A Cysteine Protease That Processes Insect Vitellin PURIFICATION AND PARTIAL CHARACTERIZATION OF THE ENZYME AND THE PROENZYME*

,

Previous work demonstrated that Vt utilization in Blattella germanica eggs is initiated at days 3-4 postovulation by proteolytic processing to a distinct set of peptides, which are then accessed by the embryo during development (9,15,16). Similar processing events are characteristic of Vt utilization in other insect species also (see Ref. 9, and references therein).
Assays of B. germanica yolk with synthetic substrates revealed that protease activity, first detectable at days 2-3 postovulation, increased in specific activity through day 5, suggesting it could be important in Vt processing (9). Greater than 90% of this proteolytic activity was inhibitable by cysteine protease inhibitors (9), suggesting that this class of proteases play a major role in the processing event. These assays identified both cathepsin L-and B-like activities (17), the former constituting more than 85% of the total. The finding that the yolk of certain B. germanica translocation heterozygotes with defective embryo development and Vt processing lack yolk protease activity also implicates them (9,18).
Yolk granules prepared from eggs 4 -6 days postovulation accumulate high concentrations of the dye acridine orange, a probe for vesicle acidity, while those from eggs at days 0 -3 do not (19,20). The acidification was determined to be the result of proton translocation, suggesting that a vacuolar ATPase was involved (20). The temporal association of Vt processing with granule acidification suggested that acidification was part of the mechanism controlling protease activation. Other experiments showed that when the yolk of freshly ovulated eggs (which contains no detectable protease activity) was acidified in vitro, Vt processing occurred (20). These facts led us to speculate that the B. germanica Vt-processing protease is stored either as an acid-activated mature enzyme or, more likely, as a proprotein that is converted to a mature, catalytically active form; in either case, the population of catalytically active molecules would be controlled in vivo by granule acidification.
As part of the overall project aimed at understanding how the Vt-processing event is controlled, the major B. germanica yolk (Vt processing) protease was purified and partially characterized. The storage (precursor) form of this enzyme was also identified, purified, and its activated form compared with the processing protease. Proprotease activation was also examined in the context of possible regulation in vivo, by acidification. A preliminary report of some of this work has been published (17).
Insects and Tissue Preparations-B. germanica, from Carolina Biological Supply Co. (Burlington, NC), were reared in the laboratory at 30°C (21). Oothecae (egg cases) were collected daily. Day zero postovulation is the first 24 h following oothecal extrusion. Manduca sexta larval muscle extract was a kind gift from Dr. Lawrence Schwartz (Department of Biology, University of Massachusetts, Amherst). Protein concentration measurements (22) used an ovalbumin standard.
Protease Assays-Protease assays were conducted as described (9) using two broad-spectrum substrates: Z-LNE and azoalbumin. Z-Phe-Arg-NMec (hydrolyzed preferentially by cathepsins L, but slowly by cathepsins B) and Z-Arg-Arg-NMec (hydrolyzed by cathepsins B only; Ref. 23) were also used. With Z-LNE, 1 unit of enzymatic activity caused an increase in absorbance of 1.0/min at 326 nm; and with azoalbumin, an increase in absorbance of 1.0/h at 366 nm. With AMC, 1 unit released 1 pmol of AMC/s from Z-Phe-Arg-NMec or Z-Arg-Arg-NMec (determined by a standard curve). AMC formation was measured at 20-s intervals by fluorescence spectrophotometry at 370 nm (excitation) and 460 nm (emission).
