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PubMed1971

A cyclic AMP-dependent protein kinase of the bovine pineal gland.

A protein kinase that is dependent on cAMP was found to be present in significant amounts in rat and bovine pineal glands. This enzyme system was isolated from bovine pineal glands and shown to consist of two proteins, a kinase and a cAMP-binding protein. The kinase primarily phosphorylates histones and may function in the control of the diurnal variation of the enzymes in the melatonin biosynthetic pathway.

Adenosine Triphosphate↗
PubMed1976

[Kinetics and regulatory properties of the disulfide reductase enzyme from mouse liver].

Disulfide reductase (DSR) of mice liver supernatant is kinetically demonstrated as associating-dissociating oligomeric protein with positive homotropic cooperativity for the substrate. Cyclic 3',5'-AMP (10(-11)--10(-5) M) activates DSR and increases V, but does not change either [S]0,5, nor nH and does not shift the plot of specific activity versus the enzyme concentration. ATP, GTP, UTP, CTP, protamine, histone, Mg2+, Ca2+, EDTA (but not adenosine, 5'-AMP, 2'3'-AMP, ADP beef serum albumin) activated DSR. The effects of different modifiers are not summed up. Preincubation is essential for the action of the majority of the activators. Heating for 8 minutes at 55 degrees C desensitized completely DSR to all the modifiers without changing its catalytic activity, [S]0,5 and nH values. Possible mechanisms of activation of DSR, especially the involvement of protein kinase, are discussed.

Animals↗
PubMed1998

Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue. III. Purification and characterisation of a calcium-regulated protein kinase.

A calcium-dependent protein kinase (PK-III), not requiring calmodulin for activity, was purified from extracts of ripening mango fruit tissue. Purification was achieved by ammonium sulfate fractionation and sequential anion exchange-, hydrophobic interaction-, dye ligand affinity- and gel filtration chromatography; which allowed a recovery of 1-5% of the total available kinase activity. The final specific activity in the presence of 1 mM Ca2+ was consistently 9 nmol min-1mg-1. The purified enzyme was a monomer with a Mr of 49000, but was resolved by denaturing electrophoresis into two related protein bands of 49 and 45 kDa. Enzyme activity was activated >30-fold by micromolar amounts of free calcium and was dependent upon millimolar Mg2+ or Mn2+ concentrations. Calmodulin (1 microM) had no effect on PK-III activity but the calmodulin antagonists, calmidazolium and chlorpromazine, inhibited PK-III in a dose-dependent manner over a range of 0 to 100 microM. The results suggest a regulatory domain that is similar to calmodulin. PK-III phosphorylated histone III-S and to a lesser extent casein, but did not phosphorylate histone II-S, phosvitin or protamine sulfate. The enzyme phosphorylated substrate proteins on either serine or threonine but not tyrosine. Some endogenous substrates and the ability to autophosphorylate were revealed by autoradiographic studies. PK-III displayed a broad pH optimum (pH 6.6-9.5), and the optimum reaction temperature with histone III-S as substrate was 35 degreesC. The kinetic reaction mechanism of PK-III was studied by using casein as substrate. The KmATP and Kmcasein of PK-III were determined as 10 microM and 1.0 mg ml-1, respectively.

Adenosine Triphosphate↗
PubMed1998

Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue. I: Purification and characterization of a calcium-stimulated casein kinase-I.

A Ca(2+)-stimulated protein kinase (PK-I), active with dephosphorylated casein as exogenous substrate, was purified from ripening mango fruit. The purification procedure involved 30-70% ammonium sulphate fractionation and sequential anion exchange-, affinity-, hydrophobic interaction- and gel filtration chromatography. PK-I was purified ca. 40-fold with an overall yield of < 1%. The final specific activity in the presence of 0.1 mM Ca2+ was 55 nmol min-1 mg-1. Analysis of the most highly purified preparations revealed a monomeric enzyme with an M(r) of 30.9 kDa and pI of 5.1. PK-I efficiently phosphorylated casein and phosvitin, but did not phosphorylate histone II-S, histone III-S, protamine sulphate or bovine serum albumin. PK-I activity was stimulated by micromolar concentrations of Ca2+ and was dependent on millimolar Mg2+ concentrations, which could not be substituted with Mn2+. PK-I activity was stimulated by, but was not dependent on Ca2+. Calmodulin and calmodulin inhibitors did not affect PK-I activity, but heparin and cAMP acted as inhibitors. The pH and temperature optima of the enzyme under standard reaction conditions were 6.5 and 35 degrees C, respectively. The kinetic reaction mechanism of PK-I was studied by using casein as substrate. Initial velocity and product inhibition studies with ADP as product inhibitor best fit an ordered bi-bi kinetic mechanism with the Mg(2+)-ATP complex binding first to the enzyme followed by binding of the protein substrate. The K(m)ATP and K(m)casein of PK-I were 9 microM and 0.26 mg ml-1, respectively. The KiADP of PK-I was 9 microM.

