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PubMed1987

Effects of glucose infusion on surfactant and glycogen regulation in fetal lamb lung.

To investigate the increased incidence of respiratory distress syndrome (RDS) that occurs in infants of diabetic mothers (IDM) with poor maternal glucose homeostasis, we infused glucose intravenously at a rate of 14 +/- 2 (SD) mg.kg-1.min-1 into eight twin and four singleton chronically catheterized fetal lambs from 112 days (0.77) gestation onward. Twelve catheterized and seven uncatheterized fetuses served as controls, including the eight twins of the glucose-treated fetuses. Glucose infusion resulted in a twofold elevation in fetal serum glucose levels and a 2.2-fold elevation in fetal serum insulin levels. Before 113 days (0.9) gestation, pulmonary disaturated phosphatidylcholine (DSPC) content was 1.5-fold higher in the glucose-infused fetuses than in the controls. However, after 0.9 gestation, pulmonary DSPC content increased 2.2-fold in the controls but did not increase significantly in the glucose-infused fetuses. In addition, the DSPC content of lung lavage was 5.0-fold higher in the controls and lung stability to air inflation was 2.0-fold greater and to deflation was 2.2-fold greater than in the glucose-infused fetuses. Pulmonary adenosine 3',5'-cyclic monophosphate-dependent protein kinase activity was also 1.5-fold higher, and pulmonary protein kinase C activity was 1.3-fold higher in the controls than in the glucose-infused fetuses. In contrast, glucose infusion was associated with a 1.8-fold increase in pulmonary glycogen content and with increased activities of glycogen phosphorylase kinase and glycogen phosphorylase. We conclude that the effects of chronic glucose infusion on fetal lamb lung DSPC and lung stability are compatible with a predisposition of the fetus to develop RDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Elucidation of conservative elements of calmodulin-dependent enzymes with the use of monoclonal antibodies.

Monoclonal antibodies against human erythrocyte membrane Ca2+-ATPase were obtained. The binding of monoclonal antibodies to the enzyme resulted in a decrease in the enzyme sensitivity to calmodulin (CaM). The effects of monoclonal antibodies on other CaM-dependent enzymes, namely, on the phosphodiesterase of cAMP, phosphorylase kinase, and Ca2+-CaM-dependent protein kinase II (PK II), were studied. It was found that all four enzymes contain a common antigenic site. However, the inhibitory effect of antibodies was observed only with respect to Ca2+-ATPase and PK II. The kinetics of the binding of monoclonal antibodies and their inhibitory action were investigated. It was shown that the antigenic site is confined to the calmodulin-binding portion of Ca2+-ATPase and PK II.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Isolation of an active form of the ATP + Mg2+-dependent protein phosphatase stimulated by the deinhibitor protein and by p-nitrophenyl phosphate.

A 75 kDa + 37 kDa-subunit high-Mr form of the ATP + Mg2+-dependent phosphatase, containing no modulator or inhibitor-1 and specific towards the beta-subunit of phosphorylase kinase, was isolated from dog liver. The phosphorylase phosphatase activity is stimulated by the deinhibitor protein, by p-nitrophenyl phosphate or by trypsin treatment. The sensitivity to modulator or inhibitor-1 can be increased dramatically by partial proteolysis. The enzyme inactivation by modulator after trypsin treatment can be reversed by protein kinase FA. The effects of p-nitrophenyl phosphate and the deinhibitor protein are synergistic, and p-nitrophenyl phosphate does not influence the sensitivity of the phosphatase to the heat-stable inhibitor proteins. These observations confirm that p-nitrophenyl phosphate stimulation is a suitable criterion for the identification of the active enzyme forms of the ATP + Mg2+-dependent phosphatase.

Adenosine Triphosphate↗
PubMed1981

Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites.

Two calcium-stimulated protein kinase activities (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylate protein I, a specific synaptic protein, have been identified in homogenates of rat brain. One of these is found in both the particulate and cytosolic fractions and phosphorylates a region of protein I that is phosphorylated in intact synaptosomes in response to calcium but not to cyclic AMP. The stimulation by calcium of the particulate enzyme and of the partially purified cytosolic enzyme requires the addition of calmodulin. It is not yet known whether the particulate and cytosolic enzymes are related. A second calcium-stimulated protein I kinase is found only in the cytosol and phosphorylates a region of protein I that is phosphorylated in intact synaptosomes in response to either calcium or cyclic AMP. The calcium stimulation of this latter kinase is probably mediated by calmodulin, judging from its inhibition by low concentrations of trifluoperazine. Both of the calcium-stimulated protein I kinases are more highly concentrated in brain than in other tissues. The two cytosolic kinases are distinguishable from each other and from myosin light chain kinase and phosphorylase b kinase by their substrate specificities and their chromatographic behavior on DEAE-cellulose.

