Protein kinase activity of purified rat liver glucocorticoid receptor

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5 m and DEAE-Sephacel. The purified GR sedimented in the 9-10S region in 5-20% sucrose gradients containing 0.10M KCl and 20mM Na2MoO4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 +/- 2,000. Affinity labeling of GR with [3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50 microM [gamma-32P]-ATP and divalent cations. Radioactive phosphate from [gamma-32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg2+ and Ca2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.     

Purification and properties of debranching enzyme from dogfish muscle.

Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.     

Purification and Characterization of a Ca2+- and Calmodulin-Dependent Protein Kinase from Rat Brain

Abstract: A Ca²⁺- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited K _m values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and K _a values of 12 n M and 1.9 μM for brain calmodulin and Ca²⁺, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca²⁺- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca²⁺- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.     

Studies on the mechanism of the conversion of cytosol δ-aminolevulinic acid synthase to the mitochondrial enzyme in the liver of the allylisopropylacetamide-treated rat

δ-Aminolevulinic acid (ALA) synthase was partially purified from liver cytosol fraction of rats treated with allylisopropylacetamide (AIA). The cytosol ALA synthase showed an apparent molecular weight of 320,000. The cytosol ALA synthase of this size dissociates into at least three protein components when subjected to sucrose density gradient centrifugation in the presence of 0.25 m NaCl: one is the catalytically active protein with an s value of about 6.4 or a molecular weight of 110,000, and the other two are catalytically inactive binding proteins showing s values of about 4 and 8, respectively. Recombination of the 6.4 S protein and the 4 S protein yielded a protein complex with an apparent molecular weight of 170,000 and recombination of all three protein components resulted in formation of the original cytosol ALA synthase. The cytosol ALA synthase also loses its binding proteins when treated with various proteases; thus, the enzyme-active protein obtained after papain digestion was very similar, if not identical, to mitochondrial ALA synthase. When treated with trypsin, however, the cytosol ALA synthase was converted to an enzyme showing an apparent molecular weight of 170,000, which probably represents the complex of the mitochondria-type enzyme and the 4 S binding protein. The cytosol ALA synthase tends to aggregate to form a dimer with an apparent molecular weight of 650,000–700,000. The aggregated form of the cytosol ALA synthase was less susceptible to trypsin digestion. Hemin strongly stimulated dimer formation of the cytosol ALA synthase and the aggregate produced by contact with hemin was very tight and did not easily dissociate into its respective protein components by sucrose gradient centrifugation or even after treatment with trypsin. The possible mechanisms of the conversion of cytosol ALA synthase to the mitochondrial enzyme and also of the inhibition by hemin of the intracellular translocation of ALA synthase are discussed.     

Purification of glycogen debranching enzyme from porcine brain: evidence for glycogen catabolism in the brain

Amylo-1,6-glucosidase from porcine brain was purified to homogeneity by ammonium sulfate fractionation, followed by sequential steps of liquid chromatography on DEAE-Sephacel, Sephacryl S-300, and Super Q. The purified enzyme had both maltooligosaccharide transferase and amylo-1,6-glucosidase activities within a single polypeptide chain, and the combination of these two activities removed the branches of phosphorylase limit dextrin. Based on these results, the purified enzyme was identified as a glycogen debranching enzyme (GDE). The molecular weight of the brain GDE was 170,000 by gel-filtration and 165,000 by reducing SDS-PAGE. The pH profile of maltooligosaccharide transferase activity coincided with that of the amylo-1,6-glucosidase activity (pH optimum at 6.0). The existence of GDE as well as glycogen phosphorylase in the brain explains brain glycogenolysis fully and supports the hypothesis that glycogen is a significant source of energy in this organ.     

Immunochemical analysis of porcine cardiac C-protein

C-protein has been isolated from pig heart and its immunochemical properties studied. It is extracted with myosin, and separated from the myosin on a DEAE-Sephadex column. The amount of C-protein recovered from crude myosin is approx. 3.5%. The molecular weight of C-protein is 150,000. Anti-C-protein serum reacts with crude myosin and purified C-protein but not with purified myosin in immunodiffusion plates. Cardiac C-protein does not react with anti-skeletal white muscle C-protein serum. Immunoblotting experiments show that anti-cardiac C-protein serum reacts with a Mr = 150,000 component in myofibrils or crude myosin. C-protein is located in the A-band, except the M-line region, of the myofibrils. These results indicate that C-protein is an intrinsic component of the thick filaments in pig heart myofibrils.     

M-protein in chicken cardiac muscle.