Synthesis of Sepharose-Gly-Phe-Glycinal Semicarbazone-A modification of the procedure of Rich et al. (24) was employed at room temperature. Z-Gly-Phe-glycinal semicarbazone (48 mg) was dissolved in 1 ml of 10% (w/w) formic acid in tetrahydrofuran. Palladium black (48 mg), in 1 ml of water, was added, and the deblocking reaction was monitored by TLC (24). After 30 min, the suspension was filtered and the filtrate was concentrated at 20°C and lyophilized, affording a syrup (56 mg) of Gly-Phe glycinal semicarbazone. Activated Sepharose 4B was equilibrated overnight in 1 mM HCl at 4°C and then washed in a column (0.8 cm diameter; bed volume 3 ml) with 60 ml of 0.1 M NaHCO 3 , pH 8.0. Gly-Phe-glycinal semicarbazone (56 mg), in 5 ml of methanol, was diluted with 3 ml of 0.1 M NaHCO 3 , pH 8.0. The mixture was combined with the activated Sepharose in a small tube and incubated overnight on a nutator at 20°C, transferred back to the column, and washed with 100 ml of 50% aqueous methanol and 100 ml of water. It was agitated again for 4 h in 10 ml of 0.13 M ethanolamine buffer, pH 9.0 at 20°C and then washed with 100 ml of water containing 0.1% azide, prior to storage at 4°C.
Preparation of Yolk Extracts-Seventy oothecae (approximately 2.1 g; day 6 for protease, day 0 for proprotease,) stored up to 3 weeks at Ϫ20°C, were thawed in a 12-ml centrifuge tube, suspended in 6.2 ml of ice-cold CBS (10 mM citrate buffer, pH 5.5, containing 0.15 M NaCl and 1 mM EDTA; protease) or HBS (10 mM Hepes, pH 7.5, containing 0.15 M NaCl and 1 mM EDTA; proprotease). Buffers at neutral pH were required for proprotease purification to preclude protease activation. Oothecae were squashed against the tube's wall with a spatula and removed. The yolk extract was centrifuged at 6,600 ϫ g for 2 min. A floating lipid layer was discarded, and the supernatant was clarified by centrifugation at 13,000 ϫ g for 10 min. Protease activity was stable at pH 5.3 for at least 1 week at 4°C but decreased rapidly at pH 7.0. Although thiol reagents stimulated activity about 20% during assay, they increased the rate of deactivation during storage. Crude Vt was precipitated from ruptured oothecae in water, egg cases were separated, and Vt was removed by centrifugation at 6,000 ϫ g. Crude Vt was dissolved in HBS.
All purification steps were carried out at 4°C unless noted. The initial step, Sephacryl S-300 HR column chromatography, was employed to purify protease, proprotease, and protease-free Vt. Clarified supernatant (6 ml) containing about 450 mg of protein (day 6) or 1000 mg of protein (day 0) was chromatographed on a Sephacryl column (91 ϫ 2.6 cm diameter) equilibrated in, and then eluted with CBS (protease) or HBS (proprotease and Vt), respectively, using a peristaltic pump at 3 ml/min. Protease Purification-Fractions (8 ml) from Sephacryl chromatography were assayed for protease activity with Z-LNE, for protein content, and for absorbance at 280 nm. Active fractions were combined and concentrated using an Amicon filtration unit and a YM 10 membrane. At this stage, the enzyme retained full activity after 48 h at 4°C and pH 5.3. Chilled Sepharose-Gly-Phe-Glycinal semicarbazone (3 ml) was equilibrated in CBS, combined with protease ultrafiltration retentate (6 ml, 24 mg of protein) and incubated overnight to assure maximum sample absorption. The mixture was then poured into a 0.8-cm diameter column and washed with 10 ml of modified CBS fortified to 0.5 M in NaCl followed by 10 ml of CBS. Approximately 84% of the enzyme units (Z-LNE) were retained by the support. Column contents were incubated overnight in CBS containing 4 mM HgCl 2 at 4°C to dissociate the enzyme, which was then eluted with this buffer at room temperature. One aliquot of each fraction was assayed for protein content. A second was incubated in 0.125 M acetate buffer, pH 4.2, containing 40 mM ␤-MSH (final concentrations) for 10 min at room temperature to reactivate the enzyme, which was assayed with Z-LNE. Active fractions were pooled and concentrated with a centriprep tube (M r 10,000 cutoff) and stored at 4°C in CBS containing 4 mM Hg 2ϩ .
