Purification and characterization of a kinase-associated, myofibrillar smooth muscle myosin light chain phosphatase possessing a calmodulin-targeting subunit.

A myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) was purified from turkey gizzard myofibrils, and it was found to be closely associated with the myosin light chain kinase (MLCKase). For this reason we have named this phosphatase the kinase- and myosin-associated protein phosphatase (KAMPPase). Subunits of the KAMPPase could be identified during the first ion exchange chromatography step. After further purification on calmodulin (CaM) and on thiophosphorylated regulatory myosin light chain affinity columns we obtained either a homogenous preparation of a 37-kDa catalytic (PC) subunit or a mixture of the PC subunit and variable amounts of a 67-kDa targeting (PT) subunit. The PT subunit bound the PC subunit to CaM affinity columns in a Ca2+-independent manner; thus, elution of the subunits required only high salt concentration. Specificity of interaction between these subunits was shown by the following observations: 1) activity of isolated PC subunit, but not of the PTC holoenzyme, was stimulated 10-20-fold after preincubation with 5-50 μM of CoCl2; 2) the pH activity profile of the PC subunit was modified by the PT subunit (the specific activity of the PTC holoenzyme was higher at neutral pH and lower at alkaline pH); and 3) affinity of the holoenzyme for unphosphorylated myosin was 3-fold higher, and for phosphorylated myosin it was 2-fold lower, in comparison with that of the purified PC subunit. KAMPPase was inhibited by okadaic acid (Ki = 250 nM), microcystin-LR (50 nM) and calyculin A (1.5 μM) but not by arachidonic acid or the heat-stable inhibitor (I-2), which suggested that this is a type PP1 or PP2A protein phosphatase.

A myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) was purified from turkey gizzard myofibrils, and it was found to be closely associated with the myosin light chain kinase (MLCKase). For this reason we have named this phosphatase the kinase-and myosin-associated protein phosphatase (KAMPPase). Subunits of the KAMPPase could be identified during the first ion exchange chromatography step. After further purification on calmodulin (CaM) and on thiophosphorylated regulatory myosin light chain affinity columns we obtained either a homogenous preparation of a 37-kDa catalytic (PC) subunit or a mixture of the PC subunit and variable amounts of a 67-kDa targeting (PT) subunit. The PT subunit bound the PC subunit to CaM affinity columns in a Ca 2؉ -independent manner; thus, elution of the subunits required only high salt concentration. Specificity of interaction between these subunits was shown by the following observations: 1) activity of isolated PC subunit, but not of the PTC holoenzyme, was stimulated 10 -20-fold after preincubation with 5-50 M of CoCl 2 ; 2) the pH activity profile of the PC subunit was modified by the PT subunit (the specific activity of the PTC holoenzyme was higher at neutral pH and lower at alkaline pH); and 3) affinity of the holoenzyme for unphosphorylated myosin was 3-fold higher, and for phosphorylated myosin it was 2-fold lower, in comparison with that of the purified PC subunit. KAMPPase was inhibited by okadaic acid (K i ‫؍‬ 250 nM), microcystin-LR (50 nM) and calyculin A (1.5 M) but not by arachidonic acid or the heat-stable inhibitor (I-2), which suggested that this is a type PP1 or PP2A protein phosphatase.
Contraction of smooth muscle is regulated by a myosinlinked regulatory system that involves phosphorylation of the 20-kDa light chain of myosin (ReLC) 1 by myosin light chain kinase (MLCKase) (1). This phosphorylation is a prerequisite for the actin-activated MgATPase activity of myosin, which is considered essential for the initiation of smooth muscle contraction (2,3). Furthermore, phosphorylation of the ReLC influences the assembly and stability of myosin filaments (4,5), which must also be related to tension development in vivo. Consistent with such a regulatory system, it has also been shown that MLCKase and its activator, calmodulin (CaM), are tightly bound to filamentous myosin (6), but their affinity for monomeric or folded myosin is low (7). However, it should be noted that the high affinity binding and high content of ML-CKase and CaM in filamentous myosin are not generally recognized (see Ref. 8).
