumin-Cys34-S-S-Cys or albumin-Cys34-SH was modified with equimolar amounts of Hcy-thiolactone and analyzed by anion exchange HPLC. Unmodified albumin-Cys34-S-S-Cys was eluted from the HPLC column at 13.2 min (Fig. 3A, broken trace). After 4 h of modification, about 50% of albumin-Cys34-S-S-Cys was N-homocysteinylated and eluted from the column as a new peak at 12.6 min, before the peak of unmodified form (Fig. 3A, thin trace). After 20 h of modification, all albumin-Cys34-S-S-Cys disappeared, and only a single peak of N-homocysteinylated albumin-Cys34-S-S-Cys at 12.6 min was observed (Fig. 3A, thick trace). Both native and N-homocysteinylated albumin-Cys34-SH were eluted from the anion exchange HPLC column at 12.6 min (not shown).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3.
Anion exchange HPLC analysis of Hcy-thiolactonemodified albumin-Cys34-S-S-Cys. Albumin-Cys34-S-S-Cys was modified with Hcy-thiolactone or [35S]Hcy-thiolactone at 37 °C and analyzed by anion exchange HPLC. A, protein profiles after 0 (broken trace), 4 (thin trace), and 22 h (thick trace) of modification with Hcy-thiolactone. Alb, albumin. B shows protein profiles of the 10-h reaction with Hcy-thiolactone after an overnight incubation without (dotted line) and with a 2-fold molar excess of cysteine (solid line). C, protein (upper panel) and 35S (lower panel) profiles after 4 h of modification with [35S]Hcy-thiolactone.

 
Altered chromatographic behavior of albumin-Cys34-S-S-Cys after modification with Hcy-thiolactone could be due to two alternative mechanisms. First, blocking an {epsilon}-amino group of lysine residue(s) by N-homocysteinylation may have changed the charge of albumin, thus weakening its binding to the anion exchange column. However, this seems unlikely because similar modification of albumin-Cys34-SH did not affect its elution position from the column. Instead, N-homocysteinylation of albumin-Cys34-S-S-Cys may have caused regeneration of a free thiol at Cys34 as a result of thiol-disulfide exchange between a free thiol of newly incorporated Hcy and the disulfide at Cys34 (Reaction 3).

(REACTION 3)

This interpretation is supported by an observation that regeneration of a disulfide at Cys34 by incubation of N-(Hcy-S-S-Cys)-albumin-Cys34-SH with 2-fold molar excess of cysteine also restored its stronger binding to the anion exchange column (Fig. 3B).

To examine more directly whether N-(Hcy-SH)-albumin-Cys34-S-S-Cys and N-(Hcy-S-S-Cys)-albumin-Cys34-S-S-Cys are formed in significant amounts during N-homocysteinylation of albumin-Cys34-S-S-Cys, [35S]Hcy-thiolactone was used. If formed, N-([35S]Hcy-SH)-albumin-Cys34-S-S-Cys and N-([35S]Hcy-S-S-Cys)-albumin-Cys34-S-S-Cys would co-elute with albumin-Cys34-S-S-Cys from the anion exchange HPLC column. However, as shown in Fig. 3C, only a single peak of radioactivity (at 12.6 min), corresponding to N-([35S]Hcy-S-S-Cys)-albumin-Cys34-SH, was observed. There was no distinct 35S-radioactivity peak at the elution position of albumin-Cys34-disulfide (at 13.2 min). This result demonstrates that a single species, N-([35S]Hcy-S-S-Cys)-albumin-Cys34-SH, is predominant during N-homocysteinylation of albumin-Cys34-S-S-Cys; other products are not observed. The absence of N-([35S]Hcy-SH)-albumin-Cys34-S-S-Cys suggests that its conversion to N-([35S]Hcy-S-S-Cys)-albumin-Cys34-SH is much faster that the N-homocysteinylation reaction. The absence of N-([35S]Hcy-S-S-Cys)-albumin-Cys34-S-S-Cys suggests that the thiol-disulfide exchange between N-([35S]Hcy-S-S-Cys)-albumin-Cys34-SH and albumin-Cys34-S-S-Cys is not favored thermodynamically.

