Ca2(+)-dependent protein phosphatase which dephosphorylates regulatory light chain-a in scallop smooth muscle myosin

Ca2(+)-dependent protein phosphatase was purified from scallop adductor smooth muscle by a combination of DEAE-Toyoperal 650S ion exchange chromatographies and gel filtration on Sephacryl S-300. The phosphatase consisted of two subunits having molecular weights of 60 and 19 kDa. Phosphorylated regulatory light chain-a (RLC-a) was dephosphorylated by this phosphatase both in free and bound states in myosin prepared from the opaque portion of scallop smooth muscle (opaque myosin). The dephosphorylation was activated by Ca2+. The half maximal activation was a 1 microM free Ca2+ in the presence of calmodulin and 7 microM free Ca2+ in the absence of calmodulin. Opaque myosin phosphorylated at the heavy chain was not dephosphorylated with this phosphatase. p-Nitrophenyl phosphate was dephosphorylated. In addition to Ca2+, the phosphatase activity for RLC-a was activated by Mn2+, while p-nitrophenylphosphatase activity was activated by Mg2+ more strongly than by Mn2+. The pH-activity curves showed a maximum at pH 7 in the presence of Mn2+, but at around pH 8 in the presence of Mg2+. This phosphatase is similar to phosphatase 2B or calcineurin. The possible regulatory function of this phosphatase in scallop catch muscle is discussed.     

Purification and characterization of a phosphoprotein phosphatase that dephosphorylates myosin and the isolated light chain from chicken gizzard smooth muscle

A phosphatase that dephosphorylates myosin and the isolated light chain has been purified to near homogeneity from chicken gizzard smooth muscle. The molecular weight of the enzyme was estimated to be 100,000 and 35,000 under native and denatured conditions, respectively. It requires Mg2+ or Mn2+. The activity was measured quantitatively with a coupled enzyme system with the aid of myosin light chain kinase. The Vm and Km were determined to be 23.4 mumol/mg/min and 4.2 microM, respectively, with the isolated light chain as substrate under the optimal conditions (5 mM Mg2+ at pH 8.45). The specific activity with myosin as substrate at a concentration of 0.9 microM was found to be 1.25 mumol/mg/min, which was about one-fifth of the activity for the isolated light chain under the same conditions. The phosphatase seems to be specific to gizzard myosin. It may play an important role in the regulation of the myosin-actin interaction in smooth muscle.     

Influence of heavy metal ions on the ATPase activity of actomyosin complex and myosin subfragment-1 from smooth muscle of the uterus

The effect of divalent cations--Co2+, Cu2+, Mn2+ and Ni2+ (5 mM) on the activity of actomyosin complex ATPase and ATPase of subfragment-1 (S1,head) of myosin from smooth muscle of the uterus was studied. It has been shown that Co2+, Mn2+ and Ni2+ inhibited, while Cu2+ activates the enzyme activity of both actomyosin and myosin S1. Mg and Mn ions had practically no effect on the emission intensity of eosin Y associated with actomyosin, while one could observe the most marked suppression of emission of related fluorescent probe in the presence of Cu cations and less pronounced suppression in the presence of Co2+. In the presence of Mn, Co and Ni cations the average hydrodynamic diameter (HD) of actomyosin complex and of subfragment-1 of the smooth muscle of the uterus is virtually identical to the HD in the presence of Mg2+. In the presence of Cu cations there is a considerable (ten-fold) increase in the size of the protein particles that may be a result of their aggregation. The results obtained evidence for the significant changes in the structure and function of the actomyosin complex of the myometrium in the presence of heavy metals and allow us to assume that the target of the effect of these metals on the contractile proteins is a subfragment-1 of myosin, where the active site of ATPase and actin-binding sites are localized.     

