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ATPASE, MYOKINASE, 5'ADENYLIC ACID DEAMINASE ACTIVITY AND SYNERESIS OF THE MYOFIBRILLS OF THE DYSTROPHIC MOUSE.
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[Evolution of some enzymes of energetic metabolism during embryonic and post-natal development of rat skeletal muscles].
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Functional compartmentation of cyclic AMP and protein kinase in heart.
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Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases.
A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.
Defective facilitated diffusion of nucleosides, a primary mechanism of resistance to 5-fluoro-2'-deoxyuridine in the HCT-8 human carcinoma line.
In vitro resistance of HCT-8 cells to 5-fluoro-2'-deoxyuridine (FdUrd) has been obtained after a stepwise increase (up to 1 microM) in the concentration of the nucleoside in the culture medium over a period of 6 months. With a clonogenic assay, the toxicities of 17 antineoplastic agents on HCT-8-sensitive and -resistant cells were compared. Resistant cells were 700-fold resistant to FdUrd and showed different degrees of cross-resistance to several purine and pyrimidine nucleoside analogues; no cross-resistance was noted to base analogues and other cytotoxic drugs. The activities of FdUrd phosphorylase, 5'-fluorouridine kinase, 5-fluorouridine phosphorylase, 5-fluorouracil phosphoribosyltransferase, and thymidylate synthase were not significantly different in the sensitive and resistant cell lines. Mixing experiments indirectly excluded the possible elevation of the level of cytoplasmic phosphatases. The activity of FdUrd kinase in sensitive cell extracts was no more than twice that of resistant cells, and the affinities of this enzyme for FdUrd and thymidine at 0.1 to 50 microM were similar in both cell lines. However, cultures of this line failed to accumulate 5-fluoro-2'-deoxyuridylate at concentrations of FdUrd that resulted in substantial accumulation of the nucleotide in the sensitive line. These contrasting data suggested a defect in the facilitated diffusion of the analogue. The entrance of free nucleoside and its subsequent phosphorylation were compared in the two lines over short (2 to 40 s) and longer time periods at 25 degrees C and at 4 degrees C over a range of extracellular FdUrd concentrations (0.1 to 10 microM). Rapid entrance of the nucleoside into sensitive cells was observed, but entry was not detectable in resistant cells. Dipyridamole and nitrobenzylthioinosine inhibition as well as high-performance liquid chromatography analysis confirmed that data obtained from the sensitive cell line during the first 40 s primarily reflected facilitated diffusion of free nucleoside.
Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes.
Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.
Effects of the specific cAMP antagonist, (Rp)-adenosine cyclic 3',5'-phosphorothioate, on the cAMP-dependent protein kinase-induced activity of hepatic glycogen phosphorylase and glycogen synthase.
The cAMP-dependent protein kinase-induced effects on phosphorylase and glycogen synthase activities and glucose production were studied in hepatocytes isolated from fed rats in the presence of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS. Incubation of hepatocytes with (Sp)-cAMPS or glucagon, both of which lead to cAMP-dependent protein kinase activation, resulted in a concentration-dependent increase in glycogen phosphorylase activity and a decrease in glycogen synthase activity. Incubation of hepatocytes with the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, in the absence of an agonist, had no significant effect on phosphorylase or glycogen synthase activities. Incubation of hepatocytes with a half-maximally inhibitory concentration of (Rp)-cAMPS shifted the agonist-induced activation curves for phosphorylase and the agonist-induced inhibition curves for glycogen synthase to 5-fold higher concentrations for both (Sp)-cAMPS and glucagon. Phosphorylase activity was very sensitive to the rapid, concentration-dependent inhibition by (Rp)-cAMPS of agonist-induced activation of cAMP-dependent protein kinase. The effects on phosphorylase activity were observable in 30 s and were concentration-dependent with half-maximal inhibition at 10 microM, similar to that observed for cAMP-dependent protein kinase. In contrast, glycogen synthase activity was less sensitive to (Rp)-cAMPS inhibition of agonist-induced activation of cAMP-dependent protein kinase. The effects on glycogen synthase activity lagged behind those on phosphorylase activity and the concentration dependence did not parallel the cAMP-dependent protein kinase effect, but was shifted to higher concentrations of (Rp)-cAMPS with half-maximal inhibition at 60 microM. Glucose (10 to 40 mM) increased the sensitivity of glycogen synthase to (Rp)-cAMPS inhibition of cAMP-dependent protein kinase over a narrow range of agonist concentration, but had no significant effect throughout most of the agonist-induced activation range. Thus, the diastereomers, (Sp)- and (Rp)-cAMPS, influence glycogen metabolism and the glycogenolytic enzymes through their modulation of cAMP-dependent protein kinase levels.
Hormonal regulation of phosphorylase phosphatase activity in rat liver.