Animals↗
PubMed1983

Phosphorylation of a 43 kdaltons protein from rat heart by a calmodulin-dependent protein kinase.

The Ca2+ -dependent phosphorylation of proteins has been recognized as a major regulatory mechanism of biological processes. In the heart, protein kinases that are activated by Ca2+ include phosphorylase kinase, myosin light chain kinase, phospholamban kinase [review in 4], and the kinases responsible for phosphorylation of endogenous proteins in the membrane [11] and soluble [6] fractions of the cell. All of these Ca2+-dependent enzymes require the presence, either as an enzyme subunit or as a cofactor, of calmodulin, a Ca2+-binding protein which is involved in various other Ca2+-requiring reactions or processes [review in 3]. We demonstrate here the presence, in the rat heart, of a soluble calmodulin-dependent protein kinase which seems different from those already described in this tissue. The substrate for this enzyme is a 43 kdaltons protein, present in the same soluble fraction.

Animals↗
PubMed1982

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain.

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic-AMP-dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N-terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr = 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB2 (apparent Mr = 24 000) at the C-terminal end of the molecule. The sequence of the N-terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5, 1a and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N-terminus of CB2 respectively. Site 1a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites 1a and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N-terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C-terminal to site 3c while chymotrypsin cleaves N-terminal to site 3a has formed the basis of a simple procedure for determining the state of phosphorylation of the seven serine residues in vivo [Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem. 124, 47-55].

Amino Acid Sequence↗
PubMed1984

Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon.

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P-labeled glycogen synthase was purified from extracts of control or hormone-treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P-labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB-1) and 28000 (CB-2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB-2 phosphorylation was increased by all three hormones while CB-1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic-AMP-dependent protein kinase nor three different Ca2+-dependent enzymes (phosphorylase kinase, calmodulin-dependent protein kinase, and protein kinase C) were effective in phosphorylating CB-2. The protein kinases most effective towards CB-2 were the Ca2+ and cyclic-nucleotide-independent enzymes casein kinase II (PC0.7) and FA/GSK-3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK-3, is involved or phosphatase activity is regulated, or both.

Animals↗
PubMed1986

Mechanisms involved in calcium-mobilizing agonist responses.

Many hormones and neurotransmitters exert their biological effects by increasing the levels of Ca2+ and 1,2-diacylglycerol in their target cells. Major agonists that act in this way are epinephrine and norepinephrine, acetylcholine, vasopressin, cholecystokinin, and angiotensin II. These and other Ca2+-mobilizing agonists may also produce effects that are not mediated by Ca2+ or diacylglycerol, but involve separate receptors and an increase or decrease in cyclic AMP. The general mechanisms by which Ca2+-mobilizing agonists induce their physiological responses are depicted in Fig. 12. These responses appear to involve an initial mobilization of Ca2+ from endoplasmic reticulum and perhaps other intracellular Ca2+ stores, followed by alterations in the flux of Ca2+ across the plasma membrane. The Ca2+ changes are consistently associated with increased turnover of cellular phosphoinositides. The most rapid response is breakdown of phosphatidylinositol 4,5-P2 in the plasma membrane, and there is much evidence that this involves a guanine-nucleotide-binding regulatory protein similar to those involved in the regulation of adenylate cyclase. Myo-inositol 1,4,5-P3 produced by phosphatidylinositol 4,5-P2 breakdown rapidly releases Ca2+ from endoplasmic reticulum, and it is likely that it is the long-sought second message for the Ca2+-dependent hormones. 1,2-Diacylglycerol, the other product of phosphatidylinositol 4,5-P2 breakdown, also acts as a second message in that it activates protein kinase C, a Ca2+-phospholipid-dependent protein kinase, by lowering its requirement for Ca2+. The cellular substrates for protein kinase C and its role in the different physiological responses to the Ca2+-mediated agonists are currently being defined. The major intracellular target for Ca2+ is the Ca2+-dependent regulatory protein calmodulin. This binds Ca2+ with high affinity, and the resulting complex interacts with a variety of enzymes and other cellular proteins, modifying their activities. A major target is the multifunctional calmodulin-dependent protein kinase that phosphorylates and alters the activities of many proteins, for example, glycogen synthase and tyrosine hydroxylase. Calcium ions may also stimulate calmodulin-dependent protein kinases that are more specific, such as phosphorylase kinase and myosin light-chain kinase. Other important Ca2+-calmodulin targets are the microtubule-associated proteins, but it is likely that many more will be found.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Phosphorylation of glycogen synthase by cyclic AMP-independent casein kinase-2 from rabbit skeletal muscle.

