Purification and characterization of rabbit liver calmodulin-dependent glycogen synthase kinase

A rabbit liver cAMP-independent glycogen synthase kinase has been purified 4500-fold to a specific activity of 2.23 mumol of 32P incorporated per min per mg of protein using ion exchange chromatography on DEAE-Sephacel and phosphocellulose, gel filtration chromatography on Sepharose 6B, and affinity chromatography on calmodulin-Sepharose. This synthase kinase, which was completely dependent on the presence of calmodulin (apparent K0.5 = 0.1 microM) and calcium for activity, also catalyzed the phosphorylation of purified smooth muscle myosin light chain but not of smooth muscle myosin. Using 0.5 mM ATP, a maximal rate of phosphorylation of glycogen synthase was achieved in the presence of 10 mM magnesium acetate with a pH optimum of 7.8. Gel filtration experiments indicated a Stokes radius of about 70 A and sucrose density gradient centrifugation data gave a sedimentation coefficient of 10.6 S. A molecular weight of approximately 300,000 was calculated. A definitive subunit structure was not determined, but major bands observed after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate corresponded to a doublet at 50,000 to 53,000. The calmodulin-dependent glycogen synthase kinase incorporated about 1 mol of 32P per mol of synthase subunit into sites 2 and 1b associated with a decrease in the synthase activity ratio from 0.8 to about 0.4. The calmodulin-dependent glycogen synthase kinase may mediate the effects of alpha-adrenergic agonists, vasopressin, and/or angiotensin II on glycogen synthase in liver.     

Ca2+-stimulated phosphorylation of muscle glycogen synthase by phosphorylase b kinase.

Phosphorylase b kinase from rabbit muscle phosphorylates glycogen synthase purified from the same tissue. The reaction is markedly stimulated by Ca2+ and results in a decrease in the synthase %I activity. Phosphorylase b kinase action leads to the incorporation of phosphate (0.6 to 0.8 mol/mol of subunit) preferentially into a single cyanogen bromide fragment of synthase (fragment III). Cyclic AMP-independent synthase kinase also shows a specificity for the site(s) contained in fragment III whereas the cyclic AMP-dependent protein kinase exerts a preference for the site(s) located in a distinct cyanogen bromide fragment (fragment II). A Ca2+-stimulated endogenous kinase also results in the phosphorylation of fragment III and can be attributed to the presence of phosphorylase b kinase. The finding of a Ca2+-stimulated phosphorylation of glycogen synthase has important implications for the regulation of glycogen metabolism and particularly those processes thought to be controlled by cytoplasmic Ca2+ concentration.     

Purification and characterization of high Ca2+-requiring neutral proteases from porcine and bovine brains

Porcine and bovine brain high Ca2+-requiring neutral proteases were purified to homogeneity by the same isolation procedures, and their properties were compared. A high degree of similarity existed between the two proteases. The purification procedures included ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on phenyl-Sepharose CL-4B, second DEAE-cellulose chromatography, second phenyl-Sepharose CL-4B chromatography, and gel filtration on Ultrogel AcA 34. Both purified enzymes were composed of Mr 75,000 and 29,000 subunits, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzymes required 250 microM Ca2+ for half-maximal activity and 700 microM Ca2+ for maximal activity. Sr2+ and Ba2+, but not Mg2+ or Mn2+, also activated both enzymes but not as effectively as Ca2+. Both enzymes displayed maximum activity at pH 7.5-8.0. Leupeptin, antipain, and trans-epoxysuccinyl-L-leucylagmatine inhibited both enzymes. Neurofilament triplet proteins and microtubule-associated proteins were extensively hydrolyzed by both proteases, but tubulin and actin were not hydrolyzed. The amino acid compositions of the two proteases were very similar. Antisera against bovine brain protease cross-reacted with porcine brain protease when examined by immunoelectrotransfer blot techniques.     

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.     

