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At least 937 records · Page 52Linked to original sources
PubMed1985

Cyclic nucleotide-independent protein kinase from rat liver. Purification and characterization of a multifunctional protein kinase.

A rat liver cAMP-independent protein kinase that phosphorylates peptide b of ATP-citrate lyase (Ramakrishna, S., Pucci, D. L., and Benjamin, W. B. (1983) J. Biol. Chem. 258, 4950-4956) has been purified to apparent homogeneity. The molecular weight, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sucrose density gradient, and by gel filtration, was found to be 36,000. This protein kinase phosphorylates in vitro ATP-citrate lyase, acetyl-CoA carboxylase, and glycogen synthase and does not phosphorylate phosphorylase, phosphorylase kinase, histone, phosvitin, and casein. It has Fa (activity factor) activity stimulating the ATP X Mg-dependent phosphatase and is therefore named a multifunctional protein kinase. This kinase differs from glycogen synthase kinase-3 with regard to substrate specificity, kinetic parameters, and physicochemical properties.

Animals↗
PubMed1979

A comment on the functional specificities of cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Cyclic AMP-dependent and cyclic GMP-dependent protein kinases (protein kinases A and G, respectively) utilize the same phosphate acceptor proteins when assayed in in vitro systems. Nevertheless, protein kinase A phosphorylates preferentially free histone, whereas protein kinase G greatly favors the histone which is associated with polydeoxyribonucleotide. On the other hand, when cytoplasmic soluble substrates such as phosphorylase kinase are used, the reactions are always more favorable for protein kinase A rather than for protein kinase G. Available evidence implies that the topographic relationship between enzyme and substrate may be an important determining factor for the functional specificities of these two classes of protein kinases.

Cyclic AMP↗
PubMed1988

Protein kinases 1988: a current perspective.

This review focuses on several recent developments in the field of protein kinases. In the area of protein serine/threonine kinases, much has been learned recently about protein kinase C structure and function. Novel lipid mediators, both stimulatory and inhibitory, have been discovered, and kinase has been shown to be an increasingly large family of gene products. Heterogeneity of cellular localization and function has been documented. Calcium/calmodulin-dependent protein kinases are now believed to consist of at least five enzymes, which range from those with extreme substrate specificity such as phosphorylase kinase and myosin light-chain kinases to calcium calmodulin kinase II, with several known substrates. Several of these enzymes appear to be important in synaptic transmission and, for calcium/calmodulin kinase III, in the regulation of protein synthesis. Several new examples of pseudosubstrate prototopes as endogenous kinase inhibitors have been described, including regions intrinsic to kinase primary sequences, which could serve as constitutive inhibitors of enzyme activity. In the field of protein tyrosine kinases, new enzyme species are being discovered at a rapid rate. There are several well-documented examples of kinase autophosphorylation on tyrosine leading to stimulation of catalytic activity. For the growth factor receptors with intrinsic protein tyrosine kinase activity, it now seems clear that kinase catalytic activity is necessary for most hormone effects on cells, with the general exceptions of ligand binding and, possibly, receptor cycling. Finally, several groups have recently described a close association between protein tyrosine kinases and a phosphatidylinositol kinase activity, a link that might eventually explain some of the initial steps in signal transduction that occur after kinase activation.

Animals↗
PubMed1985

An assessment of the importance of intralysosomal and of alpha-amylolytic glycogenolysis in the liver of normal rats and of rats with a glycogen-storage disease.