Chicken cardiac muscle myofibrils lack a visible M-line. Antibodies against chicken breast muscle M-protein, an M-line component with M_r = 165 000, were used to demonstrate the presence of a similar protein in chicken heart muscle. The immunoreplica technique showed the heart protein to have about the same molecular weight as the breast muscle M-protein on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS). Positive staining within the H-zone was observed when the indirect immunofluorescence technique was used to localize the M-protein in isolated heart myofibrils. This result was confirmed by electron microscopic investigations on longitudinal sections of antibody-incubated heart muscle fiber bundles showing the antibody against M-protein to be bound within a region corresponding to the M-line region of breast muscle myofibrils.     

Isolation and properties of platelet myosin light chain kinase.

A protein kinase which phosphorylates the 20 000-dalton light chain of platelet myosin has been isolated from human blood platelets and purified approximately 600-fold. Elution of a 7.5% polyacrylamide gel following electrophoresis of the partially purified enzyme yielded a single peak of kinase activity which could be aligned with a protein band on a stained gel. Assuming a globular shape, a native molecular weight of 83 000 (+/- 10%) was determined by gel filtration on Bio-Gel P-200. The kinase requires Mg2+ for activity and is not sensitive to the removal of trace Ca2+. The enzyme purified from human platelets phosphorylates the 20 000-dalton light chain of mouse fibroblast and chicken gizzard myosin, but does not phosphorylate human skeletal and cardiac myosin.     

Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart

The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and κ-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K_0.5 = 93 +/- 5 nM), glycogen synthase (K_0.5 = 127 +/- 10 nM), and κ-casein (K_0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 μM) but a low affinity for κ-casein (21 +/- 2 μM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.     

Isolation and characterization of the 165 000 dalton protein component of the M-line of rabbit skeletal muscle and its interaction with creatine kinase

The M-line protein component of molecular weight 165 000 was isolated and purified from rabbit skeletal muscle using ion exchange chromatography. Gel electrophoresis, in the presence and absence of sodium dodecyl sulfate, revealed the protein to be homogeneous. Sodium dodecyl sulfate gel electrophoresis and low speed sedimentation equilibrium studies in 0.5 M KCl, 50 mM potassium phosphate gave a molecular weight of 165 000 suggesting the protein to be made up of a single polypeptide chain. Circular dichroism spectra revealed the presence of two negative dichroic bands located at 216 and 208 nm, indicative of the presence of some beta-structure. Ellipticity values at these two wavelengths were --6500 +/- 400 and --7500 +/- 400 deg . cm2 . dmol-1, respectively. Addition of 165 000 component lowered the enzymatic activity of creatine kinase M-line protein and the nature of the inhibition was found to be a competitive one. When the protein was mixed with creatine kinase in a 1 : 1 mole ratio in a medium consisting of 0.2 M KCl, 25 mM Tris, 1 mM dithiothreitol (pH 8.0), low speed sedimentation equilibrium studies gave a molecular weight of 260 000 +/- 10 000 for the complex, indicative of an interaction of the two components of the M-line.     

The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1.

The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the beta isoform of protein phosphatase-1 (PP1) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1 beta with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.     

H-protein and X-protein. Two new components of the thick filaments of vertebrate skeletal muscle

With a view to obtaining a more complete view of the composition and structure of the thick filaments of vertebrate skeletal muscle, we have isolated and characterized two new myofibrillar components, H-protein and X-protein. These were purified by hydroxyapatite column chromatography of an impure C-protein preparation itself made from impure myosin extracted from rabbit back and leg muscles. H-protein is the protein responsible for band H on sodium dodecyl sulphate/polyacrylamide gel electrophoresis of crude myosin. X-protein, although present in such preparations in significant quantities, was not detected previously since it is difficult to resolve from C-protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Physical-chemical parameters have been determined for the new proteins and compared with those of C-protein. The apparent chain weight of H-protein estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis is 69,000, whereas that of X-protein (152,000) is only slightly greater than that of C-protein (140,000). The molecular weights of H- and X-proteins determined by sedimentation equilibrium centrifugation show that the molecules contain only a single polypeptide chain. The circular dichroism spectra indicate that the proteins have low alpha-helical contents. Both proteins, particularly H-protein, have a high proline content. Although X-protein is of similar chain weight to C-protein, the two show distinct differences in other properties. The sedimentation coefficient of X-protein is markedly lower than that of C-protein, suggesting X-protein is a more asymmetrical molecule. The amino acid compositions, although broadly similar, also show clear differences. Antibodies to H-protein, X-protein and C-protein have been raised in goats and shown not to cross-react.     