Purification and characterization of a cytosolic protein-tyrosine kinase from porcine spleen

A cytosolic protein-tyrosine kinase has been highly purified from porcine spleen using [Val5]angiotensin II as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, Sephacryl S-200, casein-Sepharose 4B, heparin-Sepharose CL-6B and anti-(4-aminobenzyl phosphonic acid)--Sepharose 4B. Analysis of the most highly purified preparation by SDS/PAGE revealed a major silver-stained band of molecular mass 40 kDa. The 40-kDa cytosolic protein-tyrosine kinase was purified approximately 10,000-fold with an overall yield of about 7%. It had autophosphorylation activity which was carried out by intramolecular catalysis. The stoichiometry of phosphate incorporation was about 1 mol phosphate/mol enzyme. In the autophosphorylation reaction, the apparent Km value for ATP was relatively low, 0.35 microM; Mn2+ was slightly preferred to Mg2+ as divalent cation. [Val5]Angiotensin II phosphorylation activity of the 40-kDa kinase increased with the amount of phosphate incorporated into the enzyme. A phosphate exchange reaction was observed during the autophosphorylation. These results suggest that the 40-kDa kinase described here is a different type of protein-tyrosine kinase than the enzymes so far reported.     

Purification and characterization of glycogen synthase from a glycogen-deficient strain of Saccharomyces cerevisiae

Chromatography of wild-type yeast extracts on DEAE-cellulose columns resolves two populations of glycogen synthase I (glucose-6-P-independent) and D (glucose-6-P-dependent) (Huang, K. P., Cabib, E. (1974) J. Biol. Chem. 249, 3851-3857). Extracts from a glycogen-deficient mutant strain, 22R1 (glc7), yielded only the D form of glycogen synthase. Glycogen synthase D purified from either wild-type yeast or from this glycogen-deficient mutant displayed two polypeptides with molecular masses of 76 and 83 kDa on sodium dodecyl sulfate-gel electrophoresis in a protein ratio of about 4:1. Phosphate analysis showed that glycogen synthase D from either strain of yeast contained approximately 3 phosphates/subunit. The 76- and 83-kDa bands of the mutant strain copurified through a variety of procedures including nondenaturing gel electrophoresis. These two polypeptides showed immunological cross-reactivity and similar peptide maps indicating that they are structurally related. The relative amounts of these two forms remained constant during purification and storage of the enzyme and after treatment with cAMP-dependent protein kinase or with protein phosphatases. The two polypeptides were phosphorylated to similar extent in vitro by the catalytic subunit of mammalian cyclic AMP-dependent protein kinase. Phosphorylation of the enzyme in the presence of labeled ATP followed by tryptic digestion and reversed phase high performance liquid chromatography yielded two labeled peptides from each of the 76- and 83-kDa subunits. Treatment of wild-type yeast with Li+ increased the glycogen synthase activity, measured in the absence of glucose-6-P, by approximately 2-fold, whereas similar treatment of the glc7 mutant had no effect. The results of this study indicate that the GLC7 gene is involved in a pathway that regulates the phosphorylation state of glycogen synthase.     

Ca2+, calmodulin-dependent phosphorylation of glycogen synthase by a brain protein kinase

The inactivation of rec BC nuclease activity and simultaneously the separation of 3 DNA-dependent ATPases and an ATP-independent DNases specific for single-stranded DNA have been observed after DEAE-cellulose chromatography of cell extracts from Escherichia coli. Two of the ATPases catalyze the strand separation of duplex DNA. Reconstitution of ATP-dependent DNase activity has been carried out by the combination of the separated enzymes.     