Proprotease Purification-Following Sephacryl chromatography, 30 l of each fraction were acidified as described above for 30 min to activate the putative proenzyme. Control aliquots were incubated in HBS containing ␤-MSH. Fractions containing acid-activable protease were pooled. The active pool from the previous step (100 ml, 10 mg of protein) was chromatographed on a Mono Q HR 5/5 column, equilibrated with HBS, using a Pharmacia model LCC-500 FPLC system at room temperature. A segmented elution gradient (maximum NaCl concentration 1 M) was programmed at a flow rate of 1.0 ml/min. Fractions of 1.9 ml were assayed with Z-Phe-Arg-NMec before and after acid activation, and for protein content. The Mono Q pool (13 ml, 0.5 mg of protein) which was approximately 0.25 M in NaCl, was chromatographed on a phenyl Sepharose CL-4B column (0.8 cm diameter, 2 ml bed volume) in HBS containing 0.25 M NaCl. The column was given washes (2 ml each) of HBS containing 0.1 M NaCl and 0.05 M NaCl, respectively. Proprotease was eluted using a step gradient with 1 ml each of 10, 8, 6, 4, and 2 mM Hepes, pH 7.5. The column was then washed with 5 ml of water. Fractions (0.8 ml) were assayed for acidactivable protease, conductivity, and protein content.
Stability of proprotease in 1% SDS at pH 7.5 permitted preparative SDS-PAGE (Bio-Rad Prep Cell, model 491) to be used as an alternative last step. The separation gel (1 ϫ 2.8 cm diameter) was composed of 5% acrylamide in 10 mM sodium phosphate buffer pH 7.5, containing 2.65% cross-linker. The sample buffer was 20 mM phosphate, pH 7.5, containing 0.13% SDS, 33% glycerol, and 0.08 mg/ml bromphenol blue. The electrode buffers were 6.7 mM phosphate, pH 7.5 (lower chamber), and 6.7 mM phosphate, pH 7.5, containing 0.067% SDS (upper chamber). Samples (2-5 mg of protein) were mixed with an equal volume of sample buffer and electrophoresed at 30 mA and 110 V with an elution rate at 1 ml/min, and fractions of 3.2 ml were collected.
Protease generated from acidification of proprotease was always stabilized in either 0.25 M acetate buffer, pH 4.2, or 0.25 M citrate buffer, pH 5.3, each containing 40 mM ␤-MSH and used directly in experiments, avoiding prolonged storage. The most purified preparations of protease and proprotease were used in all subsequent studies.
pH Optimum-To determine the enzyme's pH optimum, 0.25 M citrate buffer was used between pH 1.5 and 6.5, and 0.25 M Tris from pH 7.0 to 9.0. Protease was reactivated (specific activity 342 units (Z-LNE)/mg of protein) and 0.02 g of protein was assayed in 1 ml of each buffer containing 0.05% Brij 35 and 5 M Z-Phe-Arg-NMec. Activated proprotease (0.25 g of protein) was assayed in the same manner.
Protease Classification-Yolk extract, purified proprotease, or protease were incubated individually with the inhibitors listed in Table I in CBS for 15 min at 30°C. Proprotease, 10 l (11 g of protein) was activated for 1 h prior to treatment and assay for residual activity. Control preincubations lacked inhibitor. To differentiate cathepsins B and L, the protease's reactivity with Z-Arg-Arg-NMec and stability in 3 M urea (23) were determined.
Proteolytic Processing of Vt in Vivo-Lipid-free yolk extract was prepared in HBS at various times following oothecal extrusion. One aliquot was assayed for protease with Z-LNE and for protein concentration. A second was combined with an equal volume of HBS containing a mixture of 8 mM Pefabloc SC, 2 mM pepstatin, and 40 M E-64, and its peptide composition analyzed by SDS-PAGE in 10%, 15%, and 20% polyacrylamide gels.
Proteolytic Processing of Vt in Vitro-Ten l of the Hg 2ϩ form of the protease was reactivated (specific activity 212 units of Z-LNE/mg) and mixed with 10 l of CBS, pH 5.3, and incubated under toluene vapors with 300 g of protease-free Vt at 30°C. Five-l aliquots were withdrawn periodically and analyzed by SDS-PAGE. The Hg 2ϩ protease, incubated with ␤-MSH-free CBS, pH 5.3, prior to addition of proteasefree Vt, served as a control.