Adenosine Triphosphate↗
PubMed1981

Isolation and characterization of two adenosine 3',5'-monophosphate-dependent protein kinases from bovine adrenal cortex.

Bovine adrenocortical cytosol contains two cAMP-dependent protein kinases separable by diethylamino-ethyl-cellulose chromatography. The kinases exhibit behavior characteristic of type I and type II enzymes i.e. the enzyme eluting at 80 mM NaCl is activated by NaCl and histone, whereas the enzyme eluting at 140 mM NaCl is not. In addition, a third cAMP-binding protein eluting from DEAE at 120 mM NaCl has been isolated and tentatively identified as type I regulatory subunit. A method is described for the isolation of the catalytic and regulatory subunits of both enzymes from the same starting material using cGMP to dissociate the enzyme subunits and elute the regulatory subunits from N6-aminoethyl-cAMP-Sepharose 4B. The isolated proteins were homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis. Using this technique, molecular weight values of 44,000 for the catalytic subunits, 51,000 for the cAMP-binding protein and type I regulatory subunit, and 56,000 for the type II regulatory subunit were obtained. Molecular weights obtained by sucrose density gradient centrifugation on the cGMP-dissociated holoenzymes were 95,000 and 101,000 for regulatory subunits from type I and II enzymes and 34,000 and 33,000 for their respective catalytic subunits. The apparent Km and Vmax values for catalytic subunits I and II were similar when histone, casein, or ATP was used as substrate. The Vmax for protamine phosphorylation was 3-fold higher with catalytic subunit II. Phosvitin was not a substrate for either subunit. The cAMP-binding protein and regulatory subunit I were able to recombine with, i.e. inhibit, either catalytic subunit. At a molar ratio of 4:1, inhibition was total. The type II regulatory subunit was considerably less effective. Preference for a particular catalytic subunit was not evident in the case of inhibition by either regulatory subunit or the cAMP-binding protein.

Adrenal Cortex↗
PubMed1989

Binding of protein kinase C to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide through its ATP binding site.

Protein kinase C (PKC) is a Ca2+- and phospholipid-dependent protein kinase which has been implicated as a key enzyme in the regulation of cellular growth. The naphthalenesulfonamide W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] is representative of a number of cationic amphiphilic inhibitors of PKC which appear to inhibit PKC by interacting with the acidic phospholipid cofactor of the enzyme, according to kinetic studies. In a previous report, we demonstrated that PKC binds directly to W7 when the naphthalenesulfonamide is immobilized on agarose. In the present report, we have defined the mechanism of the binding of PKC to W7-agarose, and its relevance to the inhibitory mechanism of the naphthalenesulfonamide. We demonstrate that PKC bound W7-agarose through the catalytic domain of the enzyme. An active catalytic fragment of PKC was generated by limited proteolysis, and we found that this fragment bound W7-agarose and coeluted with intact PKC upon the addition of Triton X-100. W7 inhibited PKC activity by two different mechanisms. As previously reported, W7 inhibited PKC by interacting with the phospholipid cofactor of the enzyme (IC50 = 260 microM). However, at higher concentrations of W7, we found that this naphthalenesulfonamide inhibited PKC by serving as a competitive inhibitor with respect to the substrate ATP, according to a kinetic analysis of the inhibition of the active catalytic fragment of PKC by W7. W7 inhibited the active catalytic fragment of PKC as well as PKC-catalyzed phosphorylation of protamine sulfate, a reaction which is independent of Ca2+ and phospholipid, with similar potencies. Consistent with the kinetic evidence that W7 serves as a competitive inhibitor of PKC with respect to ATP, we found that, in the presence of 10 mM MgCl2, 1 mM ATP was sufficient to elute PKC from W7-agarose. Thus, naphthalenesulfonamide PKC inhibitors may include both agents which primarily function by interacting with the phospholipid cofactor of the enzyme and agents which primarily serve as active site inhibitors of PKC.

Adenosine Triphosphate↗
PubMed1991

Protein phosphorylation and dephosphorylation in type II pneumocytes.