Phosphorylation of glycogen synthase by the Ca2+- and phospholipid-activated protein kinase (protein kinase C)

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been found to phosphorylate and inactivate glycogen synthase. With muscle glycogen synthase as a substrate, the reaction was stimulated by Ca2+ and by phosphatidylserine. The tumor-promoting phorbol esters 12-O-tetradecanoyl phorbol 13-acetate was also a positive effector, half-maximal activation occurring at 6 nM. Phosphorylation of glycogen synthase, but not histone, was partially inhibited by glycogen, half-maximally at 0.05 mg/ml, probably via a substrate-directed mechanism. The rate of glycogen synthase phosphorylation was approximately half that for histone; the apparent Km for glycogen synthase was 0.25 mg/ml. Protein kinase C also phosphorylated casein, the preferred substrate among the individual caseins being alpha s1-casein. Glycogen synthase was phosphorylated to greater than 1 phosphate/subunit with an accompanying reduction in the -glucose-6-P/+glucose-6-P activity ratio from 0.9 to 0.5. Phosphate was introduced into serine residues in both the NH2-terminal and COOH-terminal CNBr fragments of the enzyme subunit. The two main tryptic phosphopeptides mapped in correspondence with the peptides that contain site 1a and site 2. Lesser phosphorylation in an unidentified peptide was also observed. Rabbit liver and muscle glycogen synthases were phosphorylated at similar rates by protein kinase C. The above results are compatible with a role for protein kinase C in the regulation of glycogen synthase as was suggested by a recent study of intact hepatocytes.     

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

Purification and characterization of calmodulin from porcine renal medulla

A calcium sensitive phosphodiesterase (PDE) activated by an endogenous calmodulin was identified in the cytosolic fraction of porcine renal medulla. The PDE and calmodulin were separated from each other by DEAE-cellulose column chromatography. Calmodulin was purified from a heat-treated supernatant by column chromatography with DEAE-cellulose and hydroxylapatite. The purified renal calmodulin has a molecular weight of 17,500, is heatstable, and has a pI of 4.2. Activation of the renal PDE by calmodulin was immediate and stoichiometric. The renal calmodulin and PDE cross react with bovine brain calmodulin and PDE, indicating a lack of tissue and species specificity. Thus, renal calmodulin is very similar to bovine brain calmodulin. However, renal calmodulin did not affect detergent-solubilized or membrane-bound renal adenylate cyclase or the antidiuretic hormone-stimulated activity of the enzyme. These results suggest that calmodulin may function in the renal medulla to regulate cAMP levels by stimulation of PDE but not adenylate cyclase. However, the ubiquitous distribution of calmodulin in eukaryotic cells and its effects on a number of other enzymes allow the possibility that calmodulin may have a role in renal function other than cAMP metabolism.     

Purification and properties of cAMP independent glycogen synthase kinase and phosvitin kinase from human leukocytes

Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had M_r 250 000, 7.2S and the peak A enzyme M_r 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl₂. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K _m ^app 4.2 m for muscle glycogen synthease I and K _m ^app 45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca²⁺, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP adenosine cyclic 3:5-monophosphate - DTT dithiothreitol - EGTA ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid - PMSF phenylmethylsulfonylfluoride - PKI protein kinase inhibitor - RI ratio of independence for glycogen synthase - SDS sodium dodecyl sulphate     

Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver

Summary Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000–52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.     

Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Purification and some regulatory properties of glycogen synthase I from rabbit renal medulla

1. Glycogen synthase I (activity ratio approximately equal to 1) was purified over 10,000-fold from rabbit renal medulla. 2. The purified synthase was stimulated about 1.5-fold by glucose-6-P and other divalent anions when assayed at pH 7.7 and near saturating UDPGlc. When assayed at physiological UDPGlc (75-100 microM), the enzyme was stimulated about 5-fold by glucose-6-P. 3. At pH 7.7 the activation by either Na2SO4 or glucose-6-P was due to an increase in V and a decrease in S0.5 for UDPGlc. At pH. 6.9, activation was due to a decrease in S0.5. 4. At low UDPGlc, synthase activity was inhibited by adenine nucleotides and the inhibition was partially relieved by glucose-6-P, UDP inhibited in a competitive manner with respect to UDPGlc. 5. These results suggest that the activity of renal medullary synthase I may be regulated by cellular metabolites.     

Ca2+, calmodulin-dependent phosphorylation, and inactivation of glycogen synthase by a brain protein kinase

Glycogen synthase from skeletal muscle was phosphorylated by a Ca2+, calmodulin-dependent protein kinase from brain, with concomitant inactivation. About 0.7 mol phosphate/mol subunit was sufficient for a maximal inactivation of glycogen synthase. Further phosphorylation of the enzyme had no effect on the activity. The concentrations required to give half-maximal phosphorylation and inactivation of glycogen synthase were 1.1 and 0.5 microM for Ca2+, and 22 and 11 nM for calmodulin, respectively. The molar ratio of the subunit of the protein kinase to calmodulin was 2-3:1 for half-maximal phosphorylation and inactivation of glycogen synthase. The Km values for glycogen synthase and ATP were 3.6 and 114 microM, respectively, for phosphorylation. Phosphate was incorporated into sites Ia, Ib, and 2 on glycogen synthase, and site 2 was the most rapidly phosphorylated. These results indicate that the brain Ca2+, calmodulin-dependent protein kinase is probably involved in glycogen metabolism in the brain as a glycogen synthase kinase.     

Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma

The Ca²⁺/Mg²⁺ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca²⁺ (Ka = 0.4 mM) and Mg²⁺ (Ka = 0.2 mM) as well as by other cations in the order Ca^2– > Mg²⁺ > Mn²⁺ > Sr²⁺ > Ba²⁺ > Ni²⁺ > Cu²⁺. The ATPase activity in the presence of Ca²⁺ was markedly inhibited by Mg²⁺, Mn²⁺, Ni²⁺ and Cu²⁺ whereas the monovalent cations such as Na⁺ and K⁺ were without effect. The enzyme did not exhibit Ca²⁺ stimulated Mg²⁺ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca²⁺ or Mg²⁺ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca²⁺ metabolism in the myocardium is discussed.     

Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin.

L-Arginine-derived nitric oxide acts as an inter- and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine-inducible enzyme in macrophages and a constitutive, Ca2(+)-regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was identified as an NO synthase with a specific NO-chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO-sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.     

Purification to homogeneity, characterization and monoclonal antibodies of phospholipid-sensitive Ca2+-dependent protein kinase from spleen.

A phospholipid-sensitive Ca2+-dependent protein kinase was purified to homogeneity, for the first time, from extracts of pig spleen, employing the steps of DEAE-cellulose, octyl-agarose, Sephacryl S-200 and phosphatidylserine-Affigel 10 affinity chromatographies. The purified enzyme appeared as a single protein band on both analytical (non-denaturing) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, having a minimum mol.wt. of 68 000 +/- 200. The molecular weight of the enzyme was also determined to be 74 500 +/- 4600 by gel filtration and 80 000 based on its sedimentation coefficient (5.52 S) and Stokes radius (3.52 +/- 0.09 nm), indicating that the enzyme was a monomeric protein. The frictional ratio (f/f0) of the enzyme was 1.24, indicating it was non-globular in shape. The enzyme had a pI of 5.3, and a pH optimum of 6.5 for its reaction. Amino acid analysis indicated that the enzyme apparently was not similar to myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) or cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The enzyme had an apparent Km for ATP of 7.5 microns. Histone H1 and myelin basic protein were effective substrates for the enzyme, with apparent Km values of 0.3 and 0.2 microns, and Vmax, values of 0.06 and 0.09 mumol/min per mg of enzyme respectively. The enzyme activity was dependent on both phosphatidylserine (apparent Ka = 6.25 micrograms/ml) and Ca2+ (apparent Ka = 160 microns). Calmodulin was unable to substitute for the phospholipid as a cofactor, nor was it a subunit of the enzyme. Sr2+ and Ba2+ could partially mimic Ca2+ to activate the enzyme in the presence of phosphatidylserine. An endogenous substrate protein (mol.wt. 41 000) for the enzyme was found in the total, solubilized fraction of pig spleen. Monoclonal antibodies against the enzyme interacted similarly with the homogeneous and impure enzyme; the antibodies, however, did not bind to cyclic nucleotide-dependent protein kinases.