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1.
Hepatic phosphorylase deficiency: a biochemical study   总被引:1,自引:0,他引:1  
Two boys with hepatomegaly had increased glycogen content in the liver and no activity of liver phosphorylase, even in the presence of 5′ AMP. The biochemical differences between phosphorylase- and phosphorylase b kinase deficiency are discussed, and a differential diagnostic procedure proposed.  相似文献   

2.
Perfused rat hearts were treated with isoprenaline (10?6M) or ouabain (5.5 × 10?6M). The phosphate contents of troponin-I and myosin P light chains were established by radiolabelling with 32P; in the case of the light chains, direct chemical analysis of total and of specifically alkali-labile phosphate was also performed. Addition of isoprenaline caused phosphorylation of both troponin-I and myosin P light chains, reaching a maximum increment, after several minutes, of 1 mol/mol and 0.30 mol/mol, respectively. The Mg2+-ATPase activities, at saturating Ca2+ concentrations, of natural actomyosin isolated from treated hearts were significantly depressed, and an inverse correlation was established between the phosphate content of troponin-I and the Vmax[Ca2+] of this ATPase activity. The Ca2+ sensitivity of the Ca2+Mg2+-ATPase was also decreased. These changes were all reversed by an incubation permitting dephosphorylation of proteins by endogenous phosphatases.Treatment of hearts with ouabain caused no increment in troponin-I phosphorylation, but increased the P light chain phosphate content to a maximum of 0.30 mol/mol after some minutes. A positive correlation was evident between phosphate content of the light chains (in all experiments) and the maximum myosin Ca2+-ATPase activities. In addition, the Vmax[ATP] of the Ca2+Mg2+-ATPase of natural actomyosin was increased when light chain phosphorylation had occurred in the absence of troponin-I phosphorylation. P-light chain phosphorylation did not affect the Ca2+ sensitivity of Ca2+Mg2+-ATPase activity.We suggest that the effects of phosphorylation of troponin-I are to diminish thin filament sensitivity to Ca2+, and to decrease the efficiency of the transduction process along neighbouring actin monomers, such that the number of actin-myosin crossbridge interactions is decreased even in the presence of Ca2+ excess. Phosphorylation of P light chains of myosin has an activating effect on myosin Ca2+-ATPase activity, as well as on the rate of cross-bridge formation.  相似文献   

3.
Phosphorylation of rabbit skeletal muscle myosin in situ   总被引:4,自引:0,他引:4  
Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.  相似文献   

4.
With muscle glycogen phosphorylase a and b, 5-thio-D-glucose is a non-competitive inhibitor toward phosphate where it has a Ki of 13 mM and 5.1 mM, respectively, and produces a mixed type of inhibition when glycogen is the substrate.5-Thio-D-glucose enhances diaphragm phosphorylase phosphatase activity to the same extent as D-glucose, yet the thioanalog does not affect phosphorylase b kinase. Thus, the action of 5-thio-D-glucose on glycogen degradation proceeds by inhibition of phosphorylase a and b and by inactivation of phosphorylase a through converting it to the b form.  相似文献   

5.
Glycogen synthase phosphatase has been purified from bovine heart. This preparation catalyzes conversion of synthase D into I and phosphorylase a into b and is able to dephosphorylate synthase D, phosphorylase a, active phosphorylase kinase, and phosphorylated histone and casein. The activity of phosphatase was assayed with synthase D, phosphorylase a, and histone as substrates after chromatography on Sephadex G-100, after sucrose gradient centrifugation, and after isoelectric focusing in a sucrose gradient. In all cases no separation of enzyme activity was observed with the above substrates. The phosphatase activity on all substrates was lost at the same rate by heat denaturation. These results indicate that this enzyme preparation contains a single phosphoprotein phosphatase which is responsible for the activity observed on the above substrates.  相似文献   