To determine whether the thiol-disulfide exchange occurs in trans between different albumin molecules, preparations of N-(Hcy-SH)-albumin-Cys34-SH and albumin-Cys34-S-S-Cys were used. When analyzed separately by anion exchange HPLC, N-(Hcy-SH)-albumin-Cys34-SH (Fig. 4, thin trace) and albumin-Cys34-S-S-Cys (Fig. 4, broken trace) were eluted at 9.9 and 10.25 min, respectively. However, when N-(Hcy-SH)-albumin-Cys34-SH was incubated with equimolar amounts of albumin-Cys34-S-S-Cys for 4 h at 37 °C and analyzed by anion exchange HPLC, only a peak at 9.9 min was observed (Fig. 4, thick trace) and the peak of albumin-Cys34-S-S-Cys at 10.25 min essentially disappeared. This shows that albumin-Cys34-S-S-Cys was converted to albumin-Cys34-SH. Thus, the thioldisulfide exchange occurs in trans.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4.
Intermolecular thiol-disulfide exchange in N-homocysteinylated albumin (Alb). Equimolar amounts of albumin-Cys34-S-S-Cys and N-(Hcy-SH)-albumin-Cys34-SH were incubated for 4 h at 37 °C and analyzed by anion exchange HPLC. Albumin-Cys34-S-S-Cys (broken trace) elutes at 12.0 min. N-(Hcy-SH)-albumin-Cys34-SH (thin trace) elutes at 9.1 min. A peak of albumin-Cys34-S-S-Cys disappears when both forms are incubated together (thick trace).

 
To determine whether a thiol of Cys34 in N-(Hcy-SH)-albumin-Cys34-SH is required for the thiol-disulfide exchange, albumin was first treated with IAA to block the Cys34 thiol and then N-homocysteinylated. This yielded an albumin derivative containing a single free thiol on N-linked Hcy, N-(Hcy-SH)-albumin-Cys34-S-IAA. When analyzed by anion exchange HPLC, N-(Hcy-SH)-albumin-Cys34-S-IAA (Fig. 5, thin trace) was eluted at 13.3 min, essentially identical to the retention time of albumin-Cys34-S-S-Cys (Fig. 5, broken trace). However, when N-(Hcy-SH)-albumin-Cys34-S-IAA was mixed in a 1:1 molar ratio with albumin-Cys34-S-S-Cys and analyzed by anion exchange HPLC, a new peak of albumin-Cys34-SH (eluting at 12.9 min) was observed (Fig. 5, thick trace). This shows that albumin-Cys34-S-S-Cys was converted to albumin-Cys34-SH as a result of a thiol-disulfide exchange with a thiol of N-linked Hcy in N-(Hcy-SH)-albumin-Cys34-S-IAA. Control experiments have shown that albumin-Cys34-S-IAA and N-(Hcy-S-IAA)-albumin-Cys34-S-IAA did not participate in the thiol-disulfide exchange with albumin-Cys34-S-S-Cys, as expected. IAA derivatives of different N-homocysteinylated forms of albumin eluted at 13.3 min, a retention time characteristic of albumin with substituted thiol of Cys34 (not shown). Taken together, these experiments suggest that a thiol of Cys34 in N-(Hcy-SH)-albumin-Cys34-SH is not required for the thiol-disulfide exchange involving N-linked Hcy.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5.
A thiol of N-linked Hcy in albumin (Alb) participates in thiol-disulfide exchange. Equimolar amounts of albumin-Cys34-S-S-Cys and N-(Hcy-SH)-albumin-Cys34-S-IAA (containing Cys34 thiol blocked with IAA) were incubated separately or together for 4 h at 37 °C and analyzed by anion exchange HPLC. Albumin-Cys34-S-S-Cys (broken trace) and N-(Hcy-SH)-albumin-Cys34-S-IAA (thin trace) elute at 13.3 min. A peak of albumin-Cys34-SH appears at 12.9 min when both forms of albumin are mixed together (thick trace).