Purification and characterization of a multisubunit phosphatase from turkey gizzard smooth muscle. The effect of calmodulin binding to myosin light chain kinase on dephosphorylation

A phosphatase that is active in dephosphorylating the isolated 20,000-Da light chain of myosin, as well as the enzyme myosin light chain kinase, has been purified to apparent homogeneity from turkey gizzards. The enzyme has a molecular weight of 165,000 by sedimentation-equilibrium centrifugation under nondenaturing conditions and is composed of three subunits (Mr = 60,000, 55,000, and 38,000) in a 1:1:1 molar ratio. The properties of the holoenzyme, as well as the purified catalytic subunit (Mr = 38,000) were compared using myosin light chains, intact myosin, and myosin light chain kinase as substrates. Although the holoenzyme is active in dephosphorylating the isolated myosin light chains and the enzyme myosin light chain kinase, the holoenzyme does not dephosphorylate myosin. On the other hand, the catalytic subunit of the holoenzyme dephosphorylates all three substrates. When myosin light chain kinase, which has been phosphorylated at two sites is used as substrate, both sites are rapidly dephosphorylated by the phosphatase in the absence of bound calmodulin. If calmodulin is bound to the diphosphorylated kinase, only one site is dephosphorylated. Interestingly, the single site dephosphorylated when calmodulin is bound to myosin light chain kinase is the site that is not phosphorylated when the calmodulin-myosin kinase complex is phosphorylated by cAMP-dependent protein kinase.     

Identification of a calmodulin-dependent protein kinase in the cardiac cytosol, which phosphorylates phospholamban in the sarcoplasmic reticulum

A multifunctional calmodulin-dependent protein kinase in the canine cardiac cytosol was purified to near homogeneity. The purified enzyme inactivated glycogen synthase by means of phosphorylation. The enzyme also phosphorylated phospholamban and several other proteins. In view of its physicochemical properties and substrate specificity, the enzyme differed from myosin light chain kinase and phosphorylase kinase, and was considered to belong to a class of similar calmodulin-dependent protein kinases from brain, liver, and skeletal muscle. The results suggest that the enzyme mediates multiple Ca2+-dependent functions in the heart.     

Glycogen phosphorylase from Neurospora crassa: purification of a high-specific-activity, non-phosphorylated form.

A highly active glycogen phosphorylase was purified from Neurospora crassa by polyethylene glycol fractionation at pH 6.16 combined with standard techniques (chromatography and salt fractionation). The final preparation had a specific activity of 65 +/- 5 U/mg of protein (synthetic direction, pH 6.1, 30 degrees C) and was homogeneous by the criteria of gel electrophoresis, amino-terminal analysis, gel filtration, and double immunodiffusion in two dimensions. The enzyme had a native molecular weight of 180,000 +/- 10,000 (by calibrated gel filtration and gel electrophoresis) and a subunit molecular weight of 90,000 +/- 5,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each subunit contained one molecule of pyridoxal phosphate. No phosphoserine or phosphothreonine was detected by amino acid analysis optimized for phosphoamino acid detection. The enzyme isolated from cells grown on high-specific-activity 32Pi (as sole source of phosphorus) contained one atom of 32P per subunit. All the radioactivity was removed by procedures that removed pyridoxal phosphate. Thus, the enzyme could not be classified as an a type (phosphorylated, active in the absence of a cofactor) or as a b type (non-phosphorylated, inactive in the absence of a cofactor). The level of phosphorylase was markedly increased in mycelium taken from older cultures in which the carbon source (glucose or sucrose) had been depleted. The polyethylene glycol fractionation scheme applied at pH 7.5 to mycelial extracts of younger cultures (taken before depletion of the sugar) resulted in co-purification of glycogen phosphorylase and glycogen synthetase.     

Identification of disulfide-linked transforming growth factor-beta 1-specific binding proteins in rat glomeruli

We have identified two distinct classes of transforming growth factor-beta (TGF-beta)-binding proteins by affinity labeling rat glomeruli with 125I-TGF-beta 1 and 125I-TGF-beta 2. The first type consists of a group of proteins that bind TGF-beta 1 but do not bind TGF-beta 2. When 125I-TGF-beta 1 affinity-labeled glomeruli were separated under nonreducing conditions, four prominent bands with Mr values of 320,000, 260,000, 170,000, and 90,000 were observed. Following reduction, the 320,000 and 170,000 bands yielded only a 100,000 band, the 260,000 complex yielded bands of 200,000, 100,000, and 85,000, and the 90,000 band migrated with an Mr of 85,000. Binding of 125I-TGF-beta 1 to these proteins was unaffected by the addition of as much as a 1,000-fold excess of TGF-beta 2. The second type of glomerular TGF-beta-binding protein consists of Mr 160,000-200,000 and 280,000 proteins that bind both TGF-beta 1 and beta 2. Digestion of these affinity-labeled proteins with heparitinase and chondroitinase resulted in a decrease of approximately 40,000 in their apparent molecular weights. Glomerular TGF-beta 1-binding proteins are distinct from previously described TGF-beta-binding proteins in their specificity for TGF-beta 1 and their formation of disulfide-linked multimers. The TGF-beta 1/beta 2-binding proteins share some properties of the previously described type III TGF-beta receptor.     

The calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Purification, subunit structure and substrate specificity

A calmodulin-dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified approximately equal to 5000-fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0-33%), and chromatographies on phosphocellulose, calmodulin-Sepharose and DEAE-Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of approximately equal to 3%. The Km for glycogen synthase was 3.0 microM and the V 1.6-2.0 mumol min-1 mg-1. The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other. Following incubation with Mg-ATP and Ca2+-calmodulin, the purified protein kinase underwent an 'autophosphorylation reaction'. The reaction reached a plateau when approximately equal to 5 mol of phosphate had been incorporated per 58 000-Mr subunit. Both the 58 000-Mr and 54 000-Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity. The calmodulin-dependent protein kinase initially phosphorylated glycogen synthase at site-2, followed by a slower phosphorylation of site-1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl-CoA carboxylase and ATP-citrate lyase. These findings suggest that the calmodulin-dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo. Glycogen synthase kinase-4 is an enzyme that resembles the calmodulin-dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site-2), but not glycogen phosphorylase. Glycogen synthase kinase-4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin-dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase-4 is not a proteolytic fragment of the calmodulin-dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+-calmodulin.     

Purification and subunit composition of guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung.

The guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung was purified to apparent homogeneity by affinity chromography using 8-2-aminoethylthio-cGMP coupled to Sepharose 4B. The kinase activity was purified approximately 6000-fold with an overall recovery of approximately 20%. The product isolated by affinity chromatography contained both cGMP-binding and cGMP-dependent histone kinase activity, indicating that the enzyme was not dissociated into regulatory and catalytic components by the immobilized cGMP derivative. The enzyme had a molecular weight of approximately 165,000 and a sedimentation coefficient of 7.8 S. The purified kinase displayed several characteristics similar to that of the partially purified enzyme including specificity for cGMP and stimulation by high concentrations of magnesium. On sodium dodecyl sulfate gels, only one major polypeptide chain was present having a molecular weight of approximately 81,000. This subunit bound 1 mol of cGMP and exhibited cGMP-dependent protein kinase activity. It is proposed that the native enzyme consists of two identical subunits (Mr=81,000), each of which binds cGMP and catalyzes protein phosphorylation.     

Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity

Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.     

Purification and characterization of a calcium-calmodulin-dependent phospholamban kinase from canine myocardium

A Ca2+-calmodulin-dependent protein kinase was purified to apparent homogeneity from the cytosolic fraction of canine myocardium, with phospholamban as substrate. Purification involved sequential chromatography on DEAE-cellulose, calmodulin-agarose, DEAE-Bio-Gel A, and phosphocellulose. This procedure resulted in a 987-fold purification with a 5.4% yield. The purified enzyme migrated as a single band on native polyacrylamide gels, and it exhibited an apparent molecular weight of 550,000 upon gel filtration. Gel electrophoresis under denaturing conditions revealed a single protein band with Mr 55,000. The purified kinase could be autophosphorylated in a Ca2+-calmodulin-dependent manner, and under optimal conditions, 6 mol of Pi was incorporated per mole of 55,000-dalton subunit. The activity of the enzyme was dependent on Ca2+, calmodulin, and ATP.Mg2+. Other ions which could partially substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations were Sr2+ greater than Mn2+ greater than Zn2+ greater than Fe2+. The substrate specificity of the purified Ca2+-calmodulin-dependent protein kinase for cardiac proteins was determined by using phospholamban, troponin I, sarcoplasmic reticulum membranes, myofibrils, highly enriched sarcolemma, and mitochondria. The protein kinase could only phosphorylate phospholamban and troponin I either in their purified forms or in sarcoplasmic reticulum membranes and myofibrils, respectively. Exogenous proteins which could also be phosphorylated by the purified protein kinase were skeletal muscle glycogen synthase greater than gizzard myosin light chain greater than brain myelin basic protein greater than casein. However, phospholamban appeared to be phosphorylated with a higher rate as well as affinity than glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)