Cyclic AMP-independent casein kinase-2 from rabbit skeletal muscle has been extensively purified by procedures including phosphocellulose, Bio-Gel A-1.5m, casein-Sepharose, and DEAE-cellulose column chromatographies. The casein kinase and glycogen synthase kinase activities are co-purified throughout the purification. The casein kinase-2 has Mr approximately equal to 135,000 as determined by glycerol density gradient centrifugation and by gel filtration. Analysis of the purified kinase by gel electrophoresis in the presence of sodium dodecyl sulfate reveals the presence of two major protein bands having Mr = 42,000 and 27,000 in a ratio of 0.85:1. The kinase phosphorylates glycogen synthase, casein, and phosvitin either in the presence of ATP or GTP; however, the Km values for GTP are slightly higher than those of ATP. The activity of this kinase is inhibited by heparin but is not affected by the addition of cyclic AMP, heat-stable inhibitor of cyclic AMP-dependent protein kinase, Ca2+, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or the combination of Ca2+ and calmodulin. The phosphorylation of the synthase by the kinase results in the incorporation of approximately 0.8 mol of PO4/subunit and a reduction in the synthase activity ratio from 0.82 to 0.6. The extent of phosphorylation of the synthase catalyzed by casein kinase-2 is similar to that by phosphorylase kinase; however, the sites phosphorylated by these two kinases are different. The relationships of casein kinase-2 to the cyclic AMP-independent synthase kinases that have been described by other laboratories and to the casein kinases isolated from other tissues are discussed.

Animals↗
PubMed1989

Isolation and characterisation of calcineurin from adrenal cell cytoskeleton: identification of substrates for Ca2+-calmodulin-dependent phosphatase activity.

Ca2+-calmodulin-dependent protein phosphatase activity is found in cytoskeletons of Y-1 mouse adrenal and bovine fasciculata cells. The activity is inhibited by three inhibitors of calmodulin (trifluoperazine, W-7 and pimozide) with EC50 in the low micromolar range. Protein phosphatase activity is inhibited by vanadate, fluoride, Zn2+ and pyrophosphate, stimulated by Mn2+ and found to be tightly bound to the cytoskeleton. Substrates for endogenous phosphatase activity were defined by one- and two-dimensional polyacrylamide gels. Phosphatase activity was seen with proteins that are substrates for both cyclic AMP-dependent and cyclic AMP-independent kinase enzymes. One specific Ca2+-calmodulin-dependent phosphatase, namely calcineurin, was purified to near homogeneity from cytoskeletons of Y-1 cells. The enzyme was found to be a heterodimer (MW 61,000 and 16,000) and the smaller subunit was shown to cross-react with antibodies raised against calcineurin from bovine brain. The purified enzyme catalyzes dephosphorylation of proteins (phosphorylase kinase and casein), phosphoamino acids (tyr greater than thre greater than ser) and a synthetic substrate (p-nitrophenyl phosphate). In addition, a new application of membrane transfer was devised by which the purified enzyme was incubated with a Western blot of cytoskeleton following incubation with [32P]ATP. This method defined four specific substrates of the enzyme (MW 150,000, 55,000, 35,000 and 30,000). Anti-calcineurin revealed that only a single Ca2+-calmodulin-dependent phosphatase is found in adrenal cell cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Effects of prostaglandin E1 in diabetic heart.

Isolated perfused hearts from normal and alloxan-diabetic rats were studied to determine the effects of prostaglandin E1 (PGE1) on phosphorylase activation in the insulin-deficient state. Perfusion of hearts from normal and diabetic rats with 3 X 10(-5) M PGE1 for the final 2 min resulted in activation to the same extent of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation, cAMP-sensitive protein kinase, and phosphorylase kinase. Although phosphorylase activation was somewhat suppressed in both the normal and diabetic heart, PGE1 produced a 36% increase in phosphorylase a in normal hearts and a 44% increase in phosphorylase a in diabetic hearts. The decreased effectiveness of phosphorylase activation by PGE1 appears to be located beyond activation of phosphorylase kinase and perhaps involves an alteration in phosphorylase sensitivity to phosphorylase kinase. Further, the activation of phosphorylase by phosphorylase kinase is hypersensitive in hearts of diabetic rats, perhaps due to a diabetes-related alteration in calcium metabolism.

Alprostadil↗
PubMed1989

A female case of type VIII glycogenosis who developed cirrhosis of the liver and hepatocellular tumor.