Relaxation of smooth muscle is associated with dephosphorylation of myosin (for review, see Refs. 3, 9, and 10), and therefore it was clear very early that another regulatory enzyme, a myosin light chain phosphatase (MLCPase), must be responsible for relaxation in vivo. Several phosphatases have been isolated from smooth muscle, which could act to dephosphorylate myosin or its ReLC. Many of these enzymes seem to be cytosolic, because they have been purified from whole smooth muscle extracts. For example, only two of the four protein phosphatases identified by Pato et al. (11,12) in the cytosol of turkey gizzard exhibited high activity toward native myosin and bound tightly to myosin, suggesting that they may be localized on the thick filaments. The properties of these and other phosphatases with respect to the effects of divalent cations and substrate specificity have been investigated. The emphasis in all of these studies has been to establish the differences among the various phosphatases, although from the introduced classification (13) and subunit composition, the similarities were also apparent. This aspect of the MLCPase identification has not been investigated. As a result, the phosphatase specifically responsible for the dephosphorylation of myosin in vivo has not yet been identified.
It might be expected that a myofibrillar phosphatase that is tightly associated with the contractile apparatus, such as the enzyme copurified with MLCKase (14), would be the most likely to serve as the phosphatase responsible for the dephosphorylation of myosin in vivo. In the present report, we characterize a holoenzyme of this MLCPase, which, as we show, is bound to calmodulin in a Ca 2ϩ -independent manner. Formation of a complex between the holoenzyme and MLCKase is addressed in the accompanying report (15).

MATERIALS AND METHODS
Chemicals and Other Reagents-[␥-32 P]ATP was purchased from Du-Pont NEN and diluted with cold ATP of special grade (catalog number 519 987; Boehringer Mannheim) to the required specific activity and concentration (16). Q-Sepharose CL-6B, Sephacryl S-100 HR, and CNBr-activated Sepharose 4B-CL were obtained from Pharmacia Fine * These studies were supported by grants from the Austrian Science Foundation and from the East-West Program of the Austrian Ministry for Science and Research. 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.
‡ To whom correspondence should be addressed: Institute of Molecular Biology, Billrothstrasse 11, A-5020 Salzburg, Austria. The buffer (AA) used throughout this study had the following composition: 60 mM KCl, 2 mM MgCl 2 , 0.5 mM dithiothreitol, and 10 mM imidazole with pH adjusted to 7.5 at 4°C. It was used, with the necessary additions, during protein purification, activity measurements, binding experiments, and all other procedures.
Protein Preparations-Turkey gizzard MLCKase (14), myosin (17), CaM (18), and ReLC (19) were prepared as described previously. 32 P labeling of the isolated ReLC, used as the substrate, was carried out by adding 25 nM of purified MLCKase and 35 nM of CaM to the light chain at a 500 M concentration. After 90 min, the phosphorylation reaction was terminated by placing the tube in boiling water for 6 min, and the radioactive ATP was removed by exhaustive dialysis against our AA buffer. The 32 P-labeled preparations of ReLC were stored at Ϫ30°C, and before use they were usually diluted 3-10-fold with the AA buffer to obtain the required level of cpm.
Phosphatase and Kinase Activity Measurements-Before any type of measurements, purified KAMPPase preparations were first diluted, to the required concentrations, with AA buffer containing 0.1% of bovine serum albumin added as an inert protein carrier. Phosphatase activity was measured by the release of 32 P from 32 P-ReLC used as a substrate as described previously (14). The assays were carried out at 25°C in the AA buffer, and concentrations of the substrate ( 32 P-ReLC) were in the range of 50 -150 M.
MLCKase assays were carried out with [␥-32 P]ATP using intact myosin or its isolated ReLC as substrates as described previously (16,18). Details of specific assays are given in the corresponding figure legends.