Mechanisms of glycogenolysis have been investigated in a comparative study with Wistar rats and gsd rats, which maintain a high glycogen concentration in the liver as a result of a genetic deficiency of phosphorylase kinase. In Wistar hepatocytes the rate of glycogenolysis, as modulated by glucagon and by glucose, was proportional to the concentration of phosphorylase a. In suspensions of gsd hepatocytes the rate of glycogenolysis was far too high as compared with the low level of phosphorylase a; in addition, only a minor fraction of the glycogen lost was recovered as glucose and lactate, owing to the accumulation of oligosaccharides. When the gsd hepatocytes were incubated in the presence of an inhibitor of alpha-amylase (BAY e 4609) glycogenolysis and the formation of oligosaccharides virtually ceased; the production of glucose plus lactate, already modest in the absence of BAY e 4609, was further decreased by 40%, owing to the suppression of a pathway for glucose production by the successive actions of alpha-amylase and alpha-glucosidase. Evidence was obtained that gsd hepatocytes are more fragile, and that amylolysis of glycogen occurred in damaged cells and/or in the extracellular medium. This may even occur in vivo, since quick-frozen liver samples from anesthetized gsd rats contained severalfold higher concentrations of oligosaccharides than did similar samples from Wistar rats. However, administration of a hepatotoxic agent (CCl4) caused hepatic glycogen depletion in Wistar rats, but not in gsd rats. The administration of phloridzin and of vinblastine, which have been proposed to induce glycogenolysis in the lysosomal system, did not decrease the hepatic glycogen level in gsd rats. Taken together, the data indicate that only the phosphorolytic degradation of glycogen is metabolically important, and that alpha-amylolysis is an indication of an increased fragility of gsd hepatocytes, which becomes prominent when these cells are incubated in vitro.

Animals↗
PubMed1999

Leptin enhances glycogen storage in hepatocytes by inhibition of phosphorylase and exerts an additive effect with insulin.

The effects of the adipocyte-derived hormone leptin on glucose metabolism in hepatocytes were investigated. Incubation of hepatocytes from Wistar rats with leptin for 16 h caused a dose-dependent increase in incorporation of [14C]glucose into glycogen, with a maximal effect at 30 nmol/l leptin. This effect of leptin was observed over a range of glucose concentrations (10-25 mmol/l) and was associated with stimulation of net glycogen deposition. It was not counteracted by mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, indicating that it is not due to increased gluconeogenic flux. Leptin also enhanced the short-term stimulation of glycogen synthesis by insulin. These effects of leptin were associated with inhibition of phosphorylase a, which occurred after 4 h of exposure to leptin. Culture with leptin for 16 h did not affect the activities of glucose-6-phosphatase or glucokinase or the activation state of glycogen synthase. Leptin did not affect glycolysis determined from the detritiation of [3-(3)H]glucose. The inhibitory effects of leptin on phosphorylase a were counteracted by short-term incubation with glucagon but were additive with the inhibitory effects of insulin and also with the inhibition caused by resorcinol (25 pmol/l), which inhibits phosphorylase kinase by a mechanism analogous to the antidiabetic drug proglycosyn. These results show that leptin has additive effects with insulin in inhibiting phosphorylase and stimulating glycogen storage in hepatocytes, indicating that these hormones act by separate but convergent mechanisms. It is concluded that the primary action of leptin in hepatocytes is to enhance glycogen storage. This may be an important compensatory mechanism for the inhibition of insulin secretion.

Animals↗
PubMed1975

Regulation of platelet phosphorylase.

A sensitive fluorimetric enzyme assay was developed for study of activation of glycogen phosphorylase (EC 2.4.1.1) in intact platelets and in platelet extracts. Activity was calculated as AMP independent (activity in the absence of AMP), total (activity in the presence of 1 mM AMP), and AMP dependent (difference between AMP independent and total). The following observations were made with intact rat platelets. (1) Stimulation of platelets with thrombin caused a 7-fold increase in total activity, with increases in both AMP-dependent and AMP-independent activities. Maximum activation was obtained within 10 s after addition of thrombin. (2) The divalent cation ionophore A23187 caused a similar, though less pronounced, activation of phosphorylase. (3) Acceleration of glycogenolysis by inhibition of respiration with cyanide caused similar changes in phosphorylase activity but with the maximum effect observed only after 45 s. (4) Dibutyryl cyclic AMP had two effects; it partially activated phosphorylase and blocked further activation by thrombin, but not A23187. Similar effects were observed with human platelets, but low resting levels of phosphorylase activity could not be maintained so that changes were not as large as with rat platelets. Experiments with extracts of rat platelets gave the following results. (1) Phosphorylase activity in many extracts of non-stimulated platelets could be increased by incubation with Mg2+-ATP and Ca2+; ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) partially inhibited. (2) In some extracts there was essentially no activation by incubation with Mg2+-ATP and Ca2+, but addition of cyclic AMP GAVE PARTIAL ACTIVATIon while addition of rabbit muscle phosphorylase kinase gave full activation. (3) Incubation of extracts of thrombin-stimulated platelets caused conversion of AMP-dependent to AMP-indeptndent activity. It is concluded that platelet phosphorylase exists in an inactive and two active forms. Conversion of the inactive to the active forms and of the AMP-dependent to the AMP-independent form is catalyzed by a kinase(s) that requires Ca2+ for full activity and is activated through a cyclic AMP-mediated process. The major change following physiological stimulation is an increase in both active forms, with little change in their ratio.

