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PubMed1989

Effect of zinc or copper deficiency on erythrocyte purine nucleoside phosphorylase and pyruvate kinase activity in the rat.

The effect of deficiencies of zinc or copper in the rat on the activity of purine nucleoside phosphorylase and pyruvate kinase in erythrocytes, liver and muscle was investigated. The data showed no effect of either zinc or copper deficiencies on the activity of these enzymes in the tissues tested. It is concluded that these enzymes are not useful markers for either zinc or copper deficiency.

Animals↗

PubMed1987

Protein phosphatases of the guinea-pig parotid gland.

The nature of protein phosphatases of the guinea-pig parotid gland was investigated. The protein phosphatases were characterized by (a) the use of five different 32P-labelled substrate proteins (phosphorylase a, histone H2B, casein, and the alpha and beta subunits of phosphorylase kinase), (b) their behaviour during ion-exchange chromatography, (c) their relative molecular mass distribution during gel filtration, (d) their sensitivity towards inhibition by inhibitor 2, (e) their ability to be stimulated by protamine and (f) by their behaviour during freezing and thawing in the presence of 2-mercaptoethanol. The following results were obtained. 1. The 'cytosol' (100,000 X g supernatant) contains protein phosphatases of the types 1, 2A and 2B. 2. On the basis of inhibition with inhibitor 2 (1.2 micrograms/ml) the 'cytosolic' phosphorylase phosphatase activity consists to about 40% of protein phosphatase 1 and to about 60% of protein phosphatase 2A. 3. In the cytosol about 80-90% of the protein phosphatases 1 and 2A exist in an inactive state. 4. A 5-10-fold activation can be achieved by ethanol precipitation, which results in the generation of a mixture of forms of low apparent molecular mass of about 30 kDa. 5. Microsome-associated phosphorylase phosphatase activities can be extracted in a highly active state by detergent (1% Triton X-100) or by 0.8 M NaCl. 6. Activity measurements in the presence of inhibitor 2 (1.2 micrograms/ml) indicate that the microsomal activities consist to about 75% of protein phosphatase 1 and to about 25% of protein phosphatase 2A. Activities corresponding to protein phosphatases 2B and 2C could not be detected. 7. The 'microsomal' protein phosphatase activities exhibit lower apparent molecular masses (70 kDa and 30 kDa) than the 'cytosolic' protein phosphatases (about 260 kDa). 8. After ethanol treatment of the microsomal protein phosphatases only activities with apparent molecular masses of about 30 kDa can be detected. These share several similarities with the ethanol-treated cytosolic protein phosphatases. 9. Both cytosolic and microsomal protein phosphatases display activity towards histone H2B and casein.

Animals↗

PubMed1978

The role of calcium and cyclic adenosine 3',5'-monophosphate in the regulation of glycogen metabolism in phagocytozing human polymorphonuclear leukocytes.

Incubation of human polymorphonuclear leukocytes in a glucose-free Krebs-Ringer bicarbonate buffer for 2 h resulted in glycogen depletion, decreased phosphorylase activity and increased synthase-R activity. Addition of dialyzed latex particles to starved leukocytes revealed a very rapid ingestion rate (half-maximal ingestion within 30 s). This uptake is followed by glycogenolysis associated with an immediate two-fold increase in phosphorylase a activity and a synthase-R to -D conversion within 30 s. Furthermore, in rapid time-course experiments with phagocytozing cells we found that the concentration of cyclic AMP increased by 93% within 15 s and returned to baseline values at 1 min. In a medium without added calcium and with 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid, phagocytosis was blocked, cyclic AMP formation decreased by 50% and phosphorylase activation was abolished, but the conversion of synthase-R to -D was preserved. Addition of calcium ions to cells suspended in a calcium-free buffer without added latex results in phosphorylase activation and glycogenolysis, but not in cyclic AMP increase or synthase-R to -D conversion. Measurements of 45Ca efflux during phagocytosis suggest an initial increase in cytosolic calcium obtained by a release of membrane-bound 45Ca. Activation of phosphorylase during phagocytosis is thus presumably due to an increase in cytosol Ca2+ and subsequent activation of phosphorylase kinase, and is independent of the simultaneous increase in concentration of cyclic AMP. Phosphorylation of synthase R to the D form does not depend on the presence of Ca2+ in the extracellular phase.