Binding Experiments-The affinity of KAMPPase for myosin filaments was determined by using the sedimentation method (20). In our case, increasing amounts of myosin were added to 50 l of a KAMPPase preparation, and, after centrifugation, the KAMPPase activity in the supernatants was measured. Concentration of the phosphatase was chosen to ensure that not more than 30% of the substrate was dephosphorylated in the controls (no myosin present). The reciprocal of the fraction of free KAMPPase was plotted against the myosin concentration. This was a little more convenient than the original procedure.
Ligand Coupling and Other Standard Procedures-The coupling of the ligands to CNBr-activated Sepharose 4B was done as recommended by the manufacturer (Pharmacia, Uppsala, Sweden). The unphosphorylated ReLC was coupled to the Sepharose, and this was thiophosphorylated for 60 -90 min by adding 0.25 mM [␥-S]ATP (Boehringer Mannheim) and 20 -40 nM of CaM•MLCKase. The gels were packed into a column approximately 0.9 ϫ 60 cm. Stability of the column was such that it could be used for four to six applications with 2-3 rethiophosphorylations within a few months.
SDS-polyacrylamide gel electrophoresis was performed in 8 -18% gradient acrylamide minislab gels according to the procedure of Matsudaira and Burgess (21) in the buffer system of Laemmli (22) with some improvements described recently in detail by Sobieszek (23). Protein concentration was determined by the methods of Gornall et al. (24) and Bradford (25) by using Bio-Rad protein standard. Unless otherwise stated, all purification steps were carried out on ice or in the cold room (4°C).

Initial Purification
Steps-The 40 -55% ammonium sulfate fraction obtained from the myofibrillar MLCKase and MLCPase extract was purified by chromatography on a Q-Sepharose column rather than on previously used DEAE-Sepharose 6B-CL. The advantage of the Q-Sepharose column was that tropomyosin was separated from the MLCPase. As shown in Fig. 1A, tropomyosin coeluted with the MLCKase and not with the MLCPase activity peak as it does from DEAE-Sepharose column. Activity measurements showed that, for both columns, most of the kinase activity could be separated from the phosphatase, but the reverse was not true; the phosphatase was always heavily contaminated by the kinase. The first peak from the Q-Sepharose column contained essentially homogenous MLCKase (with the residual tropomyosin), while the phosphatase constituted only a small portion of the total protein in the second peak (Fig. 1B). Significantly, this peak always included CaM. The separation of tropomyosin from the MLCPase is important because the viscosity of tropomyosin could impair further purification of the enzyme. The purification factor for the ionic exchange column was about 800-fold (80% yield), making it possible to detect, on SDS-PAGE, the bands that correlated with the MLCPase activity ( Fig. 1A; i.e. PT and PC).
In the initial attempts, the Q-Sepharose fractions that contained the highest MLCPase activity were subjected to a standard purification on a ␥SP-ReLC affinity column. Unexpectedly, the phosphatase that eluted at 180 or 360 mM salt was composed of three subunits with molecular masses of 37, 67, and 130 kDa (Fig. 2, lane a). From the absence of the 130-kDa band in the low ionic strength washes from this column and the presence of high MLCKase activity in the eluate, the 130-kDa band was identified as MLCKase. Further attempts to purify this kinase/phosphatase fraction on hydroxylapatite and/or ion exchange chromatography produced no separation of these enzymes.
Because of the high affinity of the kinase for CaM and the independence of the phosphatase activity from this activator, the alternative purification step used was CaM affinity chromatography. As shown in Fig. 3, however, the MLCKase still contained some MLCPase activity, and the expected complete separation of these two enzymes was again not achieved. It was clear from this and from other attempts of the type shown in Fig. 3, that the MLCKase always and to a variable extent, copurified with the phosphatase. The opposite was not true, and the "breakthrough" phosphatase that was not bound by the CaM affinity column was typically free of the kinase activity. The fractions bound by the column and eluted at high salt concentration always contained both MLCPase and MLCKase activities. Moreover, the same was true for the standard MLCKase preparations (e.g. see Fig. 3) eluted from the CaM affinity column in the absence of Ca 2ϩ . Because the apparent very tight association of the MLCPase activity with the kinase also was observed with MLCKase bound to myosin filaments (see below), we refer to this MLCPase as the kinase-and myosin-associated protein phosphatase (KAMPPase). The properties of the complex formed between the two key regulatory enzymes of smooth muscle contraction is the subject of the companion report (15). The present article focuses on the characteristics of the KAMPPase holoenzyme (PTC) and its isolated catalytic subunit (PC).