Electrophoresis-SDS-PAGE was conducted by a modification (25) of the Laemmli method (26) at 75 mA/gel as described previously (16). Nondenaturing PAGE was run at 200 V by a modification (Sigma bulletin no. MKR-137) of Bryan's procedure (27). Staining with silver or colloidal Coomassie Blue (Pro-Blue) reagents was done according to the manufacturer's instructions.
Electroblotting-The procedure of Burnette (28) was used as described (16) using Ponceau S to locate peptides. To detect biotinylated peptides, membranes were blocked in 5% nonfat milk, washed in phosphate-buffered saline containing 0.05% Tween 20, and incubated with streptavidin-HRP. Membranes were then incubated with shaking for 1 min in Luminol reagent following the manufacturer's instructions and exposed to x-ray film in the dark room, generally for 15 s to 2 min.
Biotinylation-Peptides were labeled with the active site probe biotinyl-Phe-Ala-CHN 2 , using a method modified from Cullen et al. (29). Sample (25-250 g of protein) was combined with 40 l of 100 mM acetate buffer, pH 4.2, containing 10 mM ␤-MSH, 1 mM EDTA, and 0.1% Brij 35. Biotinylated probe (5 l, 6 mM in methanol) was added, and the mixture was incubated at 4°C overnight. The reaction was stopped by heating 10 min in a boiling water bath with an equal volume of sample buffer (26). To distinguish between specific and nonspecific labeling, samples were first preincubated for 10 min at 30°C with 100 M E-64. Biotinylation of covalently bound sugars was done before and after endo H treatment. Following SDS-PAGE and electroblotting on a polyvinylidene difluoride membrane, oligosaccharides were visualized using the Glyco Track™ system following the manufacturer's instructions.
Deglycosylation of Proteins-Purified protease, proprotease, or Vt (5 l, 15 g of protein in HBS, containing 40 mM ␤-MSH) were each combined with 5 l of 100 M E-64 in 0.25 M citrate buffer, pH 5.5, and incubated at 4°C overnight to preclude proteolysis. Mixtures were added to 5 l of 1% aqueous SDS and heated 5 min in a boiling water bath. SDS was sequestered by adding 5 l of 0.5 M citrate buffer, pH 5.5 containing 14% Triton X-100. Five milliunits of endo H (30) were added, and the tubes were incubated at 37°C overnight under toluene vapors. Buffer was substituted for endo H in controls. Reactions were stopped by heating in a boiling water bath with an equal volume of SDS-PAGE sample buffer.
Proprotease Activation-In vivo, the course of proprotease activation was monitored in yolk extracts from eggs at days 0 -6 postovulation prepared in HBS. Samples were incubated with biotinyl-Phe-Ala-CHN 2 with, or without, prior treatment with E-64, as described above. Analysis of peptides was conducted using SDS-PAGE and electroblotting. In vitro, proprotease (4 l, 15 g of protein) was mixed with 4 l of 0.25 M citrate buffer, pH 5.3, or with 4 l of HBS, pH 7.5 (control). The same amount of proprotease in HBS, pH 7.5, was also incubated individually with 2 l of E-64 (40 M), Pefabloc SC (4 mg/ml), pepstatin (14 g/ml), or EDTA (4 mM) at room temperature for 1 h before addition of 2 l of 0.5 M citrate buffer, pH 5.3. Reaction mixtures were incubated at 30°C for an additional 12 h and then heated in a boiling water bath for 1 min with SDS-PAGE sample buffer. Following SDS-PAGE, gels were stained with colloidal Coomassie Blue.
Action of Purified Day 6 Protease on Day 0 Proprotease-Aliquots of purified proprotease (1.1 g of protein) and purified protease (1.7 ng of protein) were brought to 100 l with 0.25 M citrate buffer, pH 6.0. Aliquots of 10 l were withdrawn every 20 min and assayed with Z-Phe-Arg-NMec. Controls contained water substituted for proprotease or protease.