Calcium↗

PubMedDate not supplied

[Comparative study of the physiological and biochemical characteristics of haploid and diploid Pichia pinus strains in their growth process on a glucose medium].

The economic coefficient, the growth rate, the quantitative content of bound amino acids, and the activities of hexokinase, pyruvate kinase, phosphorylase, malate dehydrogenase and glucose-6-phosphate dehydrogenase were studied comparatively in the haploid and diploid strains of Pichia pinus in the process of their growth in a medium containing glucose. An increase in the content of DNA in the cells was found to be accompanied with an increase of the specific growth rate at the exponential phase of growth and an increase in the activities of hexokinase and glucose-6-phosphate dehydrogenase; however, it had virtually no effect on the economic coefficient, the amino acid composition of protein, and the activities of pyruvate kinase and malate dehydrogenase.

Amino Acids↗

PubMed1985

Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver.

A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.

Animals↗

PubMed1992

Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle.

In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.

Animals↗

PubMed1983

Purification and characterization of phosphorylase B from Ascaris suum.

Glycogen phosphorylase b (EC 2.4.1.1) has been purified from the muscle of the roundworm, Ascaris suum. The 223-fold purified enzyme was shown to be homogenous by high performance liquid chromatography (HPLC), gel filtration column chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. The apparent native molecular weight of the enzyme determined by size exclusion chromatography by HPLC and gel filtration corresponded to 200 000 and 199 000, respectively. The subunit molecular weight of the enzyme was determined to be 100 000 by electrophoresis in the presence of SDS. Therefore, the enzyme appears to be a dimer with identical or near identical subunits. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol subunit and exhibited an absorbance index E1% 280 of 13.8. The apparent isoelectric point of the enzyme is 5.53. The enzyme, inactive in the absence of AMP, can be converted to the active form by rabbit muscle phosphorylase kinase and MgATP. The molecular weight of the activated form of the enzyme is 200 000. Kinetic studies showed apparent Km values of 0.17% for glycogen, 36 mM for Pi and 52 mM for glucose-1-P. The apparent Ka for AMP was 0.22 mM.

Animals↗

PubMed1993

Phosphorylation of linker histones by cAMP-dependent protein kinase in mitotic micronuclei of Tetrahymena.

Linker histones (LHs) in transcriptionally inactive, mitotically dividing micronuclei of Tetrahymena thermophila, alpha, beta, gamma and delta, are highly phosphorylated in vivo. Analysis of the derived sequences of these LHs suggests that none of these polypeptides contain sites of phosphorylation by p34cdc2, the kinase thought to play an essential role governing the entry of all cells into mitosis. Surprisingly alpha, beta, gamma and delta each contain sites for phosphorylation by cyclic AMP-dependent kinase (PKA). p34cdc2 kinase phosphorylases H1 in vitro but fails to phosphorylate alpha, beta, gamma and delta. Conversely, PKA phosphorylates each of the micronuclear LHs but is unable to phosphorylate macronuclear H1. Micronuclear LHs labeled in vivo with [32P]phosphate were purified by reverse phase HPLC. Phosphoamino acid analysis showed that all four micronuclear LHs are phosphorylated exclusively on serine residues in vitro. Cyanogen bromide mapping of alpha, beta, gamma and delta labeled in vivo or in vitro by PKA indicates that each LH is phosphorylated only on peptides that contain either optimum (RR/KXS) or less optimum (RXXS) PKA sequences. This study suggests that PKA or a PKA-like activity(ies), but not p34cdc2 kinase, is(are) responsible for the in vivo phosphorylation of LHs in the mitotic micronucleus of Tetrahymena. We suggest that, at least in Tetrahymena, PKA-driven phosphorylation or dephosphorylation plays a significant role in the control of mitotic processes such as chromosome condensation.