Protein phosphorylation and dephosphorylation are a major mechanism for regulating cellular activity. Substantial evidence exists for ascribing a key role of protein phosphorylation and dephosphorylation in the regulation of surfactant secretion from type II pneumocytes, yet understanding of the specific molecular mechanisms is generally lacking. Herein, we report two-dimensional electrophoretic mapping of proteins phosphorylated in type II pneumocytes in primary culture, the response to protein kinase C simulation with phorbol ester, and the response to protein phosphatase inhibition with okadaic acid. Exposure of cells for 15 min to phorbol ester at a concentration (10(-4) M) which maximally stimulated both translocation of protein kinase C from cytosol to membranes and surfactant secretion increased phosphorylation (50-80% compared with control) of three specific proteins (50 kDa, pI 5.8 and 5.7; 25 kDa, pI 5.7). Exposure of cells for 2.5 h to okadaic acid (10(-6) M), a concentration that inhibited 90% of protein phosphatase activity, resulted in greatly increased phosphorylation (200-1,500% compared with control) of five specific proteins (50 kDa, pI 5.7 and 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). Combined treatment with okadaic acid and phorbol ester resulted in further increases (145-3,080% compared with control) in phosphorylation of four specific proteins (50 kDa, pI 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). We conclude that these respective proteins comprise major substrates for protein kinase C-dependent phosphorylation and for protein phosphatases in type II pneumocytes in primary culture. Furthermore, we speculate that these proteins will prove to play key roles in the regulation of type II pneumocyte function.

Animals↗
PubMed1987

The polycation-stimulated protein phosphatases: regulation and specificity.

Four classes of protein phosphatases are presumed to play an important role in dephosphorylating the major proteins involved in the control of general metabolism. Based on the enzyme-directed regulation of activity they have been classified as ATP,Mg-dependent-, polycation-stimulated-, Mg2+-dependent protein phosphatases and calcineurin. We have recently purified from rabbit skeletal muscle four distinct PCS protein phosphatases, classified according to the apparent molecular weight of the native enzymes in gel filtration at an early stage of the purification as: PCSH (390 kDa), PCSM (250 kDa) and PCSL (200 kDa) phosphatases. The PCSH phosphatase could be resolved into a 3(65:55 35 kDa)-subunit PCSH1 phosphatase and a 2(65:35 kDa)-subunit PCSH2 enzyme probably derived from the PCSH1 phosphatase, both characterized as specific deinhibitor phosphatases. PCSM phosphatase, a 3(72:65 35 kDa)-subunit enzyme, shows a high degree of stimulation with a low concentration optimum of polycations and is sensitive to a Ca2+-dependent protease, which brings about a five- to ten-fold increase in inhibitor-1 phosphatase activity. PCSL phosphatase is characterized by a 2(65:35 kDa)-subunit structure, a low intrinsic deinhibitor phosphatase activity and a low degree of stimulation of phosphorylase phosphatase activity requiring high concentrations of polycations. At low concentrations of polycations the stimulation of phosphorylase phosphatase activity of the PCS enzymes is enzyme-directed, since it occurs at concentrations far below the substrate concentration. The degree of stimulation is also typical for each type of enzyme (PCSM greater than PCSH1 greater than PCSH2 greater than PCSL greater than PCSC) and dependent on the polycation used; at the optimum concentration the most effective polycations (polylysine, protamine, histone H1) stimulate the phosphorylase phosphatase activity to about the same extent. Polycation concentrations above the optimum are less effective on phosphorylase phosphatase activity and can even become inhibitory to the basal activity. Whether this effect is enzyme- or substrate-directed (or both) is not known. The stimulation by polycations could be completely lost following preincubation of the PCS phosphatase with polycations. This deactivation is time-, temperature- and concentration-dependent. However the polycations did not affect the basal phosphorylase phosphatase activity. In addition to phosphorylase a and inhibitor-1, casein, myosin light chains and phosphorylase b kinase (alpha-subunit) are choice substrates for these enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain.

A novel serine/threonine protein phosphatase is identified, and the catalytic subunit, obtained from a detergent extraction of the pellet generated by a 100,000 x g centrifugation of a whole bovine brain homogenate, is purified and characterized. The protein phosphatase, designated as PP3, has a Mr of 36,000, does not require divalent cations for activity, is stimulated rather than inhibited by inhibitor 2, is inhibited by both okadaic acid and microcystin-LR with an intermediate IC50 compared to type 1 and type 2A protein phosphatases, and preferentially dephosphorylates the beta subunit of phosphorylase kinase. Substrate specificity, immunoblotting with type-specific antisera, and the amino acid sequences of peptides derived from PP3 indicate that PP3 is not an isoform of any known serine/threonine protein phosphatase.

Amino Acid Sequence↗
PubMed1975

Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.