The effect of glucagon and insulin on rat liver phosphorylase phosphatase activity in vivo was investigated. The activity of phosphatase was found to decrease following the administration of glucagon and increase with insulin in a reversible manner. No change was detected in the activity of heat-stable phosphatase inhibitors in the hormone-treated samples. Liver protein kinases (regulatory subunit of cAMP-dependent protein kinase and/or Ca2+-dependent phosphorylase kinase) are suggested to regulate the activity of hepatic phosphorylase phosphatase (type 1 and 2A).
Incorporation characteristics of uracil, uridine, and orotic acid into ribonucleic acid of neoplastic cells.
Incorporation of uracil and uridine into ribonucleic acid (RNA) was compared among the ascitic and solid forms of Ehrlich mouse tumor, Morris hepatoma, Rhodamine sarcoma, gastric cancer and ulcer from human patients, and several normal rat tissues. Of these cells tested, the cells of Ehrlich ascites and solid tumors, human gastric cancer and ulcer, and certain tissues of a normal rat showed a considerably high activity. Furthermore, Ehrlich ascites tumor cells indicating a high incorporation activity was also high in activities of both phosphorylase and kinase for uridine, while Rhodamine sarcoma as a representative having a low incorporation activity was considerably low in these two enzymic activities. RNA synthesis from uridine phosphates by Rhodamine sarcoma was maintained to a fairly high extent contrary to its low activities of the phosphorylase and the kinase. Consequently, the low utilization of uracil and uridine by certain tumors was suggested to be due to the extremely low activities of both enzymes.
In vivo developmental modifications of the expression of genes encoding muscle-specific enzymes in rat.
cDNA clones for rat muscle-type creatine kinase and glycogen phosphorylase and aldolase A were isolated from a rat muscle cDNA library. An additional clone recognizing an unidentified 2.7-kilobase pair mRNA species was also isolated. These cDNA clones were used as probes to investigate the expression of the corresponding mRNAs during muscle development. Two aldolase A mRNA species were detected, one of 1650 bases expressed in non-muscle tissues, fetal muscle, and adult slow-twitch muscle, the other of 1550 bases was highly specific of adult fast-twitch skeletal muscle differentiation. These aldolase A mRNAs were shown by primer extension to differ by their 5' ends. The accumulation of muscle-type phosphorylase and creatine kinase and muscle-specific aldolase A mRNA accumulation during muscle development seems to be a coordinate process occurring progressively from the 17th day of intrauterine life up to the 30th day after birth. In contrast, the 2.7-kilobase pair RNA species is maximally expressed at the 1st week after birth as is the neonatal form of myosin heavy chain mRNA.
The role of the cyclic AMP-dependent protein kinase in the glucagon-stimulated phosphorylation of ATP-citrate lyase.
We have examined the mechanism whereby glucagon stimulates the phosphorylation of ATP-citrate lyase in intact rat hepatocytes. Purified ATP-citrate lyase is phosphorylated in vitro by the catalytic subunit of the cyclic AMP-dependent protein kinase, in a reaction wherein 2-3 mol phosphate/mol lyase are incorporated, at an initial rate that approaches that observed for mixed histone. This reaction is completely abolished by the protein kinase inhibitor protein. Limited tryptic digestion of ATP-citrate lyase phosphorylated in vitro by the cyclic AMP-dependent protein kinase yields a pattern of 32P-labeled peptides, indistinguishable from those observed in parallel digests of lyase isolated from 32P-labeled, glucagon-stimulated hepatocytes. Phosphorylase b kinase catalyzes the incorporation of 1 mol phosphate/mol lyase, albeit at less than 1/160 the rate observed for phosphorylase b. The phosphorylation of purified ATP-citrate lyase is also catalyzed by homogenates of hepatocytes. This reaction is stimulated by cyclic AMP. At 30 degrees C, in the presence of maximally stimulating concentrations of cyclic AMP, the addition of excess protein kinase inhibitor protein inhibits the phosphorylation of ATP-citrate lyase by 67%. Thus, hepatocytes contain both cyclic AMP-dependent and cyclic AMP-independent ATP-citrate lyase kinase activities. Pretreatment of hepatocytes with glucagon (10(-8) M for 2 min) prior to homogenization results in activation of an endogenous hepatocyte ATP-citrate lyase kinase, as well as histone kinase and phosphorylase b kinase; the glucagon-stimulated increment in lyase kinase (and histone kinase) is observed only when homogenates are assayed in the absence of added cyclic AMP, and is completely abolished by an excess of the protein kinase inhibitor protein. We conclude that the glucagon-stimulated phosphorylation of ATP-citrate lyase in intact hepatocytes is catalyzed directly by the cyclic AMP-dependent protein kinase.
Subunit sizes of muscle proteins, as determined by sodium dodecyl sulphate gel electrophoresis.
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On the mechanism of inactivation of muscle glycogen phosphorylase by insulin.