Amino Acid Sequence↗

PubMed1975

Guanosine 3':5'-monophosphate-dependent protein kinase from bovine cerebellum. Purification and characterization.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was assayed with calf thymus histone as substrate and partially purified from the soluble fraction of bovine cerebellum. The enzyme was selectively activated by cyclic GMP at lower concentrations; the Ka value for cyclic GMP was 1.7 times 10- minus 8 M whereas that for adenosine 3':5'-monophosphate (cyclic AMP) was 1.0 times 10- minus 6 M. The Km value for ATP was 1.0 times 10- minus 5 M. A high concentration of Mg-2+ (100 mM) was needed for maximum stimulation by cyclic GMP and maximum reaction rate. The pH optimum was 7.5 to 8.0. The isoelectric point was pH 5.7. The molecular weight was about 140,000 as estimated by gel filtration. The enzyme was unable to activate muscle glycogen phosphorylase kinase, and was clearly distinguishable from cyclic AMP-dependent protein kinase in kinetic and catalytic properties. Comparative data on cyclic GMP-dependent and cyclic AMP-dependent protein kinases in this tissue are presented.

Adenosine Triphosphate↗

PubMed1976

Biochemical adaptations for flight in the insect.

1. Flight by insects is characterized by the most intense respiration known in biology and also the most controlled. Thus insect flight muscle may be the tissue of choice for the study of biochemical adaptation in the control of catabolism and biological oxidations, and many of the results obtained with insects have a significance and a relevance that transcend the boundaries between classes. In insects, such as the blowfly, flight is distinguished additionally by high wingbeat frequencies and an asynchronous type of excitation-contraction coupling. In spite of this intense muscular work, metabolic processes are not limited by the availability of oxygen. Also of importance is the morphological organization of the flight muscle and mitochondria, which have evolved ultrastructurally and biochemically into an effective catabolic machine. 2. In the fly, carbohydrate, principally glycogen, is the sole metabolic fuel; fats are not used in flight and enzymes concerned with fatty acid utilization are virtually lacking. Glycogenolysis does not lead to lactic acid; instead, the end products of glycolysis are pyruvate and alpha-glycerophosphate. The alpha-glycerophosphate cycle provides a mechanism not only for the reoxidation of glycolytically produced NADH but also for the stoicheiometric formation from each molecule of hexose equivalent of two molecules of pyruvate, which are then available for oxidation via the tricarboxylate cycle. The absence of dicarboxylate and tricarboxylate carriers from the mitochondria ensures that tricarboxylate-cycle intermediates do not exit from the mitochondrion but that pyruvate is oxidized to completion. On initiation of flight, mitochondrial oxidation of pyruvate is impeded by the lack of tricarboxylate-cycle intermediates for the generation of oxaloacetate. This is circumvented by the oxidation of proline. 3. The controls on metabolism in flight muscle, i.e. (1) glycogenolysis at phosphorylase and phosphorylase kinase, (2) glycolysis at phosphofructokinase, (3) alpha-glycerophosphate dehydrogenase, (4) proline dehydrogenase and (5) tricarboxylate cycle at isocitrate dehydrogenase, are effected by the phosphate potential and/or Ca2+. It is suggested that the metabolic changes, such as those seen in the rest-to-flight transition, are achieved by the concerted actions of these effectors at the different loci.

Adaptation, Physiological↗

PubMed1983

Phosphorylation of hormone-sensitive lipase by cyclic AMP-dependent protein kinase.

Hormone-sensitive lipase, detergent-solubilized and purified from rat adipose tissue, was phosphorylated with the catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Maximally 1.05 +/- 0.05 (mean +/- S.E. (n = 3) ) mol of phosphate/mol of hormone-sensitive lipase Mr = 84,000 subunit was incorporated. Phosphoserine was the only phosphorylated amino acid residue. A single phosphorylation site was demonstrated by digestion with Staphylococcus aureus V8 protease and trypsin that produced a single acidic phosphopeptide of about 10 amino acid residues length, which was isolated by two-dimensional electrophoresis-thin layer chromatography. Enzyme activity was enhanced, 2.5-fold against trioleoylglycerol, concomitant with phosphorylation, with half-maximal effect within 30 sec, a rate of phosphorylation of the enzyme comparable to that obtained in vivo (Nilsson, N. O., Strålfors, P., Fredrikson, G., and Belfrage, P. (1980) FEBS Lett. 111, 125-130). The initial rate of phosphorylation was approximately half that with phosphorylase kinase as substrate. The effects of modifications of hormone-sensitive lipase and of various additions and variation in pH were examined.