The case of a 17-year-old female with a rare form of type VIII glycogenosis who developed cirrhosis of the liver and hepatocellular tumor is reported. Laparoscopy showed a tumor 50 mm in diameter in the lower portion of the right lobe of the liver. The tumor was biopsied under ultrasonic guidance, and tentatively diagnosed as adenomatous hyperplasia. The patient was also diagnosed as having type VIII glycogenosis (phosphorylase kinase deficiency).

Adolescent↗
PubMed1981

Purification and characterization of smooth muscle myosin light chain kinase.

Smooth muscle myosin light chain kinase was purified from turkey gizzards. The enzyme was extracted from washed myofibrils and the final step of purification was affinity chromatography using calmodulin coupled to Sepharose 4B. The purified enzyme was characterized with respect to its physical, chemical, and kinetic properties. It has a molecular weight of 130,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 124,000 by sedimentation equilibrium centrifugation under nondenaturing conditions. It is an asymmetric molecule with a Stokes radius of 75 A, a sedimentation coefficient of 4.45 S, and a frictional coefficient of 1.85. Smooth muscle myosin light chain kinase is dependent on the calcium-binding protein calmodulin for activity. It has an apparent K0.5 for calmodulin of 10(-9) M and binds 1 mol of calmodulin/mol of myosin kinase in the presence of calcium. The binding of calmodulin increases the sedimentation coefficient from 4.45 S to 5.05 S, and the Stokes radius from 75 A to 79 A, and does not alter the frictional coefficient. The enzyme has a Km for ATP and the 20,000-dalton light chain of smooth muscle myosin of 50 microM and 5 microM, respectively. It phosphorylates the 20,000-dalton light chain of smooth muscle myosin more rapidly than the equivalent light chain from cardiac and skeletal muscles. It does not phosphorylate histones, alpha-casein, phosphorylase kinase, or phosphorylase b at a significant rate.

Amino Acids↗
PubMed1989

Structure-function relationships in cardiac troponin T.

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.

Animals↗
PubMed1985

X-linked glycogen storage disease. A cause of hypotonia, hyperuricemia, and growth retardation.

Seven male members of one family had a form of glycogen storage disease that was inherited in an X-linked recessive pattern. The clinical manifestations included hepatomegaly, delay in growth and sexual maturation, muscular weakness in childhood, and gouty arthritis. The cause of the glycogen accumulation did not appear to be a deficiency of glucose 6-phosphatase, debrancher enzyme, phosphorylase, or phosphorylase kinase. Prognosis appeared to be good although there was significant disability during childhood.

Adolescent↗
PubMed1993

Dephosphorylation of phosphorylated atrial natriuretic peptide by protein phosphatase 2A.

A phosphatase which exhibits strong activity toward phosphorylated atrial natriuretic peptide (ANP) was identified in the soluble fraction of rat brain homogenate. This ANP phosphatase has a neutral pH optimum, does not require divalent cations for activity, is inhibited by low concentrations of okadaic acid (50% inhibition at 1 nM) and preferentially dephosphorylates the alpha subunit of phosphorylase kinase. These properties are characteristic of serine/threonine protein phosphatase type 2A (PP2A). The apparent molecular mass of the ANP phosphatase (160 kDa), as estimated by gel filtration, is similar to that of the native heterotrimeric form of PP2A. In addition, phosphorylated ANP is an excellent substrate for the purified catalytic subunit of PP2A (Km = 42 microM, Vmax = 10.3 mumol x min-1 x mg-1). In contrast, protein phosphatase 2B (PP2B) has only very low ANP phosphatase activity (Km = 2.5 microM, Vmax = 0.008 mumol x min-1 x mg-1), and the catalytic subunit of protein phosphatase type 1 (PP1) as well as purified protein phosphatase type 2C (PP2C) are essentially inactive on ANP. These findings are consistent with the observation that PP2A-like activity accounts for virtually all ANP dephosphorylation in brain homogenate. While the phosphorylation of ANP in vitro by cAMP-dependent protein kinase is well documented, this is a first report on a phosphatase that efficiently can reverse this modification.

Animals↗
PubMed1977

[Inhibition of the reinitiation of meiosis in Xenopus laevis oocytes by three natural antiproteases: antipain, chymostatin, and leupeptin].

Oocytes incubated for 1 hr. in saline containing 1 mg/ml of antipain, leupeptin or chymostatin do not reinstate meiosis when progesterone or ionophore A 23187 are added. On the contrary such oocytes remain able to undergo meiosis when injected with cytoplasm of maturing oocytes (MPF), with Rabbit phosphorylase kinase or with Beef protein kinase. These results demonstrate that the loss of responsiveness to progesterone or ionophore is not due to toxic effects but rather to some interference with a key event which occurs prior to MPF action.

Animals↗