 
We next examined whether other N-homocysteinylated proteins can participate in the thiol-disulfide exchange with albumin-Cys34-S-S-Cys. An experiment with N-(Hcy-SH)-transferrin and albumin is shown in Fig. 6. The preparation of albumin (solid trace) contained albumin-Cys34-S-S-Cys and albumin-Cys34-SH, which elute from an anion exchange HPLC column with retention times of 14.0 and 13.2 min, respectively. The retention time of transferrin was 4 min. When albumin and N-(Hcy-SH)-transferrin were mixed together and analyzed by anion exchange HPLC, the peak of albumin-Cys34-S-S-Cys at 14.0 min completely disappeared (broken trace). Unmodified transferrin did not affect the elution pattern of albumin (not shown). This shows that albumin-Cys34-S-S-Cys was converted to albumin-Cys34-SH in the presence of N-(Hcy-SH)-transferrin. N-Homocysteinylated fibrinogen, antitrypsin, hemoglobin, myoglobin, and cytochrome c, but not native proteins, also removed cysteine from the albumin-Cys34-S-S-Cys disulfide with the liberation of albumin-Cys34-SH (not shown).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 6.
Thiol-disulfide exchange between N-homocysteinylated transferrin and albumin-Cys34-S-S-Cys. Equimolar amounts of albumin and N-(Hcy-SH)-transferrin were incubated together or separately and analyzed by anion exchange HPLC. Albumin (Alb) analyzed separately (solid trace) shows peaks of albumin-Cys34-S-S-Cys (at 14.0 min) and albumin-Cys34-SH (at 13.2 min). Peak of albumin-Cys34-S-S-Cys disappears when albumin and N-(Hcy-SH)-transferrin are incubated together (broken trace).

 
N-Homocysteinylation Affects Susceptibility of Albumin to Oxidation—Because N-homocysteinylation results in addition of thiol group(s) to albumin, modified albumin should become more prone to formation of intermolecular aggregates after oxidation. Indeed, treatment of N-homocysteinylated albumin with H2O2 resulted in formation of albumin aggregates (eluting as a broad peak at 18 min), which bound more strongly than monomeric forms to the DEAE column (Fig. 7A). Treatment of N-homocysteinylated and oxidized sample with DTT resulted in the disappearance of aggregates, suggesting that they were formed as a result of intermolecular disulfide bond formation. Native albumin did not form appreciable aggregates upon treatment with H2O2 (Fig. 7B), which suggests that thiols introduced by N-homocysteinylation are required for aggregation.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 7.
N-Homocysteinylation affects susceptibility of albumin to oxidation. Protein profiles from anion exchange HPLC analyses are shown. A, albumin (Alb) aggregates (4th peak eluting at 18 min, labeled N-Hcy-Alb(ox)) are present in a sample of H2O2-oxidized N-Hcy-albumin (solid trace). After DTT treatment the peak at 18 min becomes smaller, the peak of albumin-Cys34-S-S-Cys disappears (2nd peak), and the 1st peak, corresponding to albumin-Cys34-SH, becomes larger (broken trace). B, minor peak is visible at 18 min on HPLC profile of H2O2-oxidized native albumin (solid trace). After DTT treatment the minor peak at 18 min is still visible (labeled Alb(ox)), but the peak of albumin-Cys34-S-S-Cys (2nd peak) disappears, and the peak of albumin-Cys34-SH (1st peak) becomes larger (broken trace). Identity of the 3rd peak is not known.

 
N-Homocysteinylation Affects Susceptibility of Albumin to Proteolysis—Structural changes caused by N-homocysteinylation may affect proteolytic turnover of the modified protein. To determine this, we examined the susceptibility of unmodified and N-homocysteinylated forms of albumin to trypsin, chymotrypsin, elastase, and cathepsin D. As shown in Fig. 8, two major forms of circulating albumin, albumin-Cys34-S-S-Cys and albumin-Cys34-SH differed in their susceptibility to digestion by trypsin, with the former being degraded faster than the latter. The nature of a thiol bound by a disulfide linkage to Cys34 of albumin does not seem to affect its sensitivity to proteolytic digestion by trypsin; albumin-Cys34-S-S-Hcy was degraded by trypsin as efficiently as was albumin-Cys34-S-S-Cys (not shown). After N-homocysteinylation, albumin-Cys34-SH became more resistant, but the sensitivity of N-(Hcy-S-S-Cys)-albumin-Cys34-SH to trypsin was not much different from that of unmodified form (Fig. 8). N-(Hcy-S-S-Cys)-albumin-Cys34-SH and N-(Hcy-S-S-Cys)-albumin-Cys34-S-S-Cys exhibited similar sensitivities to trypsin (not shown). Similar pattern of susceptibilities to proteolysis were also observed with chymotrypsin (Fig. 8). There were only minor differences in susceptibilities of different forms of albumin to elastase or cathepsin D (Fig. 8).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 8.
N-Homocysteinylation affects susceptibility of albumin to proteolytic degradation. Albumin-Cys34-SH, N-(Hcy-SH)-albumin-Cys34-SH, N-(Hcy-S-S-Cys)-albumin-Cys34-SH, or albumin-Cys34-S-S-Cys was digested for 2 h at 37 °C with the indicated protease at an enzyme/substrate ratio 1:25. Samples were analyzed by SDS-PAGE under reducing conditions. Albumin (Alb) bands, visualized by Coomassie Blue staining, were quantified by densitometric scanning. Percentages ± S.D. of undigested albumin remaining after treatments with trypsin, chymotrypsin, elastase, or cathepsin D are shown.