Adipose Tissue↗

PubMed1998

Overexpression of protein targeting to glycogen (PTG) in rat hepatocytes causes profound activation of glycogen synthesis independent of normal hormone- and substrate-mediated regulatory mechanisms.

Protein targeting to glycogen (PTG), also known as PPP1R5, is a widely expressed member of a growing family of proteins that target protein phosphatase-1 (PP-1) to glycogen particles. Because PTG also binds to glycogen synthase and phosphorylase kinase, it has been suggested that it serves as a "scaffold" for efficient activation of glycogen synthesis. However, very little is known about the metabolic effects of PTG. In this study, we have used recombinant adenovirus to overexpress PTG in primary rat hepatocytes, a cell type with high glycogenic capacity. We find that overexpression of PTG potently activates glycogen synthesis in cultured hepatocytes. Surprisingly, the glycogenic effect of PTG is observed even in the complete absence of carbohydrates or insulin in the culture medium. Furthermore, glycogenolytic agents such as forskolin or glucagon are largely ineffective at activating glycogen degradation in PTG overexpressing hepatocytes, even though large increases in cAMP levels are demonstrated. These metabolic effects of PTG overexpression are accompanied by a 3.6-fold increase in glycogen synthase activation state and a 40% decrease in glycogen phosphorylase activity. Our results are consistent with a model in which PTG overexpression "locks" the hepatocyte in a glycogenic mode, presumably via its ability to promote interaction of enzymes of glycogen metabolism with PP-1.

3T3 Cells↗

PubMed1976

Purification and general properties of guanosine 3':5'-monophosphate-dependent protein kinase from guinea pig fetal lung.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was purified from the guinea pig fetal lung, a tissue shown to be the richest in this enzyme in all mammalian sources examined, and its general properties studied. The enzyme was purified 150-fold from crude extract by steps of pH 5.4 isoelectric precipitation, Sephadex G-200 filtration, hydroxylapatite treatment and DEAE-cellulose chromatography. The purified enzyme, free from contamination with adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase, had a specific activity at least equivalent to 600-fold purification of the enzyme from the adult lung. The pulmonary enzyme exhibited an absolute requirement of protein kinase modulator (prepared from various mammalian tissues with an exception of skeletal muscle) for its activity. Inhibitor protein of cyclic AMP-dependent protein kinase purified from rabbit skeletal muscle could not stimulate nor inhibit the cyclic GMP target enzyme, indicating the factors from mammalian sources regulating the two classes of protein kinases may not be the same. The enzyme had Ka values of 1.3 times 10(-8) and 3.3 times 10(-8) M for 8-bromo cyclic GMP and cyclic GMP, respectively, compared to 3.0 times 10(-6) M for cyclic AMP. Cyclic GMP lowered the Km of the enzyme for ATP from 6.3 times 10(-5) M in its absence to 2.1 times 10(-5) M in its presence, accompanied by an approximate doubling of the Vmax. The molecular weight of the enzyme (assayed by its catalytic and cyclic GMP-binding abilities) was estimated to be 123,000, corresponding to a sedimendation coefficient of 7.06 S, by means of sucrose density gradient ultracentrifugation. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity with optimal concentrations of about 30 and 0.7 mM, respectively. The maximal activity seen in the presence of Mg2+, however, was nearly twice as high as that seen in the presence of Co2+. Histones were generally effective substrates for the enzyme, whereas protamine, casein, phosvitin, phosphorylase kinase, and activator protein of phosphodiesterase were not. The cyclic GMP-dependent enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent enzyme in the presence of Mg2+.

Animals↗

PubMed1979

Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids.

The proenzyme of a Ca2+-dependent protease-activated protein kinase previously obtained from mammalian tissues (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) was enzymatically fully active without limited proteolysis when Ca2+ and a membrane-associated factor were simultaneously present in the reaction mixture. The activation process was reversed by removing Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. An apparent Ka value for Ca2+ was less than 5 x 10(-5) M. Other divalent cations were inactive except for Sr2+, which was 5% as active as Ca2+. The factor was almost exclusively localized in membrane fractions of various tissues including brain, liver, kidney, skeletal muscle, blood cells, and adipose tissue. It was easily extractable with chloroform/methanol (2:1), and was recovered in the phospholipid fraction. In fact, this membrane factor could be replaced by chromatographically pure phosphatidylinositol, phosphatidylserine, phosphatidic acid, or diphosphatidylglycerol. Phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were far less effective under the comparable conditions. Ca2+-dependent modulator protein was unable to support enzymatic activity. The enzyme thus activated showed an ability to phosphorylate five histone fractions and muscle phosphorylase kinase, and appeared to possess multifunctional catalytic activities.