6.
Carnosine (β-alanyl-L-histidine) activates rabbit muscle phosphorylase a in the presence and absence of AMP and phosphorylase b in the presence of AMP in a biphasic manner with a maximal activation at about 50mM carnosine and with phosphorylase b showing a greater degree of activation than phosphorylase a. Anserine (β-alanyl-L-Nπ-methyl-histidine) activates phosphorylase a to a lesser extent than carnosine up to a concentration of 90mM, whereas with phosphorylase b a weak activation below 30mM and a concentration-dependent inhibition above this concentration occurs. These effects are specific for the dipeptides and are not shown by their constituent amino acids. Carnosine and anserine activate phosphorylase a in the presence of the allosteric inhibitors ATP, D-glucose and caffeine, and the inhibition of phosphorylase b by anserine is also observed in the presence of these inhibitors.  相似文献   

7.
Regulation of pyridoxal 5'-phosphate metabolism in liver   总被引:4,自引:0,他引:4  
The pyridoxal 5′-phosphate content of liver in vivo and of hepatocytes in vitro remains unaltered in the presence of excess unphosphorylated vitamin B6 precursors. Studies with isolated hepatocytes and subcellular fractions show that while product inhibition of pyridoxine phosphate oxidase does not limit synthesis sufficiently to account for the phenomenon, inhibition of phosphatase activity produces striking increases in pyridoxal 5′-phosphate concentration. Protein-binding protects it against degradation by the phosphatase. The data suggest that protein-binding and the enzymatic hydrolysis of pyridoxal 5′-phosphate, synthesized in excess, act jointly to preserve the constancy of the cellular content of this coenzyme.  相似文献   

8.
Adenosine monophosphofluoridate has been synthesised and purified to remove all contaminating AMP. This AMP analogue fails to activate glycogen phosphorylase b, even at high concentration, but inhibits the AMP activation with a Ki value of 3 mM. Activation of phosphorylase b by adenosine phosphoramidate has been re-investigated in the light of these findings and a purified sample of this nucleotide analogue has been shown to produce little or no activation of the enzyme. These findings are interpreted in terms of an absolute requirement of the nucleotide activatorsite in phosphorylase for a nucleotide with a dianionic phosphate. The implications of this for the role of the phosphate moiety in the proposed mechanism of activation are discussed.  相似文献   

9.
Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase a and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.  相似文献   

10.
Human fibrinogen was shown to be a substrate of the catalytic subunit of pig muscle cyclic 3′,5′-AMP-stimulated protein kinase in vitro. Maximally at least 6 mol of (32P)phosphate per mol of fibrinogen was bound, preferentially to the α-chain.  相似文献   

11.
In skeletal muscle of animals with the phosphorylase b kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase b to a in the presence of Ca++, Mg++, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase b. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase b converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA1 (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca++. Thus the phosphorylase b kinase in skeletal muscle of animals with the phosphorylase b kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.  相似文献   

12.
The molecular basis of the developmental increase in AMP deaminase activity in chick muscle was investigated with a view toward determining whether isozymes of AMP deaminase exist in embryonic avian muscle and, if so, whether a stage-specific isozyme transition occurs during myogenesis in vivo and in vitro. Under specified conditions, AMP deaminase isozymes in adult chicken brain and muscle may be distinguished on the basis of differences in relative substrate specificities for 5′-dAMP and 5′-AMP (expressed as a ratio of the rates observed with these compounds; i.e., dAMPAMP ratios), as well as by differential immunoinactivation by antibody directed against breast muscle AMP deaminase. It was found that the AMP deaminase(s) that is (are) present in 6-day embryos is (are) catalytically and immunologically similar to the enzyme in adult brain. With mixtures of known amounts of adult muscle and brain enzymes, values for the dAMPAMP ratio (as well as the fraction of uninactivated AMP deaminase at antibody excess) were proportional to the fraction of muscle isozyme present. Standard curves constructed from these data were used to determine that the fraction of adult muscle-like AMP deaminase in developing muscle, as assessed by dAMPAMP ratios (and differential immunoinactivation), on days 6, 8, 10, and 15 were 23 (28), 55 (65), 83 (85), and 93% (96), respectively, Thus, parallel results were obtained for the two techniques, and the isozyme transition is virtually complete by the 15th day of incubation. Primary muscle cultures were used to investigate the isozyme transition of AMP deaminase during myogenesis in vitro. Comparison of the data obtained from primary muscle cultures treated with bromodeoxyuridine, cytosine arabinoside, and fluorodeoxyuridine with data from control cultures showed that biochemical differentiation of AMP deaminase in vitro could be attributed to the muscle cell. Also, the isozyme composition changed from a small percentage of adult muscle-like isozyme at the time of plating, to approximately 100% by the 6th day of culture.  相似文献   