Catalytic Subunit and KAMPPase Holoenzyme-The breakthrough fractions from the CaM affinity column of the Q-Sepharose KAMPPase always contained a relatively high level of phosphatase activity. The amounts of the bound and unbound activities were variable and depended on the amount of proteolytic degradation. This, in turn, introduced some variability in appearance of the KAMPPase on SDS-PAGE during purification.
The breakthrough phosphatase was subjected to three purification steps, which included a long ␥SP-ReLC affinity column (Fig. 4A) and a short DEAE-Sepharose column (Fig. 4B) followed by a 0.6 ϫ 110-cm Sephacryl S-100HR gel filtration column. If a relatively higher degree of proteolysis had occurred, this three-step column procedure resulted in a homog-enous KAMPPase preparation, which exhibited only one subunit of 37 kDa (Fig. 2, lanes b and c). With less proteolytic degradation, the analogous preparation contained an additional subunit of 67 kDa (Fig. 2, lane f). The relative ratio of the 37-and 67-kDa subunits, therefore, varied from preparation to preparation and depended on the extent of proteolytic degradation that occurred. Unless otherwise indicated, the complex composed of these two subunits was used throughout the experiments described below. We refer to it as the KAMPPase holoenzyme or simply KAMPPase.
The purified 37-kDa subunit contained all of the KAMPPase activity and was therefore identified as the catalytic (PC) subunit of the KAMPPase. It eluted as a single peak at a position corresponding to ϳ67 kDa when run on a calibrated Sephacryl S-100 HR or AcA54 column equilibrated with AA buffer, containing or not containing 0.3 M NaCl. The purified PC subunit showed a closely spaced doublet on SDS-PAGE (Fig. 2, lane b), which would be consistent with the dimer formation. However, a single band of this size was observed in the initial purification steps (Fig. 1A). The doublet could then result from a slight proteolytic degradation of the subunit.
CaM Affinity-bound Phosphatase-After extensive washing at low ionic strength, and before EGTA elution, the CaM affinity column was eluted at 180 or 360 mM salt concentration, both in the presence of 0.2 mM calcium (Fig. 3). The composition of the eluted peak was complex and, as before, as a result of proteolysis, the band patterns differed from preparation to preparation. Nevertheless, the 37-and 67-kDa subunits were always present as the main protein bands (Fig. 2, lanes d  and e).
Further purification of the CaM affinity-bound 37-67-kDa complex on the short DEAE-Sepharose column produced a relatively homogenous preparation of the PTC holoenzyme (Fig. 2,  lane f). The two subunits comigrated during the elution, and their positions correlated well with the KAMPPase activity. The same was true during the subsequent purification by gel filtration (see above), which served to remove some minor impurities, e.g. tropomyosin (Fig. 2, compare lanes f and g). No difference was noted between this KAMPPase and the one purified from the breakthrough fraction, provided that both contained the PT subunit.
Localization of Myofibrillar MLCPase on Myosin-We have previously shown (6) that MLCKase and CaM are very tightly bound to filamentous smooth muscle myosin. As a result, the addition of Ca 2ϩ and ATP to purified myosin leads to a very rapid phosphorylation of the ReLC. In the same experiments, we also showed that the MLCPase was also associated with the same type of (filamentous) myosin preparation. As shown in Fig. 5A, after initial rapid phosphorylation by the kinase, the myosin became dephosphorylated by the endogenous phosphatase. Both enzymes were simultaneously active, and the resulting phosphorylation rate and extent depended only on the kinase and the phosphatase activities associated with the myosin. However, as soon as ATP was depleted, the kinase could not act, and the phosphatase action became apparent (Fig. 5A). For longer incubation times, this resulted in biphasic phosphorylation curves (see Ref. 15). Correspondingly, the duration and the levels of phosphorylation could be increased by adding phosphatase inhibitor (microcystin-LR) or decreased after adding purified KAMPPase (Fig. 5A) or the PC subunit. This demonstrates clearly that the KAMPPase was very active toward phosphorylated filamentous myosin and could be responsible for the dephosphorylation of myosin filaments in vivo.