Adenine Nucleotides↗
PubMed1997

Tissue and subcellular distributions, and characterization of rat brain protein phosphatase 2A containing a 72-kDa delta/B" subunit.

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

Animals↗
PubMed2004

Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa.

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.

Animals↗
PubMed2003

Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status.

OBJECTIVE: To test, both in semen and washed-sperm fractions, whether hyaluronic acid (HA) binding is restricted to sperm that have completed cellular maturation. DESIGN: Comparisons of sperm in semen and in HA-bound sperm fractions. SETTINGS: University-based diagnostic and research andrology laboratory. PATIENT(S): Semen samples originated in men being tested for infertility. INTERVENTION(S): The attributes of sperm maturity were tested by immunocytochemistry with creatine kinase and HspA2 antisera (highlights cytoplasmic retention in diminished-maturity sperm), aniline blue chromatin staining (detects persistent histones), pisum sativum lectin staining (reveals acrosomal integrity), and the FertiLight viability kit (highlights viable and nonviable sperm). RESULT(S): All markers of sperm maturity and immaturity supported the hypothesis that HA-bound sperm are mature. Nonbinding sperm exhibited cytoplasmic and nuclear properties of diminished maturity. The acrosomal status of HA-bound sperm was either unreacted or slightly capacitated, but not acrosome reacted. Only viable sperm exhibited HA binding. CONCLUSION(S): Sperm that are able to bind to HA are mature and have completed the spermiogenetic processes of sperm plasma membrane remodeling, cytoplasmic extrusion, and nuclear histone-protamine replacement. Hyaluronic acid-bound sperm show unreacted acrosomes. These studies provide further insights into the relationship between spermiogenesis and sperm function.

Acrosome↗
PubMed1982

Studies on endogenous phosphorylation of chicken erythrocyte membranes. Calcium-dependent phosphorylation of specific proteins.

In this work, endogenous phosphorylation of chicken erythrocyte membranes was investigated. The membrane proteins were rapidly phosphorylated endogenously (half maximum time was 30 s) in the presence of millimolar concentration of Mg2+ under physiological conditions. As an exogenous substrate, protamine was phosphorylated most rapidly of those tested, and histone, casein and bovine serum albumin were rather poor substrates. Cyclic nucleotides had no effect on the endogenous membrane phosphorylation. EGTA inhibited the phosphorylation of a membrane protein having an approximate molecular weight of 43000, and this inhibition was reversed by the addition of a stoichiometric amount of Ca2+. Furthermore, trifluoperazine, an inhibitor of calmodulin, was found to have the same effect as that of EGTA. The phosphorylated 43 kDa protein could be extracted from the membranes under high salt conditions, and was precipitated specifically with anti-actin antibody. These results suggest that the phosphorylation of a peripheral membrane protein (which has an approximate molecular weight of 43000) of chicken erythrocytes by membranous protein kinase(s) depends on Ca2+ and possibly on calmodulin.

Adenosine Triphosphate↗
PubMed2002

Regulatory properties of p105: a novel PKC isoenzyme in mantle tissue from marine mussels.

Previous results suggested operative similarities between Apl II from nervous cells of Aplysia californica, epsilonPKC from brain of vertebrates, and p105 from mantle tissue of Mytilus galloprovincialis Lmk., all of them belonging to the nPKC family. The optimal substrate for Apl II and p105 from mussel is protamine sulfate. In contrast, Ca2+ inhibits p105 but does not affect Apl II. As occurs in other epsilonPKC, p105 is autophosphorylable; however, in Apl II, no P-Tyr residues are detected in the most phosphorylated form. The presence of p105 in all the tissues of M. galloprovincialis studied, proves the important, yet unknown, physiological role that this enzyme must play.

Animals↗
PubMed1983

Hormonal regulation of myocardial lipolysis.

Although it has been documented that hormones promote lipolysis in the heart, an enzyme mediating this event has not been identified. We have found that perfusion of the rat heart with epinephrine, 3-isobutyl-1-methylxanthine, or dibutyryl (Bt2) cyclic AMP produced a biphasic effect on the activity of an enzyme having the properties of the intracellular fraction of lipoprotein lipase, i.e., stability in acetone:ether, pH optimum of 8.1, serum requirement, and sensitivity to heparin, NaCl, and protamine sulfate. We have termed this enzyme type L hormone-sensitive lipase (HSL). Perfusion with high concentrations of agent stimulate type L HSL activity, while perfusion with relatively low concentrations of agent inhibit enzyme activity. This inhibition is not observed if enzyme is extracted in organic solvent. The activity of type L HSL has a high negative (r greater than -0.93) relationship with the amount of triglyceride in the heart. Early attempts to purify this enzyme from control and epinephrine-stimulated hearts suggest that the enzyme can be obtained in two forms (control and activated). Although the data suggest that the mechanism of control of this enzyme is complex, it seems that the activity is controlled, in part, through cyclic AMP and protein kinase.