13.
When fasted rats were fed a chow or fat-free diet supplemented 5% with L-histidine for three days, the brain adenosine 3′:5′-monophosphate (cAMP) level increased. A 50% increase occurred in rats fed a chow diet and 20% increase in rats fed a fat-free diet. Purification of liver fatty acid synthetase and the isolation of liver apo-, holo-a and holo-b fatty acid synthetases demonstrated that L-histidine feeding caused changes in the relative amounts of these enzymes. Apo- and holo-b fatty acid synthetases increased while the holo-a form simultaneously decreased. This effect was observed in rats fed either chow or fat-free diets supplemented with L-histidine.  相似文献   

14.
Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as E.coli, but doesnot affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.  相似文献   

15.
The activities of glycogen synthase (I and total) and phosphorylase (a and total) in crude extracts of isolated extensor digitorum longus and soleus muscles of the rat incubated in vitro in the absence or presence of methadone were very low. Addition of glycogen during homogenization increased the activities of both enzymes in control muscles. Even at optimal concentrations of glycogen, however, the activities of both enzymes from methadone-treated muscles were significantly lower than their activities in control muscles. The activity of phosphoglucomutase was not altered by incubation with methadone or by homogenization with glycogen. It is suggested that the addition of optimal amounts of glycogen during extraction of the enzymes enhances the extractability of glycogen synthase and increases the activity of phosphorylase by some other mechanism and that these processes are interfered with when the muscles are pretreated with methadone.  相似文献   

16.
It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with bis-(p-nitrophenyl) phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards 5′ -dTMP-p-nitrophenyl ester, which is probably associated with intracellular membrane structure in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.  相似文献   

17.
2′ (3′)-O-ethyl-CMP was prepared by alkylation of CMP with diethylsulphate in alkaline medium and deaminated to give 2′(3′)-O-ethyl-UMP, which was phosphorylated to 2′(3′)-O-ethyl-UDP. About 90% of the product consisted of the 2′ isomer. The 2′(3′)-O-ethyl-UDP was readily polymerized by E. coli polynucleotide phosphorylase in the presence of Mn++, but not Mg++. The 3′-isomer did not seriously interfere with polymerization nor did it act as a chain terminator. The resulting poly 2′-O-ethyluridylic acid formed a helical structure with a stability much higher then that of poly (rU) or poly 2′-O-methyluridylic acid. It also complexed readily with poly (rA). Implications with regard to the role of the 2′-hydroxyl in nucleic acid conformation are discussed.  相似文献   

18.
A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p-nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5′-nucleotidase inhibitor α,β-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5′-nucleotidase. An antibody against mouse liver 5′-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5′-nucleotidase and the necessity for distinguishing between true 5′-nucleotidase and non-specific phosphatase activity is discussed.  相似文献   

19.
20.
Less cyclic adenosine 3′:5′ monophosphate (cAMP) accumulated in rat pineal gland, invivo, after two doses of l-isoproterenol (5mg/kg, i.p.) than after one dose. A single injection of l-isoproterenol decreased the ability of l-isoproterenol to activate adenylate cyclase and increased the activity of the low Km phosphodiesterase (PDE). Tolerance to l-isoproterenol-induced accumulation of cAMP in rat pineal invivo may be due to decreased responsiveness of adenylate cyclase as well as to increased activity of PDE.  相似文献   

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