At the myosin concentrations (30 -50 M) used in these ex-periments, most of the ATP hydrolysis was due to myosin ATPase. This is not the case in similar experiments with MLCKase autophosphorylation, where only the kinase is present (26). By adding different concentrations of the KAMPPase (or the PC subunit) to the kinase, we could decrease both the autophosphorylation level and the initial rate (Fig. 5B). In contrast, the addition of microcystin-LR resulted in 2-fold (or greater) increase in the autophosphorylation even for homogenous MLCKase preparations (Fig. 5B). Thus, our KAMPPase is also very active toward MLCKase autophosphorylation sites.

Divalent Cation Requirements and Inhibition by Common Protein Phosphatase Inhibitors-
The activity of the isolated PC subunit was stimulated by preincubation in micromolar concentrations of CoCl 2 . The 10 -20-fold stimulation was observed with the two substrates used and depended more on the preincubation time than on the Co 2ϩ ion concentration. It was observed only for the purified PC subunit and not for the PTC holoenzyme (Fig. 6). In agreement with a recent observation by Okubo et al. (27), the activity of the phosphatase copurified with myosin was also stimulated by CoCl 2 . The stimulation was, however, rather variable and consistently lower than that observed for the purified PC subunit.
The effects of other divalent cations on the activity of the PC subunit were also investigated (data not shown). As expected for a type PP1 phosphatase, other cations were not required for activity, although some stimulation was observed. The stimulation occurred in the 0.5-2.5 mM concentration range and amounted to not more than a 1.5-fold increase for Mn 2ϩ , Mg 2ϩ , and Ca 2ϩ . Heavy metal cations such as Ni 2ϩ inhibited the activity at approximately the same concentration range, while a 50% inhibition by Cd 2ϩ required only 50 -100 M of CdCl 2 .
Unexpectedly, the heat-stable protein inhibitor (I-2) at concentrations of up to 5 g/ml had no effect on the activity of the PTC holoenzyme (Fig. 7A) or the PC subunit. Okadaic acid, a potent and specific inhibitor of the type PP1 and PP2 protein phosphatases, inhibited the activity of both the PTC holoenzyme and the PC subunit (Fig. 7A) with an IC 50 value of about 250 nM, which is 25 times higher than that characteristic for a type 1 protein phosphatase (28,29).
We have also tested the inhibition of the KAMPPase by other potent protein phosphatase inhibitors commonly used for smooth muscle fiber experiments (Ref. 30; see also Ref. 29). One of them, arachidonic acid, had no effect on the KAMPPase activity (Fig. 7B). With calyculin A, the inhibition occurred in the 1-3 M concentration range (Fig. 7A). The most effective inhibitor was microcystin-LR (Fig. 7A), with full inhibition obtained at approximately 0.250 M. Thus, okadaic acid, calyculin A, and microcystin-LR appear to be the only inhibitors suitable for in vivo experiments on smooth muscle fibers or muscle strips.
Targeting Subunit and KAMPPase Activity-The effect of the PT subunits on the specific activity of the PC subunit was not accurately established because the PT subunit could not be completely purified without proteolytic degradation or contamination with the phosphatase. However, indirect estimations indicated a 3-4-fold stimulation of the PC subunit after the addition of partially purified PT. This stimulation depended on ionic conditions and pH. As shown in Fig. 8, the pH activity profile of the PC subunit was completely different from that of the PTC holoenzyme. Significantly, the difference amounted to a shift of the activity optimum from basic pH values to neutral, physiologically relevant values in the case of PTC. The increase FIG. 6. Effects of CoCl 2 on the activities of MLCPase preparations. Note that the activity of PC subunit (छ) and not of the PTC holoenzyme (ࡗ) was stimulated by CoCl 2 . The activity of the myosin endogenous phosphatase (E) was also stimulated by CoCl 2 .