Effect of Proprotease Concentration on Its Activation-Purified proprotease (1.1 g of protein) was diluted 10-, 10 2 -, or 10 4 -fold in activation buffer to give total volumes of 0.01, 0.1, and 10 ml, respectively. Aliquots of 0.5, 5, and 500 l (55 ng of proprotease each) were then removed at intervals of 10, 30, 60, 120, and 240 min and adjusted to 500 l with 0.25 M citrate buffer, pH 5.5, containing 40 mM ␤-MSH. (The uniform final volume equalized the possible effect of activation buffer on the protease assay.) This solution was assayed for protease activity with Z-Phe-Arg-NMec.

RESULTS
Protease Purification-Tests of the crude extract with inhibitors at concentrations effective with the various protease classes (31) showed that the predominant activity is a cathepsin L-like cysteine protease (Table I). Sephacryl S-300 chromatography of the extract afforded a good separation of protease from processed Vt polypeptides (Fig. 1). Three non-protein peaks (fractions 59 -65, 75-81, and 84 -96) were not investigated further. The pool of fractions 36 -58, enriched about 20-fold from the crude extract, was active with Z-Phe-Arg-NMec but not Z-Arg-Arg-NMec, also indicative of cathepsins L. This three-step procedure gave a 300-fold purification of the protease, which could be stored as the Hg 2ϩ form for up to 1 month at pH 5.3 and 4°C. Reactivated protease was stable in CBS, pH 5.0, at 4°C for at least 12 h.
Properties of the Protease- Fig. 2 Fig. 4). Previous work demonstrated that the yolk granules become acidified at days 3-4 postovulation (20), their pH decreasing from neutral to about 5.5. Thus, the profile is consistent with protease activation in vivo being regulated by granule acidification. Purified protease had the same relative susceptibility to inhibitors as the crude extract (Table I). TLCK and TPCK, inhibitors of trypsin and chymotrypsin-like serine proteases respectively (31), also inhibit some cysteine proteases (32). Protease-free Vt-When Vt is prepared by ion exchange chromatography of day 0 yolk (33), it retains some acid-activable protease, which made it unsuitable for in vitro enzyme action pattern studies (described below). Vt isolated by Sephacryl S-300 chromatography of day 0 yolk (see Fig. 6) was acceptable. It showed no evidence of degradation (by SDS-PAGE) after acidification and no cysteine protease contamination when probed with biotinyl-Phe-Ala-CHN 2 (data not shown).
Proteolytic Processing of Vt-The time course of Vt processing by the protease in vitro was compared with the in vivo pattern. Table II summarizes the results of these studies. In both situations the M r 102,000, 95,000, and 50,000 subunits of Vt were degraded completely and "limit" peptides of M r 53,000, 22,000, 21,000, and 20,000 accumulated. In addition, peptides of M r 88,000, 68,000 -77,000, 40,000 -43,000, and 30,000 were produced. Unique limit peptides in vitro were M r 88,000, 70,000, and 50,000 -53,000. Processing of the M r 50,000 subunit in vitro was obscured by other products of that approximate size, but this subunit is processed beginning at day 5 (16,18). Addition of fresh enzyme to the in vitro incubation did not alter the product distribution. Thus, despite the enzyme's activity with synthetic substrates, it catalyzes only limited proteolysis of Vt in vitro, which is what occurs in vivo.
Evidence for a Proenzyme Precursor-Previous work (20) demonstrated that Vt in day 0 yolk was processed at acidic pH.
To check for the presence of a proenzyme, reactivities of day 0 and day 6 yolk with biotinyl-Phe-Ala-CHN 2 (29) were compared FIG. 1. Sephacryl S-300 HR column chromatography of day 6 yolk extract. Extract (6 ml, 464 mg of protein) was chromatographed at 4°C on a Sephacryl S-300 HR column (91 ϫ 2.6 cm diameter. Fractions were assayed for protein content, absorbance at 280 nm, and protease activity with Z-LNE. Fractions indicated by the bracket were pooled and concentrated.

FIG. 2. SDS-PAGE analysis of the protease purification.
Samples were subjected to SDS-PAGE (10% gel, 5 g of protein/lane) and peptide bands were visualized by silver staining. Lanes 1, 2, and 3 contain samples of yolk extract, Sephacryl S-300 pool, and affinity purified protease, respectively. Stds, lanes containing polypeptide molecular size standards.