Animals↗
PubMed1995

[Interaction between glycogen phosphorylase b and creatinine kinase from rabbit skeletal muscle].

Phosphorylase b association with creatine kinase has been studied by frontal elution affinity chromatography, using CNBr-Sepharose 4B immobilized creatine kinase as the affinity matrix. The quantitative parameters of this interaction were estimated from the volumes of phosphorylase b elution at various concentrations of the enzyme. The dissociation constants for phosphorylase b complexes with immobilized creatine kinase and of the phosphorylase b complex with free creatine kinase were found to be equal to 0.49 and 0.191 microM, respectively. In the presence of AMP the interaction between the proteins became weaker. With a rise in AMP concentration from 0.02 to 0.15 mM the value of the dissociation constants increased from 1.59 up to 9.66 microM. One molecule of AMP was shown to bind on the phosphorylase b-immobilized creatine kinase complex.

Adenosine Monophosphate↗
PubMed1983

The inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.

The activity of glycogen synthase phosphatase in rat liver stems from the co-operation of two proteins, a cytosolic S-component and a glycogen-bound G-component. It is shown that both components possess synthase phosphatase activity. The G-component was partially purified from the enzyme-glycogen complex. Dissociative treatments, which increase the activity of phosphorylase phosphatase manyfold, substantially decrease the synthase phosphatase activity of the purified G-component. The specific inhibition of glycogen synthase phosphatase by phosphorylase a, originally observed in crude liver extracts, was investigated with purified liver synthase b and purified phosphorylase a. Synthase phosphatase is strongly inhibited, whether present in a dilute liver extract, in an isolated enzyme-glycogen complex, or as G-component purified therefrom. In contrast, the cytosolic S-component is insensitive to phosphorylase a. The activation of glycogen synthase in crude extracts of skeletal muscle is not affected by phosphorylase a from muscle or liver. Consequently we have studied the dephosphorylation of purified muscle glycogen synthase, previously phosphorylated with any of three protein kinases. Phosphorylase a strongly inhibits the dephosphorylation by the hepatic G-component, but not by the hepatic S-component or by a muscle extract. These observations show that the inhibitory effect of phosphorylase a on the activation of glycogen synthase depends on the type of synthase phosphatase.

Animals↗
PubMed1976

The control of glycogen metabolism in the liver.

The major factor that controls glycogen metabolism in the liver is the concentration of phorphorylase alpha. Indeed, this enzyme catalyzes the limiting step of glycogen breakdown and, by controlling the activity of synthetase phosphatase, also regulates glycogen synthesis. The formation of phosphorylase alpha is stimulated by cAMP, by glycogen, and presumably also by some still ill-defined ionic changes. The ininactivation of phosphorylase is greatly stimulated by glucose and inhibited by AMP and glycogen. Glycogen synthesis is proportional to the concentration of synthetase alpha, which in normally fed animals is formed only when most of the phosphorylase is in the beta form. The inactivation of glycogen synthetase is stimulated by cAMP, an elevated concentration of which puts a double lock on glycogen synthetase by activating phosphorylase alpha (and thereby preventing synthetase activation) and by inactivating glycogen synthetase. The effect of cAMP, 5'-AMP, glucose, and glycogen can presently be explained in molecular terms. The main missing link is in the ionic effect whose elucidation might lead to the understanding of the mode of action of insulin.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1978

[Regulation processes in biological systems. II. Role of compartmentation and of organized multienzyme systems].