 
Lys525 in Albumin Undergoes Preferential N-Homocysteinylation in Vitro—To identify site(s) susceptible to N-homocysteinylation, albumin was modified with Hcy-thiolactone at a molar ratio 1:1, reduced with DTT, acetamidated with IAA to block thiols, and digested with trypsin. Tryptic peptides were separated by reversed phase HPLC (Fig. 9A). A predominant N-Hcy-peptide (eluting in 32 min fraction) was further purified by cation exchange HPLC (Fig. 9C). N-Homocysteinylated peptides were detected by using tryptic digests of N-[35S]Hcy-albumin in parallel experiments (Fig. 9, B and D). Purified N-Hcy-peptide was subjected to MALDI-TOF mass spectrometric analysis. A single signal at m/z of 1,302.83 (Fig. 10A) was observed on mass spectra of the N-Hcy-peptide. This mass corresponds to an acetamidated and N-homocysteinylated peptide containing Lys525, 525KQTALVELVK534 (calculated mass of the derivatized peptide is 1,302.8; the site of N-homocysteinylation is in boldface). This peptide was also observed on mass spectra of unpurified tryptic digests of N-Hcy-albumin (not shown).



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 9.
Purification of a predominant N-homocysteinylated peptide from N-Hcy-albumin. Tryptic peptides were prepared from N-Hcy-albumin (A and C) or N-[35S]Hcy-albumin (B and D) and analyzed by reversed phase C18 HPLC (A and B) followed by cation exchange HPLC (C and D). Arrows in A and C indicate position of a major N-Hcy-peptide.

 



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 10.
MALDI-TOF mass spectra of a predominant Hcy-containing tryptic peptide form of N-Hcy-albumin (A) and native albumin (B). A, single signal at m/z 1,302.83 is observed for a sample of a major Hcy-containing tryptic peptide isolated from albumin modified with Hcy-thiolactone in vitro. B, a similar signal at m/z 1,302.92 is observed in a sample prepared from a tryptic digest of native albumin. The signal corresponds to an N-homocysteinylated and acetamidated peptide, 525KQTALVELVK534, from human serum albumin (site of N-homocysteinylation is in boldface).

 
Lys525 in albumin-Cys34-S-S-Cys was N-homocysteinylated significantly faster than the same lysine residue in N-Hcy-albumin-Cys34-SH. By analyzing tryptic digests on a reversed phase C18 HPLC column (Fig. 9B), we found that 9.22 ± 1.13% (n = 5) and 4.91 ± 0.92% (n = 7) of N-linked Hcy in N-[35S]Hcy-albumin-Cys34-S-S-Cys and N-[35S]Hcy-albumin-Cys34-SH, respectively, was attached to Lys525. Thus, the reactivity of Lys525 is affected by the status of Cys34.

Lys525 in Native Albumin Is N-Homocysteinylated—Although in vitro data indicated that Lys525 is a predominant site susceptible to N-homocysteinylation, it is unclear whether this site is also N-homocysteinylated in vivo. To determine this, native albumin was isolated from two human subjects (having elevated plasma total Hcy levels of 40–80 µM) and analyzed for the Lys525 modification. To facilitate detection of N-linked Hcy, native human serum albumin was enriched in N-Hcy-containing species by using thiopropyl-Sepharose. Such enriched preparations were reduced with DTT, modified with IAA, and digested with trypsin. Putative peptide containing N-homocysteinylated Lys525 was purified by HPLC and subjected to mass spectrometric analysis. A peptide with a mass of 1,302.92, corresponding to the mass of Lys525-containing peptide, was present in a MALDI-TOF spectrum (Fig. 10B). The signal at m/z 1,302.92 was about 2-fold smaller for albumin from a subject who had 40 µM tHcy compared with a subject who had 80 µM tHcy. The signal at m/z 1,302.92 was also observed in analysis of commercial native albumin but was much less intense (not shown). These data strongly suggest that Lys525 is a site of N-homocysteinylation in native albumin in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human serum albumin is the major plasma protein (30) which is also a major target for N-homocysteinylation by Hcy-thiolactone in vitro (22) and in vivo (26). The present work identifies Lys525 as a preferential site of N-homocysteinylation in human serum albumin in vitro and in vivo, and provides evidence for specific structural alterations caused by N-homocysteinylation. The findings of this work also suggest that a disulfide at Cys34, a conserved residue in albumins from various organisms, facilitates conversion of N-homocysteinylated mercaptoalbumin to a proteolytically sensitive form.