Animals↗

PubMed1986

Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase.

A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1989

Regulation of protein phosphatase-1G from rabbit skeletal muscle. 1. Phosphorylation by cAMP-dependent protein kinase at site 2 releases catalytic subunit from the glycogen-bound holoenzyme.

The glycogen-associated form of protein phosphatase-1 (PP-1G) is a heterodimer comprising a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit, the latter being phosphorylated by cAMP-dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C-terminal to site 1 in the primary structure. The sequence in this region is: (sequence; see text) At physiological ionic strength, phosphorylation of glycogen-bound PP-1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP-1G, while the phospho-G subunit remained bound to glycogen. Dissociation reflected a greater than or equal to 4000-fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate-limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti-site-1 Fab fragments to glycogen-bound PP-1G. At near physiological ionic strength, PP-1G and glycogen concentration, site 2 was autodephosphorylated by PP-1G with a t0.5 of 2.6 min at 30 degrees C, approximately 100-fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type-2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the alpha subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type-2A and type-2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10-fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP-1 activity towards the glycogen-metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen--protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin-stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP-1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.

Amino Acid Sequence↗

PubMed1993

The endogenous inhibitor of protein kinase-C in the rat ovary is a protein phosphatase.

Calcium- and lipid-dependent protein kinase (PKC) activity in the ovary of the pseudopregnant rat is masked by an endogenous inhibitor of PKC. These studies were undertaken to examine the mechanism of action of the endogenous inhibitor of PKC in the rat ovary. The addition of the phosphatase inhibitors calyculin-A (0.09 nM), microcystin-LR (6.4 nM), and okadaic acid (10 nM) resulted in the loss of PKC inhibitory activity and an increase in basal PKC activity in rat ovarian cytosol. In phosphatase assays, significant dephosphorylation of histone-III-S or myelin basic protein that had been phosphorylated by PKC occurred within 4 min after the addition of ovarian cytosol from the pseudopregnant rat. This dephosphorylation was prevented from the pseudopregnant rat. This dephosphorylation was prevented by the addition of calyculin-A (0.73 nM) and was removed by fractionation of ovarian cytosol on diethylaminoethyl cellulose. No inhibition of PKC activity was observed when the PKC-specific peptides AcMBP-(4-14) and [Ser25]PKC-(19-31) were used as the substrate for phosphorylation. In addition, rat ovarian cytosol did not exhibit phosphatase activity when the peptide AcMBP-(4-14) was used as the substrate. Addition of ovarian cytosol resulted in dephosphorylation of phosphorylase-alpha phosphorylated by phosphorylase kinase, but not dephosphorylation of histone-II-A or histone-VIII-S phosphorylated by PKA. The data suggest that the endogenous inhibitor of PKC in the rat ovary is a protein phosphatase.

Animals↗

PubMed1978

Protein kinase regulation of cardiac phosphorylase activity and contractility.

The relationship between cAMP-dependent protein kinase activity and epinephrine-produced activation of phosphorylase and increase in contractility was investigated in the intact working rat heart. Epinephrine was administered as a bolus into the superior vena cava of open-chest preparations and the hearts were rapidly frozen. cAMP increased within 5 s and returned to control within 20-30 s. Protein kinase and phosphorylase kinase activity ratios increased transiently with the same time course as that for cAMP. The phosphorylase activity ratio and the rate of left ventricular pressure development increased maximally within 15 s and returned to control in 30-60 s. Continuous infusion of epinephrine caused a sustained elevation of the protein kinase. Free catalytic protein kinase activity increased proportionately with the dose of epinephrine. The beta-adrenergic blocking agent, practolol, had no effect on the basal levels of the five parameters studied, but did prevent the epinephrine-produced increases. The results suggest that the time course of cAMP-dependent protein kinase activation is appropriate if this enzyme is to play a role in the catecholamine-induced increase in both glycogenolysis and contractility in the in vivo heart.

Animals↗