FIG. 3. SDS-PAGE and electroblotting analysis of the protease purification.
Samples were biotinylated and subjected to SDS-PAGE and electrophoretic transfer. Peptide bands were visualized with streptavidin-HRP and Luminol as described under "Experimental Procedures." Lane 1, crude extract (30 g of protein). Lane 2, S-300 pool (5 g). Lane 3, affinity purified protease (2 g). Lane 4, affinity-purified enzyme (2 g) was preincubated with 100 M E-64 prior to biotinylation. (Fig. 5). Day 6 yolk (lane 1) contains a broad reactive band at approximately M r 29,000 and a trace component at approximately M r 50,000. The M r 29,000 band's mobility matched that of the purified protease (lane 2). Day 0 yolk (lane 3) was labeled more extensively by the probe, especially the three Vt subunits at M r 102,000, 95,000, and 50,000, which have been degraded by day 6 in vivo (16). However, lane 3 also contains three probe-reactive peptides at approximately M r 40,000 (arrowheads), absent when day 0 yolk was preincubated with E-64 (lane 4). Although peptides of M r 50,000 and larger were labeled nonspecifically, only the M r 40,000 components were E-64-sensitive, suggesting that they contain active site cysteine residues. Because the protease is also resolvable into multiple bands on SDS-PAGE (lane 2 and Figs. 2 and 3), the M r 40,000 triplet was considered as the candidate proprotease.
Purification of the Proprotease-The putative proprotease was stable in day 0 yolk extracts at pH 7.5 at 4°C for at least 1 week. Values lower than 7.0 resulted in protease activation, and those higher than 8.0 resulted in irreversible inactivation. Sephacryl S-300 chromatography (Fig. 6) gave one major protein peak (fractions 21-30) and a peak of activable protease activity (fractions 31-40), which was absent before acidification. FPLC separated the putative proprotease, which eluted between 0.2 and 0.3 M NaCl, from additional proteins and proved to be a critical step in the purification (data not shown). Optimal resolution was achieved when the protein load per run was kept below 10 mg. In the final step, phenyl-Sepharose CL-4B chromatography resolved the putative proprotease, which eluted at approximately 2 mM Hepes, from trace contaminants (data not shown). The candidate proprotease consists of four peptides of M r 35,500, 37,000, 39,000, and 41,000 (Fig. 7,  lane 4), their mobilities essentially the same as those in day 0 yolk probed with biotinyl-Phe-Ala-CHN 2 (Fig. 5). The proprotease migrated as a single band on nondenaturing PAGE at each acrylamide concentration employed (data not shown), and the calculated molecular weight (27) was 45,600. After acidification, the M r 45,600 peptide disappeared but new ones were evident at M r 36,800 and 34,800. The shift in molecular weights is close to the difference between the proprotease (40,000) and the protease (29,000), obtained by SDS-PAGE.
Protease and acid-activated proprotease displayed similar pH activity profiles (Fig. 4), relative susceptibilities to numerous inhibitors (Table I), and heat inactivation characteristics (data not shown). Reactivity of the proprotease with biotinyl-Phe-Ala-CHN 2 demonstrates that the active site is accessible to the inhibitor in the proenzyme and is probably in a conformation similar to the protease (34). They retained 63% and 67% of their enzymatic activities, respectively, after exposure to 3 M urea. While cathepsins B are inactivated under these condi-   tions, cathepsins L are far less sensitive to this reagent (23). In addition, protease and activated proprotease had only 2.5% and 5.5% of the enzymatic activity, respectively, with Z-Arg-Arg-NMec, a cathepsin B substrate, compared to that with the cathepsin L substrate Z-Phe-Arg-NMec (23).
Conversion of Proprotease to Protease-In vivo, the relationship between the proprotease and the protease is seen in an experiment using yolk extracts (day 0 to day 6 postovulation) and the active site probe biotinyl-Phe-Ala-CHN 2 (Fig. 8, panels  A and B). Although numerous yolk peptides were derivatized nonspecifically, a reactive band at approximately M r 40,000, present at oothecal extrusion (panel A, lanes 0 and 1, closed arrowhead), decreased in intensity during development and was absent after day 3. However, during days 4 -6, the intensity of a doublet at M r 29,000 increased (panel A, open arrowhead). Since the staining intensity of several bands changed during the 7 days of embryo development, the specificity of derivatization was evaluated by preincubation of the yolk with E-64. With inhibitor present (panel B), no bands were detected at M r 40,000 or 29,000 kDa. Thus, these peptides contained E-64-reactive cysteine residues. Appearance of the M r 29,000 components also correlated temporally with Vt processing and protease activation in vivo (9).