The picture, emerging from the experimental results on the physical association of multienzyme systems, is that the true physiological significance of the aggregated state can be understood only if it is correlated to the structural and functional integration of the cellular metabolic framework as a whole. The enzyme clusters exhibit two distinct functional properties. The first is the spatial translocation of intermediate substrates, the effect of which may be viewed as metabolic "channelling" or "vectorial catalysis" if the enzyme clusters are arrayed in some manner in the cell. The second is the coordinate regulation which represents an efficient and economical mean of controlling two or more functionally related enzymes. The common element to these two properties is the spatial character, which is potentially present in the function and the regulation of the intermediary metabolism. The biological systems, and metabolism in particular, exhibit both stability and variability; the latter sometimes assumes the character of periodicity. Whether the oscillations have a definite importance at the level of the intermediary metabolism itself, may well be questioned; the oscillatory faculties may rather serve as elements to be used in more complex functions of the biological systems. A thorough understanding of the role of clustered multienzyme systems and of the oscillatory phenomena in cellular metabolism demands a clearer physicochemical picture of the dynamic state of the living cell than we have at present. For this reason some of the generalizations derived from in vitro studies of single, isolated enzyme activities are not justified.

Adenosine Triphosphate↗
PubMed1976

Purification, properties, and substrate specificities of phosphoprotein phosphatase(s) from rabbit liver.

The phosphoprotein phosphatase(s) acting on muscle phosphorylase a was purified from rabbit liver by acid precipitation, high speed centrifugation, chromatography on DEAE-Sephadex A-50, Sephadex G-75, and Sepharose-histone. Enzyme activity was recovered in the final step as two distinct peaks tentatively referred to as phosphoprotein phosphatases I and II. Each phosphatase showed a single broad band when examined by sodium dodecyl sulfate gel electrophoresis; the molecular weights derived by this method were approximately 30,500 for phosphoprotein phosphatase I and 34,000 for phosphoprotein phosphatase II. The s20, w value for each enzyme was 3.40. Using this value and values for the Stokes radii, the molecular weight for each enzyme was calculated to be 34,500. Both phosphatases, in addition to catalyzing the conversion of phosphorylase a to b, also catalyzed the dephosphorylation of glycogen synthase D, activated phosphorylase kinase, phosphorylated histone, phosphorylated casein, and the phosphorylated inhibitory component of troponin (TN-I). The relative activities of the phosphatases with respect to phosphorylase a, glycogen synthase D, histone, and casein remained essentially constant throughout the purification. The activities of both phosphatases with different substrates decreased in parallel when they were denatured by incubation at 55 degrees and 65 degrees. The Km values of phosphoprotein phosphatase I for phosphorylase a, histone, and casein were lower than the values obtained for phosphoprotein phosphatase II. With glycogen synthase D as substrate, each enzyme gave essentially the same Km value. Utilizing either enzyme, it was found that activity toward a given substrate was inhibited competitively by each of the alternative substrates. The results suggest that phosphoprotein phosphatases I and II are each active toward all of the substrates tested.

Adenosine Triphosphate↗
PubMed1983

The enzymes of purine salvage in Trypanosoma cruzi, Trypanosoma brucei and Leishmania mexicana.

We have previously shown the presence of various purine salvage enzymes in Trypanosoma cruzi, including phosphoribosyltransferase, aminohydrolase, kinase, phosphorylase and hydrolase activities. We now report that a similar situation occurs in Leishmania mexicana amazonensis and Trypanosoma brucei brucei. In all three organisms we found higher levels of activity for the phosphoribosyltransferase enzymes than for the nucleoside kinases, suggesting a preference for the salvage of purine bases rather than nucleosides. Similarly, absence of inosine phosphorylase activity suggests that only one route for the salvage of hypoxanthine is available to the three organisms. The most striking difference was that whereas T. cruzi and T. brucei possessed adenosine aminohydrolase activity, this was not detected in L. mexicana; instead adenine aminohydrolase activity was found. The overall similarity, as judged by the distribution of enzyme activities, of purine salvage in these three members of the kinetoplastida suggest a broad spectrum of activity for any inhibitor acting in this area; the plethora of alternative salvage pathways, however, suggests that in no case would such inhibition be cidal.

Aminohydrolases↗