Glucose 6-phosphate, an allosteric inhibitor of skeletal muscle phosphorylase b, inhibits at physiological concentrations and conditions the phosphorylation and activation of the enzyme by phosphorylase b kinase. AMP inhibits the dephosphorylation of phosphorylase a, but is without effect on the phosphorylation of phosphorylase b. Glucose 6-phosphate has no effect on the activity of phosphorylase a and does not affect its dephosphorylation by phosphatases 1 or 2A. The inhibition of the phosphorylation of phosphorylase b by glucose 6-phosphate may explain the reported decreased phosphorylation of phosphorylase in muscle following insulin treatment, which elevates intracellular levels of glucose 6-phosphate.
Altered regulation of cardiac glycogen metabolism in spontaneously diabetic rats.
Isolated perfused hearts from control Bio-Breeding/Worcester (BB/W) rats and spontaneously diabetic BB/W rats were studied to determine whether metabolic abnormalities that are expressed in alloxan-diabetic rats in the regulation of enzymes involved in glycogen metabolism could be observed in this non-chemically induced insulin-deficient rat. Perfusion of hearts from control rats with 10(-8) M insulin for 10 min resulted in activation of glycogen synthase (30% synthase I without insulin to 44% synthase I with insulin). Perfusion of hearts from BB/W diabetic rats demonstrated a lack of acute synthase activation with insulin and a 45% decrease in synthase phosphatase activity. Perfusion of hearts from BB/W diabetic rats with 0.28 microM epinephrine for 1 min resulted in a greater activation of phosphorylase (44% phosphorylase a) than that observed in BB/W control hearts (31% phosphorylase a) perfused under the same conditions. Epinephrine produced similar changes in cyclic AMP accumulation, protein kinase activation, and phosphorylase kinase activation in perfused hearts of BB/W control and diabetic rats. Further, phosphorylase phosphatase activities were not changed by epinephrine or insulin deficiency. These studies further document metabolic abnormalities in the BB/W diabetic rat that are attributable to insulin deficiency in a non-chemically induced model for insulin-dependent diabetes.
5-Fluorouracil is converted to F-nucleotides more extensively and is more cytotoxic in poorly differentiated than in well differentiated human gastric carcinoma.
The sensitivity to 5-fluorouracil (5-FU) was examined in 40 well differentiated and 50 poorly differentiated gastric cancer tissues and 15 normal tissues, using the in vitro succinate dehydrogenase inhibition (SDI) test. The tissue phosphorylating and degrading activities of 5-FU were compared in each type of tumor and in the normal tissues. Decreases in succinate dehydrogenase (SD) activity were more apparent in the poorly differentiated cancer tissues than in the well differentiated cancer tissues (p less than 0.005), and than in the normal tissues (p less than 0.001), exposed to 5-FU. The rate of sensitivity to 5-FU was higher in the poorly differentiated than in the well differentiated tissues and than in the normal tissues. The phosphorylating activities of 5-FU, in pathways involving uridine (Urd) phosphorylase and Urd Kinase, and thymidine (dThd) phosphorylase and dThd Kinase, were 1.7 fold higher in the poorly differentiated than in the well differentiated tissues and several fold higher than in the normal tissues (p less than 0.05-p less than 0.001). The degrading activity of 5-FU was similar in both types of tumor and in the normal tissues. Our findings show that 5-FU is actively metabolized to 5-FU-nucleotides in poorly differentiated tissues after incorporation into the tumor cells. 5-FU seems to have an increased susceptibility in cases of poorly differentiated gastric carcinoma.
[Metabolic myopathies in childhood. A review in summarized form].
In this review hereditary disorders of muscle metabolism are summarized. Defects in the glycogen metabolism, the glycolysis, the carnitine metabolism, the respiratory chain in the mitochondria and other rare defects are discussed.
Determination of calcium transport and phosphoprotein phosphatase activity in microsomes from respiratory and vascular smooth muscle.
1. Calcium transport into microsomal vesicles of respiratory (tracheal) smooth muscle was characterized. This calcium transport was ATP dependent and stimulated by the presence of the oxalate ion. The magnitude of transport was similar to that reported for microsomes from other types of smooth muscle. 2. Bovine and rabbit, heavy and light microsomes were isolated from respiratory (tracheal) and vascular (aortic) smooth muscle. Preincubation of these vesicles with cyclic AMP and protein kinase did not alter the transport of calcium into the vesicles. There uas no evidence of phosphate incorporation into microsomal membrane proteins. Similar results were obtained if phosphorylase b kinase replaced the combination of cyclic AMP and protein kinase during the preincubation. 3. The phosphoprotein phosphatase activity of cardiac sarcoplasmic reticulum and smooth muscle microsomes was determined. The activity of this enzyme was found to be several-fold less in the cardiac sarcoplasmic reticulum than in various smooth muscle microsome preparations.