The present data support the following mechanisms of albumin N-homocysteinylation. Hcy-thiolactone reacts with lysine residues of both major forms of circulating albumin. However, the status of Cys34 affects the rate of N-homocysteinylation; the reaction with albumin-Cys34-S-S-Cys is faster than with albumin-Cys34-SH. The reactivity of Lys525, a predominant N-homocysteinylation site, in albumin-Cys34-S-S-Cys is about 2-fold greater than in albumin-Cys34-SH. The initial product of N-homocysteinylation of albumin-Cys34-S-S-Cys (N-(Hcy-SH)-albumin-Cys34-S-S-Cys) is not observed because it undergoes a rapid thiol-disulfide exchange to form N-(Hcy-S-S-Cys)-albumin-Cys34-SH (Reaction 1). Another possible product of thiol-disulfide exchange between N-(Hcy-S-S-Cys)-albumin-Cys34-SH and albumin-Cys34-S-S-Cys, N-(Hcy-S-S-Cys)-albumin-Cys34-S-S-Cys is also not observed, suggesting that the equilibrium of the reaction is shifted far to the left. The thiol-disulfide exchange occurs in trans between different molecules of N-(Hcy-SH)-albumin-Cys34-S-S-Cys. An intramolecular thiol-disulfide exchange is unlikely, because residue Cys34 (domain IA) is located too far away from Lys525 (domain IIIB) in the structure of human serum albumin (30, 35). Facile exchange is known to occur between free reduced Hcy and albumin-Cys34-S-S-Cys (32).

N-Homocysteinylation of mercaptoalbumin affords N-(Hcy-SH)-albumin-Cys34-SH (Reaction 4), which undergoes thioldisulfide exchange with albumin-Cys34-S-S-Cys to yield N-(Hcy-S-S-Cys)-albumin-Cys34-SH (Reaction 5).

(REACTION 4)

(REACTION 5)
Thus, N-homocysteinylation of a mixture of albumin-Cys34-SH and albumin-Cys34-S-S-Cys, which is present in circulation (31), leads to a single N-homocysteinylated product, N-(Hcy-S-S-Cys)-albumin-Cys34-SH. The equilibrium is strongly shifted toward N-(Hcy-S-S-Cys)-albumin-Cys34-SH because the Cys34 thiolate anion has an unusually low pKa of ~5 (30) and thus is more thermodynamically stable than Hcy thiolate anion. The low pKa of the Cys34 thiolate also makes the thiol-disulfide exchange of N-(Hcy-SH)-albumin-Cys34-SH with albumin-Cys34-S-S-Cys thermodynamically more favored than with cystine. For example, under conditions where the thiol-disulfide exchange between N-(Hcy-SH)-albumin-Cys34-SH and albumin-Cys34-S-S-Cys went to completion (Fig. 3), the exchange with cysteine was <20% complete (not shown). Thiol-disulfide exchange reactions between albumin-Cys34-SH and cystine, homocystine, or Cys-S-S-Hcy disulfide are known to be slow, being about 20% or less complete in 4 h (32, 36).

It has been hypothesized that metabolic conversion of Hcy to Hcy-thiolactone, the reactivity of Hcy-thiolactone toward proteins, and resulting protein damage contribute to pathologies associated with elevated Hcy levels in human beings (21–23). If this hypothesis is correct, it is likely that protective mechanism(s) against Hcy-thiolactone have evolved. One possible protective mechanism can be provided by Hcy-thiolactonase/paraoxonase, a component of high density lipoprotein, which detoxifies Hcy-thiolactone by hydrolyzing it to Hcy (28, 29).