In vitro, the mobility of purified proprotease on SDS-PAGE (Fig. 9, lane pH 5.3, 0 h) shifted to that of the protease following incubation at pH 5.3 (compare pH 5.3, 12 h lane and Protease lane) but not at neutral pH (pH 7.5, 12 h). The conversion was blocked by E-64 but not by Pefabloc SC, pepstatin, or EDTA (Fig. 9), demonstrating that activation requires both acidification and participation of a cysteine protease. These results were confirmed by the experiment illustrated in Fig. 10. When proprotease and protease were incubated separately for 60 min, the increment in enzymatic activity was negligible when compared to the activity obtained upon mixing them at these individual concentrations. Taken together, the results of these several experiments indicate that the protease is derived from the putative proprotease in day 0 yolk.
Another experiment demonstrated that the activation rate is intermolecular. Protease was diluted and assayed as described under "Experimental Procedures." At dilutions of 10 4 -, 10 2 -, and 10-fold, the relative rates of activation were 1, 5.3, and 48 (data not shown). The dependence of the activation rate on enzyme concentration rules out a single intramolecular (zero order) process. The Proprotease Is a Glycoprotein-Purified proprotease reacted with biotin hydrazide, showing that the proenzyme is a glycoprotein (Fig. 11, lanes C, minus endo H). Removal of carbohydrate by endo H, indicated by a slight increase in mobility (lanes C, plus and minus endo H) suggests that most, if not all, occurs as high Man-type oligosaccharides (30), the class of oligosaccharides found in B. germanica Vt (16,35) and other insect glycoproteins (36,37). The probe also labeled the endo H-treated protein, but with less intensity than with the control, a result reflecting either incomplete removal of oligosaccharides or to the remaining single GlcNAc residues (30). Mature protease was not reactive with the probe (lanes B, minus and plus endo H), showing that the oligosaccharides are located exclusively on the pro-region of the proprotease. As expected, the Vt control was deglycosylated by endo H (lanes A, minus and plus endo H). A mobility shift in the M r 50,000 subunit was not observed.
Vt-processing Peptides Are Not Ubiquitinated-A 26 s "proteolytic complex" that degrades ubiquitinated proteins in vitro has been isolated from Drosophila melanogaster embryos, and it was suggested (38) that ubiquitination might be important in yolk polypeptide degradation. To check for this possibility in B. germanica, extracts of yolk prepared daily from eggs at days  (39) and a sample of bovine ubiquitin, which co-electrophoresed with yolk extract, served as positive controls. Although controls contained the appropriate reactive bands, no ubiquitinated peptides were detected in the extracts tested (data not shown).

DISCUSSION
Cathepsin-like cysteine proteases are derived from proprotein precursors (40). An excellent example is propapain (M r 39,000), which is processed in vivo to the cathepsin L papain (M r 24,000) (41). In addition to similarity in molecular weights with the B. germanica yolk proenzyme and protease, all four molecules have an essential thiol at the active site (34) and propapain is also converted to papain, in vitro, at acidic pH (34). Like the B. germanica proprotease, propapain is glycosylated exclusively on its propeptide region (42). Whether this modification is critical to production of a functional enzyme, as it is with propapain (42), remains to be established. Although tests with various inhibitors demonstrated that the protease is more closely related to the cathepsins L than cathepsins B (20,43,44), the distinction is not absolute. Because its molecular weight heterogeneity was retained throughout purification, degradation during the procedure is not causing the multiple bands. Other examples of mature proteases consisting of multiple peptides of similar molecular weights are found in the mosquito Culex nigripalpus (45), the bacterium Erwinia chrysanthemi (46), eggs of Ornithodorus. moubata (7,8), the silkmoth Bombyx mori (14), and human neutrophils (47).