FIG. 7. Effects of commonly used inhibitors on activity of KAMPPase.
In panel A the activity of PTC holoenzyme was inhibited by okadaic acid (ࡗ) and calyculin A (ϫ) but was unaffected by I-2 heat-stable inhibitor (छ). The activities were normalized to 28,000 cpm, and the maximal concentration of an I-2 was 5 mg/ml. In panel B, the activity was inhibited by microcystin-LR (E) but was unaffected by arachidonic acid (ϫ), also used as an MLCPase inhibitor.
in the activity at basic pH was also not observed for the endogenous myosin phosphatase. Thus, in this respect, the endogenous myosin phosphatase was similar to the PTC holoenzyme and not to the isolated PC subunit.
The effect of the PT targeting subunit on the binding of the KAMPPase to myosin was also investigated. We measured the affinities of the PC subunit and the PTC holoenzyme for myosin filaments assembled from unphosphorylated and thiophosphorylated smooth muscle myosin. The affinity of the PC subunit for unphosphorylated myosin was moderate (K d ϭ 15 M) but increased about 3-fold in the presence of the PT subunit (Fig.  9). The opposite effect was observed with thiophosphorylated myosin. The relatively high affinity of the PC subunit (K d ϭ 4 M) was 2-fold lower in comparison with that of the PTC holoenzyme (Fig. 9). Although these effects were not large, they indicate that the regulatory role of the PT subunits also includes modification of the KAMPPase affinity for the in vivo substrate (filamentous myosin).

Myofibrillar MLCPase and Purification Approaches-
The identification and partial purification of a myofibrillar smooth muscle myosin MLCPase was first described by Sobieszek and Barylko (14), although a similar phosphatase was noted earlier (31). Until recently, much effort has been made to identify and purify cytoplasmic type smooth muscle myosin phosphatases, and less attention has been paid to the myofibrillar MLCPases. From its close association with MLCKase, we suggested that myofibrillar MLCPase is more likely to be responsible for the relaxation of smooth muscle.
The present report describes the further purification and characterization of turkey gizzard myofibrillar MLCPase denoted here as KAMPPase. The KAMPPase was purified as a PTC holoenzyme composed of the two subunits or as a large multienzyme complex, which included MLCKase and CaM (15). In addition, a homogenous preparation of the PC catalytic subunit was obtained when the targeting PT subunit was degraded during purification. The phosphatase must be considered as being myofibrillar in origin because the starting material (myofibrils) was obtained by extensive fragmentation and washing of gizzard muscle, as originally described by Sobieszek and Bremel (32). The myofibrils are practically depleted of cytosolic proteins, and only tightly bound enzymes (including the phosphatase considered here) are extracted with the AA buffer containing 25 mM MgCl 2 . As originally indicated (33), adding MgCl 2 to the extraction medium represents a significant improvement of the purification procedure and is generally used in extraction of the MLCKase (e.g. see Refs. 31 and 34).
The procedure used to purify the KAMPPase included a new modified affinity chromatography approach on CaM and on ␥SP-ReLC affinity columns. The fragmentation and extensive washing prior to KAMPPase extraction (see Ref. 32) made it possible to identify the KAMPPase bands on SDS-PAGE of fractions obtained during the first purification step on the ion exchange column. The use of CaM affinity chromatography to purify MLCPase has not been reported previously and is a novel aspect of this study.
Subunit Composition and Specificity-The KAMPPase holoenzyme obtained after two affinity columns exhibited, on SDS-PAGE, the two subunits of 37 and 67 kDa. In agreement with previous reports (13,28,35,36), the 37-kDa protein was identified as the catalytic subunit. This subunit possessed all of the phosphatase activity. The role of the 67-kDa subunit was established as the CaM-targeting one because it was responsible for binding the PC subunit to the CaM affinity gel; the most illustrative data is presented in the companion paper (15).
As expected from its tight association with MLCKase, the PC subunit and the PTC holoenzyme effectively dephosphorylated not only intact myosin but also the kinase itself. The dephosphorylation rates for the three substrates investigated were similar and, for example, intact myosin was dephosphorylated only 1.5-fold faster than the isolated ReLC. The corresponding K m values were also similar (3-10 M). We demonstrated that a similar phosphatase was tightly associated with purified MLCKase and could not be completely removed even after extensive attempts to separate the two. This endogenous phosphatase effectively dephosphorylated MLCKase autophosphorylation sites but not the well characterized sites phosphorylated by cAMP-dependent protein kinase or Ca 2ϩ /CaMdependent multifunctional kinase II (26). We believe that the latter endogenous phosphatase as well as the one associated with filamentous myosin are the same or similar enzymes, possessing the same catalytic subunit.
Association with Myosin-Because the affinity of the PC subunit for myosin was only moderate (K d Ϸ 15 M), it would appear that the tight association of endogenous MLCPase with myosin requires the presence of another subunit. Our results suggest that the PT targeting subunit could fulfill this role, since the other components needed for the interaction (CaM or CaM⅐MLCKase complex) are known to be firmly incorporated into myosin filaments (see Ref. 6). Recently, Mitsui et al. (37) reported the purification of a smooth muscle phosphatase (myosin-associated protein phosphatase I) from chicken gizzard myosin. Since we have also concluded that our KAMPPase is associated with myosin, it is likely that these two phosphatases are similar if not identical. Both enzymes are tightly associated with filamentous myosin, exhibit similar size during gel filtration on Sephacryl S-100HR columns, have catalytic subunits of approximately the same size, and have a similar elution position (170 kDa) from a gel filtration column. A targeting subunit of 67 kDa, however, was not identified for the myosin-associated protein phosphatase I phosphatase (37). The 3-fold increase in the affinity of the PC subunit for myosin in the presence of the PT subunit supports our hypothesis for the existence of and the role for the PT subunit. However, an independent demonstration of the presence of this targeting subunit on myosin filaments is still lacking. The observed CaM-dependent but Ca 2ϩ -independent targeting by this subunit is also consistent with the independent binding of CaM and the kinase to myosin filaments (6).
Comparison with Other Myosin Phosphatases-During the last 10 -15 years, several cytosolic MLCPases have been purified from smooth muscle extracts, and many of them appear to be similar to our KAMPPase. Its relation to the only other myofibrillar phosphatase of Alessi et al. (38) is discussed in the companion paper (15), and it is only briefly considered here (see below). A purified aortic smooth muscle myosin phosphatase, composed of two subunits of 67 and 37 kDa (39), is structurally similar to the myosin phosphatase from cardiac muscle (40). Another phosphatase, which is composed of three subunits (67, 54, and 34 kDa), was purified from chicken gizzards by Onishi et al. (41) and by Pato and Adelstein (42). They both dephosphorylated isolated ReLC, but only the former dephosphorylates myosin. Nevertheless, they may represent the same enzyme, since both were purified by ␥SP-ReLC affinity chromatography. The same comment could be applied to the ML-CPases purified by Di Salvo et al. (43). Taken together, it appears that most smooth muscle myosin phosphatases have a catalytic subunit of about 37 kDa. Similarly, it can be concluded that a subunit of 60 -67 kDa is a constant feature of this type of phosphatase and that the band of 54 -58 kDa sometimes observed may represent a proteolytic fragment of this subunit.
During the course of the present study, two papers were published that describe another myofibrillar type of MLCPase from gizzard muscle (Ref. 38; see also Ref. 44). The holoenzyme of that enzyme is composed of a regulatory subunit of 130 kDa, a catalytic subunit of 37 kDa, and an unidentified component of 20 kDa. Differences in the methods of preparing myofibrils and in extraction conditions make it difficult to determine the relationship of that enzyme to ours. However, the absence of the 67-kDa targeting subunit indicates that the MLCPase described by Alessi et al. (38) and by Shirazi et al. (44) is different from the enzyme we have isolated.