Human serum albumin, because of its abundance and the ability to avidly react with Hcy and Hcy-thiolactone, is likely to serve an important dual protective role. Consistent with this suggestion are the observations that S-Hcy-albumin and N-Hcy-albumin account for most (>90%) of S-linked and N-linked Hcy, respectively, present in human plasma protein and for >90% of total plasma Hcy (22, 26, 28). In addition to S-Hcy-protein (26, 31, 32) and N-Hcy-protein (12, 13, 26–28), Hcy exists in circulation as free reduced Hcy (31), Hcy-thiolactone (24, 25), and disulfide forms with low molecular weight thiols (31). Of these, Hcy-thiolactone (10, 17, 21–23, 26, 28, 29) and free reduced Hcy (37), which comprise 1.4 (24, 25) and 2% (31) of plasma total Hcy, respectively, have been suggested to be the deleterious forms of Hcy. For example, free reduced Hcy is associated with endothelial dysfunction, whereas disulfide forms of Hcy, including albumin-Cys34-S-S-Hcy, are not (37). Hcy-thiolactone induces caspase-independent vascular endothelial cell death with apoptotic features (38). Thus, the conversion of Hcy into albumin-Cys34-S-S-Hcy would prevent cellular uptake of Hcy and therefore minimize the conversion to Hcy-thiolactone. Indeed, albumin-Cys34-S-S-Hcy comprises 82%, whereas reduced Hcy accounts for only 2% of "total" Hcy in human serum (31). In addition, albumin detoxifies a significant fraction of Hcy-thiolactone by virtue of N-homocysteinylation. Plasma pool of N-linked Hcy comprises up to 25% of total plasma Hcy in humans, with N-Hcy-albumin accounting for 90% of plasma N-Hcy-protein (22, 26). Both N-linked and S-linked protein Hcy are most likely detoxified in the liver where transmethylation and trans-sulfuration pathways of Hcy metabolism are the most active (1, 2). Consistent with this suggestion are our findings that N-(Hcy-S-S-Cys)-albumin-Cys34-SH, albumin-Cys34-S-S-Cys, and albumin-Cys34-S-S-Hcy are more susceptible to proteolysis than albumin-Cys34-SH. Although N-(Hcy-SH)-albumin-Cys34-SH is resistant to proteolytic digestion, this form is unlikely to exist in circulation because it undergoes the thiol-disulfide exchange with albumin-Cys34-S-S-Cys, which converts it into proteolysis-prone form, N-(Hcy-S-S-Cys)-albumin-Cys34-SH (Reaction 5). These findings suggest that a disulfide at residue Cys34 in albumin may have an important role in facilitating proteolytic turnover of N-homocysteinylated albumin.

Different proteolytic susceptibilities of albumin-Cys34-SH and albumin-Cys34-S-S-Cys suggest that albumin adopts a different structure depending on the state of Cys34. Indeed, a structural transition in albumin dependent on the state of Cys34 has been detected by NMR spectroscopy (39). Our data suggest that N-homocysteinylation interferes with this structural transition.

Because it is a downstream metabolite that, most likely, reflects damage caused by Hcy, N-linked Hcy could be a new marker of cardiovascular risk, possibly more predictive than total Hcy. To determine this, it would be important to monitor protein N-homocysteinylation in human beings. Present methods of monitoring protein N-homocysteinylation are relatively complex and thus not very useful in a clinical setting. However, identification of Lys525 as a predominant N-homocysteinylation site in human serum albumin opens up a way of designing new diagnostic tools for monitoring cardiovascular risk associated with elevated levels of plasma Hcy. For example, specific antibodies can be raised against N-homocysteinylated peptides of albumin containing Lys525 and used to monitor the status of albumin N-homocysteinylation in human beings.


   FOOTNOTES
 
* This work was supported by grants from the National Science Foundation and the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Dept. of Environmental Chemistry, University of Lódz, 90-236 Lódz, Poland. Back

|| To whom correspondence should be addressed. Tel.: 973-972-4483 (ext. 28733); Fax: 973-972-8982; E-mail: jakubows{at}umdnj.edu.