Comparison of the properties of the protein phosphatases from avian and mammalian smooth muscles: purification and characterization of rabbit uterine smooth muscle phosphatases

Three protein phosphatases were purified to near homogeneity from rabbit uterine muscle. These enzymes are termed rabbit uterine smooth muscle phosphatase (RU SMP)-I, -II, and -IV. RU SMP-I is composed of three subunits (Mr 60,000, 55,000, and 38,000) which comigrated with the subunits of turkey gizzard smooth muscle phosphatase (TG SMP)-I. Ethanol treatment of RU SMP-I dissociated the subunits and led to the purification of its catalytic subunit (Mr 38,000), RU SMP-Ic. Structural homology between the turkey gizzard and rabbit uterine SMP-I is indicated by the cross-reactivity of RU SMP-I with the polyclonal antibodies against TG SMP-I and -Ic. Like TG SMP-II, RU SMP-II is inactive in the absence of divalent cations and can be activated by Mg2+ and Mn2+. However, their electrophoretic profiles on sodium dodecyl sulfate-polyacrylamide gel are different. RU SMP-II shows two bands (Mr 42,000 and 44,000) while TG SMP-II is monomeric (Mr 43,000). Western blot analysis revealed that the 42,000 and 44,000-Da proteins cross-react with anti-TG SMP-II antibodies, suggesting that these proteins share common structural properties. The anti-TG SMP-I and Ic antibodies do not cross-react with RU SMP-II and -IV. Likewise, the anti-TG SMP-II antibodies do not cross-react with RU SMP-I and -IV, implying that these enzymes are distinct. RU SMP-IV is composed of a catalytic subunit (Mr 40,000) and a subunit with a molecular weight of 60,000 or 58,000. All three rabbit uterine smooth muscle phosphatases dephosphorylate the isolated myosin light chains but only RU SMP-IV dephosphorylates heavy meromyosin. However, when the catalytic subunit of RU SMP-I is dissociated from the regulatory subunits, it is active toward heavy meromyosin and exhibits higher activity toward myosin light chains and phosphorylase a than its holoenzyme. The substrate specificity of these enzymes and the effects of ATP, NaF, pyrophosphate, okadaic acid, Mg2+, Mn2+, and Ca2+ on their activities are very similar to those of the turkey gizzard smooth muscle phosphatases.     

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).     

Effects of metal ions on sphingomyelinase activity of Bacillus cereus

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.     

Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase.

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5'-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.     

The divalent cation dependence of bovine brain calmodulin-dependent phosphatase

The divalent cation dependence of a calmodulin-stimulated phosphatase from bovine brain has been characterized kinetically using phosphorylated myelin basic protein and casein as substrates. At saturating concentrations of calmodulin, dephosphorylation of both myelin basic protein and casein was catalyzed 8- to 10-fold more rapidly at saturating concentrations of Mn2+ than at saturating concentrations of Ca2+. Half-maximal rates of dephosphorylation of both substrates occurred at either 15 microM Mn2+ or 1 microM Ca2+, and the Kact for each ion was not influenced appreciably by the presence of calmodulin. Half-maximal rates of dephosphorylation were observed at concentrations of calmodulin ranging from 3 X 10(-8) to 10(-6) M at saturating concentrations of divalent cations depending on the substrate used and the particular cation chosen. Trypsin treatment of the phosphatase activated the enzyme several-fold, eliminated its calmodulin dependence, but did not alter the Mn2+ concentration dependence of the activity. Ca2+ (10 microM) increased dephosphorylation rates without altering the Mn2+ concentration dependence of the phosphatase activity regardless of the presence of calmodulin. Mg2+ at millimolar concentrations did not alter the Ca2+ or Mn2+ concentration dependence of the activity. As measured without calmodulin, Ca2+ (90 microM) or Mn2+ (200 microM) produced nearly identical alterations of the far ultraviolet circular dichroic spectrum of the phosphatase.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle: effects of Sr2+ and Cd2+ on Ca2+ uptake and formation of the phosphorylated intermediate of the Ca2+,Mg2+-ATPase

The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase.     