Numerous similarities between the activated proprotease and the protease lead to the conclusion that the proprotease in day 0 yolk is the enzyme's precursor. Most importantly, degradation of Vt in vivo and by the protease in vitro is limited, and both events afford several common intermediates and products (Table II). The differences that were noted could reflect unique sets of scissile bonds being exposed to proteolysis in each case, possibly due to differences in the structures/solution conforma-tions of Vt in vivo and in vitro, or to the fact that Vt's oligosaccharides are trimmed coincident with proteolysis in vivo (16). Digestion of B. mori Vt with a purified yolk cysteine protease has been shown to give a peptide distribution similar, but not identical, to that seen in vivo (48). Electroblotting of SDS gels of yolk during embryo development (Fig. 8) demonstrated that the conversion of the putative proenzyme to the processing protease, begins at day 2-3 postovulation, which correlates with the time in embryo development when both granule acidification and protease activation occur (9,19,20).
It is now clear that yolk granule acidification is of general physiological importance and that Vt proteolysis is initiated by a developmentally regulated decrease in intragranular pH. Vt-degrading yolk proteases from O. moubata (7, 8), B. mori  11. Endo H treatment of purified proprotease and protease. Purified proprotease or protease (15 g of protein) in HBS containing 40 mM ␤-MSH were incubated overnight with an equal volume of E-64 in 0.25 M citrate buffer, pH 5.5, at 4°C to preclude proteolysis. Each mixture was then denatured, incubated without or with Endo H (5 milliunits). B. germanica Vt served as a control. SDS-PAGE, electroblotting, and detection of carbohydrate were conducted as described under "Experimental Procedures." Samples A, B, and C, B. germanica Vt, protease and proprotease, untreated (-) or treated (ϩ) with endo H, respectively. (48), Aedes aegyptii (49), and Musca. domestica (50) are all converted from proenzymes to active, mature enzymes in vitro at acid pH; and yolk granules of O. moubata (51) and the blowfly Phormia regina (20) also acidify in concert with Vt utilization. Other well documented examples of acidificationdependent Vt degradation include the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus (52) and frog Xenopus laevis (53). The pH range of acidified granules measured for the latter two examples, pH 5.6 -6.2 (52,54), is probably adequate for proteolytic processing of proenzymes and is in the range for catalytic activity of both the activated B. germanica proprotease and protease (Fig. 4). Bafilomycin sensitivity of granule acidification in X. laevis (54) is consistent with our finding (20) that granule acidification occurs by proton translocation and it provides additional evidence that a vacuolar ATPase is responsible for pumping protons into yolk granules. Although the source of the proton pump and the mechanism initiating intragranular proton accumulation remain to be determined, the present work links the Vt-processing protease of B. germanica to the acidification-dependent, proteolysis of a proprotease precursor.
The dynamics of vitellogenesis and Vt processing in B. germanica, as they are currently understood, can be summarized as follows. Vitellogenin is synthesized in, and secreted from, the fat body of vitellogenic females (35) and endocytosed by the oocytes (55). Following vitellogenin's uptake by the oocyte, fusion of small Vt-containing vesicles leads to formation of large, mature yolk granules (56), which are stored for approximately 72 h prior to acidification and initiation of Vt processing. Recently acquired immunoelectron microscopy data 2 demonstrate that proprotease is located over these mature granules from days 0 -2 postovulation.
Ultrastructural studies have also demonstrated the presence of vitellophages initially at about day 2 postovulation, primarily on the ventral periphery of the yolk mass (56). These cells then intercalate the tightly packed granules, extending filoand lamellipodia over granule surfaces. Portions are engulfed and sequestered as large vacuoles (56) in a sequence of events strikingly similar to that observed in embryogenesis of the stick insect Carausius morosus Br. (57). The B. germanica vacuoles then become vesiculated and partitioned into smaller vesicles (56) with the same size distribution as granules isolated from yolk (20). A role for vitellophages in the acidification and protease activation events remains to be established, but providing acidification machinery (e.g. a vacuolar ATPase) to the granules is one possibility.