1 The abbreviations used are: Hcy, homocysteine; DTT, dithiothreitol; N-Hcy-albumin, albumin containing Hcy bound by an amide linkage; S-Hcy-albumin, albumin containing Hcy bound by a disulfide linkage; HPLC, high performance liquid chromatography; IAA, iodoacetamide; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. Back


   ACKNOWLEDGMENTS
 
We thank Helga Refsum for kindly providing samples of human plasma from subjects with hyperhomocysteinemia. We also thank Andrzej Guranowski for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Mudd, S. H., Levy, H. L. & Krauss, J. P. (2001) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, D., eds) 8th Ed., Vol. II, pp. 2007–2056, McGraw Hill Inc., New York
  2. Rosenblatt, D. S. & Fenton, W. A. (2001) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, D., eds) 8th Ed., Vol. II, pp. 3897–3936, McGraw Hill Inc., New York
  3. Homocysteine Studies Collaboration (2002) J. Am. Med. Assoc. 288, 2015–2022[Abstract/Free Full Text]
  4. Klerk, M., Verhoef, P., Clarke, R., Blom, H. J., Kok, F. J., Schouten, E. G. & MTHFR Studies Collaboration Group (2002) J. Am. Med. Assoc. 288, 2023–2031[Abstract/Free Full Text]
  5. Anderson, J. L., Muhlestein, J. B., Horne, B. D., Carlquist, J. F., Bair, T. L., Madsen, T. E. & Pearson, R. R. (2000) Circulation 102, 1227–1232[Abstract/Free Full Text]
  6. Seshadri, S., Beiser, A., Selhub, J., Jacques, P. F., Rosenberg, I. H., D'Agostino, R. B., Wilson, P. W. & Wolf, P. A. (2002) N. Engl. J. Med. 346, 476–483[Abstract/Free Full Text]
  7. Zhang, C., Cai, Y., Adachi, M. T., Oshiro, S., Aso, T., Kaufman, R. J. & Kitajima, S. (2001) J. Biol. Chem. 276, 35867–35874[Abstract/Free Full Text]
  8. Mattson, M. P. & Shea, T. B. (2003) Trends Neurosci. 26, 137–146[CrossRef][Medline] [Order article via Infotrieve]
  9. Chen, P., Poddar, R., Tipa, E. V., Dibello, P. M., Moravec, C. D., Robinson, K., Green, R., Kruger, W. D., Garrow, T. A. & Jacobsen, D. W. (1999) Adv. Enzyme Regul. 39, 93–109[CrossRef][Medline] [Order article via Infotrieve]
  10. Jakubowski, H. (2004) Cell. Mol. Life Sci. 60, in press
  11. Jakubowski, H. (1997) in Homocysteine Metabolism: From Basic Science to Clinical Medicine (Graham, I., Refsum, H., Rosenberg, I. H. & Ueland, P. M., eds) pp. 157–165, Kluwer Academic Publishers, Norwell, MA
  12. Jakubowski, H. (2000) J. Nutr. 130, (suppl.) 377–381[Abstract/Free Full Text]
  13. Jakubowski, H. (2001) J. Nutr. 131, (suppl.) 2983–2987
  14. Jakubowski, H. (2001) Encyclopedia of Life Sciences, Vol. 18, pp. 441–449, Nature Publishing Group, London
  15. Jakubowski, H. (2001) Biomed. Pharmacother. 55, 443–447[CrossRef][Medline] [Order article via Infotrieve]
  16. Jakubowski, H. (2001) in Homocysteine in Health and Disease (Jacobsen D. W. & Carmel, R., eds) pp. 21–31, Cambridge University Press, UK
  17. Jakubowski, H. (2003) Clin. Chem. Lab. Med. 41, 1462–1466[CrossRef][Medline] [Order article via Infotrieve]
  18. Jakubowski, H. (2004) in The Aminoacyl-tRNA Synthetases (Ibba, M., Francklyn, C. & Cusack, S., eds) Landes Bioscience, Georgetown, TX, in press
  19. Jakubowski, H. & Fersht, A. (1981) Nucleic Acids Res. 9, 3105–3117[Abstract/Free Full Text]
  20. Jakubowski, H. & Goldman, E. (1993) FEBS Lett. 317, 593–598
  21. Jakubowski, H. (1997) J. Biol. Chem. 272, 1935–1941[Abstract/Free Full Text]
  22. Jakubowski, H. (1999) FASEB J. 13, 2277–2283[Abstract/Free Full Text]
  23. Jakubowski, H., Zhang, L., Bardeguez, A. & Aviv, A. (2000) Circ. Res. 87, 45–51[Abstract/Free Full Text]
  24. Jakubowski, H. (2002) Anal. Biochem. 308, 112–119[CrossRef][Medline] [Order article via Infotrieve]
  25. Daneshvar, P., Yazdanpanah, M., Cuthbert, C. & Cole, D. E. (2003) Rapid Commun. Mass Spectrom. 17, 258–262
  26. Jakubowski, H. (2002) J. Biol. Chem. 277, 30425–30428[Abstract/Free Full Text]
  27. Uji, Y., Motomiya, Y., Hanyu, N., Ukaji, F. & Okabe, H. (2002) Clin. Chem. 48, 941–944[Free Full Text]
  28. Jakubowski, H. (2000) J. Biol. Chem. 275, 3957–3962[Abstract/Free Full Text]
  29. Jakubowski, H., Ambrosius, W. & Pratt, J. H. (2001) FEBS Lett. 491, 35–39[CrossRef][Medline] [Order article via Infotrieve]
  30. Peters, T., Jr. (1996) All About Albumin, pp. 51–54, Academic Press Inc, San Diego, CA
  31. Mansoor, M. A., Svardal, A. S. & Ueland, P. M. (1992) Anal. Biochem. 200, 218–229[CrossRef][Medline] [Order article via Infotrieve]
  32. Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Budy, B., Ketterer, M. E. & Jacobsen, D. W. (2001) J. Biol. Chem. 276, 30111–30117[Abstract/Free Full Text]
  33. Jakubowski, H. (2000) J. Biol. Chem. 275, 21813–21816[Abstract/Free Full Text]
  34. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70–77[CrossRef][Medline] [Order article via Infotrieve]
  35. Zunszain, P. A., Ghuman, J., Komatsu, T., Tsuchida, E. & Curry, S. (2003) BMC Struct. Biol. 3, 6[CrossRef][Medline] [Order article via Infotrieve]
  36. Carballal, S., Radi, R., Kirk, M. C., Barnes, S., Freeman, B. A. & Alvarez, B. (2003) Biochemistry 42, 9906–9914[CrossRef][Medline] [Order article via Infotrieve]
  37. Chambers, J. C., Ueland, P. M., Wright, M., Dore, C., Refsum, H. & Kooner, J. S. (2001) Circ. Res. 89, 187–192[Abstract/Free Full Text]
  38. Mercie, P., Garnier, O., Lascoste, L., Renard, M., Closse, C., Durrieu, F., Marit, G., Boisseau, R. M. & Bolloc, F. (2000) Apoptosis 5, 403–411[CrossRef][Medline] [Order article via Infotrieve]
  39. Christodoulou, C., Sadler, P. J. & Tucker, A. (1994) Eur. J. Biochem. 94, 363–368