Animals↗
PubMed1993

Characterization of a 7.5-kDa protein kinase C substrate (RC3 protein, neurogranin) from rat brain.

A 7.5-kDa heat- and acid-stable rat brain protein kinase C (PKC) substrate was purified to near homogeneity by a two-step procedure using DEAE-cellulose and hydroxylapatite column chromatography. This 78-amino-acid protein has a sequence identical to that deduced from rat brain RC3 cDNA identified with a cortex-minus-cerebellum subtracted cDNA probe (J. B. Watson et al., J. Neurosci. Res. 26, 397-408, 1990) and exhibits extensive sequence identity to bovine brain neurogranin (J. Baudier et al., J. Biol. Chem. 266, 229-237, 1991). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis this protein, RC3, migrated as a M(r) 15-18K species in the presence of reducing agent and as heterogeneous species of M(r) 13-28K in the absence of reducing agent. Phosphorylation of RC3 by PKC alpha, beta, or gamma was stimulated by Ca2+, phospholipid, and diacylglycerol. A single site, Ser36, which is adjacent to the predicted calmodulin (CaM)-binding domain, was phosphorylated by these enzymes. Phosphorylation of RC3 by PKC or PKM, a protease-degraded PKC, was inhibited by CaM. The effect of CaM apparently targets at RC3, as phosphorylation of protamine sulfate by PKM was not inhibited by CaM. In the absence of Ca2+, RC3 formed a stoichiometric complex with CaM as evidenced by an increase in the M(r) determined by gel filtration chromatography. In the presence of Ca2+, the affinity of RC3 toward CaM is greatly reduced and Ca2+/CaM becomes less inhibitory of the PKM-catalyzed phosphorylation of RC3. Phosphorylation of RC3 by PKM prevented the interaction of this protein with CaM even in the absence of Ca2+. A 20-amino-acid synthetic peptide (AS-20F-W) containing the PKC phosphorylation site and CaM-binding domain of RC3 (Ala29-Ser48) with a substitution of Phe37 with tryptophan was used to monitor the interaction of this peptide with CaM by spectrofluorometry. In the absence of Ca2+, CaM caused negligible change in tryptophan fluorescence of the peptide; however, an enhancement and blue-shift of the emission fluorescence was observed in the presence of Ca2+. It seems that this synthetic peptide, as well as RC3 holoprotein, interacts with CaM through electrostatic interaction in the absence of Ca2+ but through hydrophobic interaction in the presence of Ca2+. In rat brain homogenate, RC3 formed a stable complex with CaM in the presence of Ca2+, as demonstrated by immunoblot analysis following gel filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1971

Metabolism of D-arabinose: origin of a D-ribulokinase activity in Escherichia coli.

The kinase responsible for the phosphorylation of d-ribulose was purified 45.5-fold from a strain of Escherichia coli K-12 capable of growth on d-arabinose with no separation of d-ribulo- or l-fuculokinase activities. Throughout the purification, the ratios of activities remained essentially constant. A nonadditive effect of combining both substrates in an assay mixture; identical K(m) values for adenosine triphosphate with either l-fuculose or d-ribulose as substrate; and, the irreversible loss of activity on both substrates, after removal of magnesium ions from the enzyme preparation, suggest that the dual activity is due to the same enzyme. A fourfold greater affinity of the enzyme for l-fuculose than for d-ribulose, as well as a higher relative activity on l-fuculose, suggest that the natural substrate for this enzyme is l-fuculose. The product of the purified enzyme, with d-ribulose as substrate, was prepared. The ratio of total phosphorous to ribulose phosphate was 1.01:1, indicating that the product was ribulose monophosphate. The behavior of the kinase product in the cysteine-carbazole and orcinol reactions, as well as the results of periodate oxidation assays, provided evidence that it was not d-ribulose-5-phosphate. Reaction of this compound with a cell-free extract of E. coli possessing l-fuculose-l-phosphate aldolase activity resulted in the production of dihydroxyacetone phosphate and glycolaldehyde. The kinase product failed to reduce 2,3,5-triphenyltetrazolium and possessed a half-life of approximately 1.5 min in the presence of 1 n HCl at 100 C. These properties suggested that the phosphate group was attached to carbon atom 1 of d-ribulose.

Antacids↗