Limited proteolytic digestion and dissociation of smooth muscle phosphatase-I modifies its substrate specificity. Preparation and properties of different forms of smooth muscle phosphatase-I

Smooth muscle phosphatase-I (SMP-I), a protein phosphatase purified from turkey gizzard smooth muscle, is composed of 2 regulatory subunits (Mr = 60,000 and 55,000) and a catalytic subunit (Mr = 38,000). Two other forms of this enzyme have been prepared and characterized. The free catalytic subunit, termed SMP-Ic, was prepared by ethanol treatment of SMP-I, and a form devoid of the 55,000-Da subunit, termed SMP-I2, was prepared by limited tryptic digestion. Exposure of SMP-I to proteases like trypsin and chymotrypsin results in a rapid degradation of the 55,000-Da polypeptide. Degradation of the catalytic subunit is observed only upon prolonged digestion. The 60,000-Da polypeptide appears to be resistant to the action of trypsin and chymotrypsin. SMP-I dephosphorylates myosin light chains but is not active toward intact myosin or heavy meromyosin. However, when the catalytic subunit is dissociated from both regulatory subunits or from the 55,000-Da polypeptide, the enzyme becomes active toward myosin suggesting that the 55,000-Da polypeptide inhibits the activity of the catalytic subunit toward myosin. In addition to alteration of the substrate specificity, the regulatory subunits also modulate the effect of divalent cations, like Mn2+, on the activity of the enzyme.     

Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes. Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.     

Escherichia coli S-adenosylhomocysteine/5''-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action.

Ca2+-activated protein phosphatase activity was demonstrated in mouse pancreatic acinar cytosol with alpha-casein and skeletal-muscle phosphorylase kinase as substrates. This phosphatase activity preferentially dephosphorylated the alpha subunit of phosphorylase kinase. After DEAE-cellulose chromatography, the Ca2+-activated phosphatase activity became dependent on exogenous calmodulin for maximal activity. Half-maximal activation was achieved at 0.5 +/- 0.1 microM-Ca2+. Trifluoperazine completely inhibited Ca2+-activated phosphatase activity, with half-maximal inhibition occurring at 8.5 +/- 0.6 microM. Mn2+, but not Mg2+, at 1 mM concentration could substitute for Ca2+ in eliciting full enzyme activation. The apparent Mr of the phosphatase as determined by Sephadex G-150 chromatography was 93000 +/- 1000. Submitting active fractions obtained after Sephadex chromatography to calmodulin affinity chromatography resulted in the resolution of a major protein of Mr 55500 +/- 300. In conclusion, Ca2+-activated protein phosphatase activity has been identified in exocrine pancreas and has several features in common with Ca2+-activated calmodulin-dependent protein phosphatases previously isolated from brain and skeletal muscle. It is possible that this Ca2+-activated phosphatase may utilize as substrates certain acinar-cell phosphoproteins previously shown to undergo dephosphorylation in response to Ca2+-mediated secretagogues.     

Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards

A phosphoprotein phosphatase that dephosphorylates smooth muscle myosin has been purified to apparent homogeneity from turkey gizzards. Smooth muscle phosphatase (SMP) IV has a molecular weight of 150,000 as determined by gel filtration on a Sephadex G-200 column and is composed of two subunits (Mr = 58,000 and 40,000). Although it is active toward a number of proteins, its activities toward the contractile proteins, intact myosin, heavy meromyosin, and isolated myosin light chains are higher than its activities toward phosphorylase alpha, histone IIA, and phosphorylase kinase. SMP-IV preferentially dephosphorylates the beta-subunit of phosphorylase kinase. The properties of the enzyme have been studied using heavy meromyosin, a soluble chymotryptic fragment of myosin, and isolated myosin light chains as substrates. SMP-IV has high affinity for both substrates and is optimally active at neutral pH. Divalent cations, Ca2+ and Mg2+, activate the dephosphorylation of heavy meromyosin but inhibit the activity toward myosin light chains. Low concentrations of ATP (1-5 mM) activate SMP-IV but concentrations higher than 5 mM are inhibitory. Inhibition of 50% of the activity of the enzyme by NaF and PPi requires concentrations higher than 10 mM. Rabbit skeletal muscle heat stable inhibitor-2 has no effect on the activity of SMP-IV toward heavy meromyosin, myosin light chains, and phosphorylase alpha.     

Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N

CPI-17 is a phosphorylation-dependent inhibitory protein for smooth muscle myosin phosphate. Phosphorylation at Thr(38), in vitro, by protein kinase C or Rho-kinase enhances the inhibitory potency toward myosin phosphatase. Phosphorylation of CPI-17 by protein kinase N (PKN), a fatty acid- and Rho-activated serine/threonine kinase, and its effect on smooth muscle myosin phosphatase activity were investigated. CPI-17 was phosphorylated by GST-PKN-CAT, a constitutively active GST-fusion fragment of PKN, to 1.46 mol of P/mol of CPI-17, in vitro. The K(m) value of CPI-17 for PKN was 0.96 microM. Phosphorylation of PKN dramatically increased the inhibitory effect of CPI-17 on myosin phosphatase activity. The major and inhibitory phosphorylation site was identified as Thr(38) using a point mutant of CPI-17 and a phosphorylation-state specific antibody. Thus, CPI-17 is a substrate of PKN and might be involved in the Ca(2+) sensitization of smooth muscle contraction as a downstream effector of Rho and/or arachidonic acid.     

Turkey gizzard smooth muscle myosin phosphatase-III is a novel protein phosphatase.

Chromatography of turkey gizzard extract on Sephacryl S-300 has been shown to fractionate the various smooth muscle phosphatases. We have previously reported the purification and characterization of three of these enzymes, termed smooth muscle phosphatase (SMP)-I, -II, and -IV. Recently, we have purified SMP-III to near homogeneity. Although all of the smooth muscle phosphatases dephosphorylate the isolated myosin light chains, only SMP-III and -IV are active toward intact myosin and, therefore, are most likely to play a direct role in the muscle contraction-relaxation process. SMP-III has a higher molecular weight (390,000), as determined by gel filtration, than the other smooth muscle phosphatases and migrates as single band with a molecular weight of 40,000 in a sodium dodecyl sulfate-polyacrylamide gel. SMP-III is immunologically distinct from SMP-I and -II. It dephosphorylates heavy meromyosin and the isolated myosin light chains at a rapid rate but has low activity toward phosphorylase alpha. The activity of SMP-III is not affected by Ca2+ but is activated by Mn2+.Mg2+ stimulates the activity toward heavy meromyosin but inhibits the myosin light chain phosphatase activity. Attempts to classify SMP-III according to the scheme proposed by Ingebritsen and Cohen (Ingebritsen T. S., and Cohen, P. (1983) Science 221, 331-338) revealed that it is resistant to the heat stable inhibitor-2, suggesting that it is a Type 2 protein phosphatase. However, SMP-III is inhibited by concentrations of okadaic acid which are characteristic of Type 1 protein phosphatases and it binds to heparin-Sepharose like other Type 1 phosphatases. But most interestingly, SMP-III does not dephosphorylate the alpha- or beta-subunits of phosphorylase kinase, a property not reported for any Ser/Thr protein phosphatase.     

The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers

We have studied the effect of myosin P-light chain phosphorylation on the isometric tension generated by skinned fibers from rabbit psoas muscle at 0.6 and 10 microM Ca2+. At the lower Ca2+ concentration, which produced 10-20% of the maximal isometric tension obtained at 10 microM Ca2+, addition of purified myosin light chain resulted in a 50% increase in isometric tension which correlated with an increase in P-light chain phosphorylation from 0.10 to 0.80 mol of phosphate/mol of P-light chain. Addition of a phosphoprotein phosphatase reversed the isometric tension response and dephosphorylated P-light chain. At the higher Ca2+ concentration, P-light chain phosphorylation was found to have little effect on isometric tension. Fibers prepared and stored at -20 degrees C in a buffer containing MgATP, KF, and potassium phosphate incorporated 0.80 mol of phosphate/mol of P-light chain. Addition of phosphoprotein phosphatase to these fibers incubated at 0.6 microM Ca2+ caused a reduction in isometric tension and dephosphorylation of the P-light chain. There was no difference before and after phosphorylation of P-light chain in the normalized force-velocity relationship for fibers at the lower Ca2+ concentration, and the extrapolated maximum shortening velocity was 2.2 fiber lengths/s. Our results suggest that in vertebrate skeletal muscle, P-light chain phosphorylation increases the force level at submaximal Ca2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament.     

Characterization of protein kinase pathways responsible for Ca2+ sensitization in rat ileal longitudinal smooth muscle

We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca2+ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 microM microcystin induced LC20 diphosphorylation and contraction of beta-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF-109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 muM, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca2+ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle.     

Purification and characterization of smooth muscle myosin-associated phosphatase from chicken gizzards.

Myosin light chain phosphatase associated with smooth muscle myosin (MAPP) was isolated from chicken gizzard. The MAPP was tightly associated with myosin and was not dissociated from myosin under the physiological ionic conditions. The phosphatase was dissociated from myosin in the presence of high MgCl2, i.e. 80 mM MgCl2. The binding site of the enzyme on the myosin molecule was the subfragment-2 region, since the enzyme did bind to the myosin rod and heavy meromyosin but not to the subfragment-1 affinity column. MAPP was purified with a heparin-Sepharose 6B column, and two activity peaks were obtained, i.e. MAPP I and MAPP II. The major activity peak, MAPP I, was further purified to homogeneity by thiophosphorylated myosin light chain-Sepharose 4B column chromatography. MAPP I was a tetramer composed of four 34-kDa subunits. The enzyme preferentially dephosphorylated the beta-subunit of phosphorylase kinase and was strongly inhibited by the heat- and acid-stable protein phosphatase inhibitor-1, whereas it was partially inhibited by the inhibitor-2. The IC50 (concentration of inhibitor giving 50% inhibition) value for the inhibition of the enzyme by okadaic acid was 70 nM which was about eight times higher than skeletal muscle type-1 and 390 times higher than type-2 protein phosphatase. These results demonstrate that the MAPP I is a type-1-like protein phosphatase, although the properties are not the same as type-I phosphatase. The properties of the myosin-associated phosphatase were distinct from the phosphatases reported previously, although some properties were similar to smooth muscle phosphatase-IV. Therefore, it is concluded that MAPP I is a novel smooth muscle protein phosphatase. Since it strongly associated with smooth muscle myosin, it is likely that MAPP I is responsible for the dephosphorylation of smooth muscle myosin in situ.     

Characterization of regulatory light chain-a myosin kinase from smooth muscle of scallop, Patinopecten yessoensis

The protein kinase that phosphorylates the regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was isolated from scallop smooth muscle (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163). The enzymatic properties of this kinase (aMK) were investigated using RLC-a as the substrate. The Km value for ATP was 6.5 microM in the presence of 27 microM RLC-a at pH 7.0, and that for RLC-a was 133 microM in the presence of 1 mM ATP. The Vm value at saturation of both RLC-a and ATP was 0.25 s-1 at pH 7.0. The pH activity curve for aMK was bell-shaped with a maximum at around pH 7.8. The aMK activity was inhibited strongly by an increase in the KCl concentration. aMK required Mg2+, but was inhibited by high concentrations of Mg2+. The optimum activity was seen at 3 mM MgCl2. The mode of inhibition of the aMK activity by Ca2+ was studied. Assuming that the binding of Ca2+ to aMK induces the inhibition, the dissociation constant of Ca2+ was estimated to be 64 microM. aMK also phosphorylated LC20 of chicken gizzard myosin at a similar rate to that for RLC-a and the DTNB light chain of rabbit skeletal muscle myosin at a more lower rate. The helix and beta-sheet contents of aMK were estimated to be 19 and 30%, respectively, from the CD spectrum.