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
A. Undas, M. Kolarz, G. Kopec, R. Glowacki, E. Placzkiewicz-Jankowska, and W. Tracz
Autoantibodies against N-homocysteinylated proteins in patients on long-term haemodialysis
Nephrol. Dial. Transplant., June 1, 2007; 22(6): 1685 - 1689.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
G. Chwatko, G. H. J. Boers, K. A. Strauss, D. M. Shih, and H. Jakubowski
Mutations in methylenetetrahydrofolate reductase or cystathionine {beta}-syntase gene, or a high-methionine diet, increase homocysteine thiolactone levels in humans and mice
FASEB J, June 1, 2007; 21(8): 1707 - 1713.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Zimny, M. Sikora, A. Guranowski, and H. Jakubowski
Protective Mechanisms against Homocysteine Toxicity: THE ROLE OF BLEOMYCIN HYDROLASE
J. Biol. Chem., August 11, 2006; 281(32): 22485 - 22492.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
A. Undas, J. Brozek, M. Jankowski, Z. Siudak, A. Szczeklik, and H. Jakubowski
Plasma Homocysteine Affects Fibrin Clot Permeability and Resistance to Lysis in Human Subjects
Arterioscler. Thromb. Vasc. Biol., June 1, 2006; 26(6): 1397 - 1404.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
H. Jakubowski
Pathophysiological Consequences of Homocysteine Excess
J. Nutr., June 1, 2006; 136(6): 1741S - 1749S.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
G. Chwatko and H. Jakubowski
Urinary Excretion of Homocysteine-Thiolactone in Humans
Clin. Chem., February 1, 2005; 51(2): 408 - 415.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/12/10864    most recent
M313268200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Glowacki, R.
Right arrow Articles by Jakubowski, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Glowacki, R.
Right arrow Articles by Jakubowski, H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement