Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase

Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The Km for glycogen synthase was close to the concentration found in heart tissue. The Km and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation. Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.     

Dephosphorylation of glycogen synthase in rat heart extracts by E. coli alkaline phosphatase

Summary Regulation of the dephosphorylation of glycogen synthase in extracts from rat heart has been studied by adding exogenous phosphatase to the extract. These experiments were possible only because the endogenous protein phosphatase activity of the extract could be inhibited by KF under conditions where alkaline phosphatase activity was not. The concentration of substrate (glycogen synthase from the heart extract) and catalyst (purified E. coli alkaline phosphatase) could be varied independently, by adding known amounts of alkaline phosphatase to the KF-containing heart extracts. Alkaline phosphatase could completely dephosphorylate glycogen synthase while phosphorylase was unchanged. The rate of dephosphorylation was proportional to both the concentration of alkaline phosphatase added to the tissue extract and the amount of glycogen synthase in the extract. The K_m for glycogen synthase was close to the concentration found in heart tissue. The K_m and the maximum rate of dephosphorylation were both dependent on the phosphorylation state of the glycogen synthase. Less phosphorylated enzyme forms were dephosphorylated faster. These results indicate the necessity for precise control of many variables in studying the rate of glycogen synthase dephosphorylation.Alkaline phosphatase-catalyzed dephosphorylation could be inhibited by physiological concentrations of glycogen. Glycogen synthase dephosphorylation in extracts from fasted-refed rats was less sensitive to glycogen inhibition than in extracts from normal animals. The phosphorylation state of the glycogen synthase in these animals was assessed by kinetic studies to show that differences in phosphorylation state probably could not account for the observations. Fasting led to a decreased rate of dephosphorylation of glycogen synthase due to both an apparent change in kinetic properties of glycogen synthase as a substrate for alkaline phosphatase, and an increased inhibitory effect of glycogen. Stable modifications of glycogen synthase caused by altered nutritional states in the animals are thought to produce these effects.%GSI represents the percentage of glycogen synthase activity that is active without glucose 6-P.     

EFFECT OF THYROID DEFICIENCY ON THE LEVELS OF SEVERAL ENZYMES IN RAT BRAIN

—Activities of acid phosphatase, alkaline phosphatase and β-glucuronidase have been estimated in the brain tissues, using various subcellular particles, in growing thyroidectomized rats and also using cytoplasmic extracts free from debris and nuclear fraction in young hypothyroid animals. Hepatic glucose-6-phosphate dehydrogenase activity was markedly reduced after thyroidectomy but the enzyme was brought back to normal levels by thyroxine treatment. There was no change, however, in the activity of neural glucose-6-phosphate dehydrogenase after thyroidectomy. In the thyroidectomized animals an increase only in the free acid phosphatase activity in the neural synaptosomes was found and this increase in activity was not counteracted by administration of thyroxine. In the hypothyroid young animal β-glucuronidase, acid phosphatase and alkaline phosphatase activities were found to be affected during development.     

Characterization and localization of alkaline phosphatase activity in rat testes

Alkaline phosphatase activity in extracts of testes of sexually immature (13 days old) and sexually mature rats has been characterized by its heat sensitivity, the extent of inhibition by homoarginine and phenylalanine, and by polyacrylamide gel electrophoresis. The testicular enzyme appears to be a liver-bone-kidney-type alkaline phosphatase. There are no significant differences in the properties of the enzyme from animals of these two ages. Spermatocytes and early spermatids contain very little alkaline phosphatase activity; the specific activity of a nonflagellate germinal cell suspension is only 1/20th that of the whole testis. Since the constant level of activity in immature and mature animals is not consistent with the enzyme activity being present only in late spermatids, we conclude that the majority of the testicular enzyme is present in nongerminal cells. The presence of alkaline phosphatase in plasma membrane purified from testes of adult rats was demonstrated.     

Effect of isoproterenol on phosphorylase activation in the hyperthyroid rat heart.

Pretreatment of rats for 3 days with triiodothyronine produced an increase in rate in the right atrium and a decrease in force of contraction in the right ventricle and Langendorff heart. Isoproterenol administration produced a time-dependent increase in rate and tension. The increase in rate was consistently greater in atria from hyperthyroid rats, and the increase in tension consistently greater in tissues from euthyroid rats. Isoproterenol also produced a time- and dose-dependent increase in phosphorylase a activity. In the isolated atria and ventricles enzyme activity was similar in the two groups. In the Langendorff hearts, however, there was an enhancement of the isoproterenol-induced increase in phosphorylase activity in hearts from hyperthyroid rats. Reduction of the coronary blood flow to the level found in euthyroid animals did not reduce the potentiation of phosphorylase activation found in hearts from hyperthyroid rats. It is concluded that the potentiation of phosphorylase activation in hearts from hyperthyroid rats is not due to the increase in coronary blood flow.     

Influence of right atrial stretch on plasma renin activity in the conscious rat

Rats were prepared with inflatable balloons at the superior vena cava - right atrium junction. After recovery 1 week later, when blood was taken from conscious, normovolaemic animals plasma renin activity was found not to be influenced by right atrial stretch. Plasma renin activity was then measured in rats in which an extracellular fluid deficit had been produced by peritoneal dialysis against a hyperoncotic, isotonic solution. Although basal plasma renin activity was elevated (6.8 +/- 0.9 from 1.5 +/- 0.2 ng X mL X h, n = 19), no depression was observed in the experimental group after 15 or 90 min of balloon inflation. In rats pretreated with isoprenaline (10 micrograms/kg body wt.) plasma renin activity was also increased over basal levels, but again balloon inflation caused no reduction in plasma renin activity. It would appear that right atrial stretch has little, if any, influence on renin release in the conscious rat.     

Insulin stimulation of heart glycogen synthase D phosphatase (protein phosphatase).

Insulin rapidly produced an increase in per cent of total heart glycogen synthase in the I form in fed rats. In fasted rats the response was diminished and delayed. In diabetic animals there was no response over the 15-min time period studied. Since synthase phosphatase activity is necessary for synthase D to I conversion, the phosphatase activity was determined in extracts from these groups of animals. In the fasted and diabetic rats phosphatase activity was less than one-half of that in fed animals. Administration of insulin to fasting animals increased synthase phosphatase activity to a level approaching that of fed animals by 15 min. In diabetic animals insulin also stimulated an increase in synthase phosphatase activity but 30 min were required for full activation. Insulin had no effect in normal fed animals. Insulin activation of synthase phosphatase activity in heart extracts from fasted animals was still present after Sephadex G-25 chromatography and ammonium sulfate precipitation. Thus insulin had induced a stable modification of the phosphatase itself or of its substrate synthase D rendering the latter a more favorable substrate for the reaction. A difference in sensitivity of the reaction to glycogen inhibition was present between fed and fasted animals. Increasing concentrations of glycogen had only a slight inhibitory effect in extracts from fed animals but considerably reduced activity in extracts from fasted animals. Insulin administration reduced the sensitivity of the phosphatase reaction to glycogen inhibition. This could explain, at least in part, the increased phosphatase activity noted in the insulin-treated, fasted rats since glycogen was routinely added to the homogenizing buffer.     

Characterization of an Escherichia coli K12 mutant that is sensitive to chlorate when grown aerobically.

1. The ;initial activity' of the pyruvate dehydrogenase enzyme complex in whole tissue or mitochondrial extracts of lactating rat mammary glands was greatly decreased by 24 or 48h starvation of the rats. Injection of insulin and glucose into starved rats 60min before removal of the glands abolished this difference in ;initial activities'. 2. The ;total activity' of the enzyme complex in such extracts was revealed by incubation in the presence of free Mg(2+) and Ca(2+) ions (more than 10 and 0.1mm respectively) and a crude preparation of pig heart pyruvate dehydrogenase phosphatase. Starvation did not alter this ;total activity'. It is assumed that the decline in ;initial activity' of the enzyme complex derived from the glands of starved animals was due to increased phosphorylation of its alpha-subunit by intrinsic pyruvate dehydrogenase kinase. 3. Starvation led to an increase in intrinsic pyruvate dehydrogenase kinase activity in both whole tissue and mitochondrial extracts. Injection of insulin into starved animals 30min before removal of the lactating mammary glands abolished the increase in pyruvate dehydrogenase kinase activity in whole-tissue extracts. 4. Pyruvate (1mm) prevented ATP-induced inactivation of the enzyme complex in mitochondrial extracts from glands of fed animals. In similar extracts from starved animals pyruvate was ineffective. 5. Starvation led to a decline in activity of pyruvate dehydrogenase phosphatase in mitochondrial extracts, but not in whole-tissue extracts. 6. These changes in activity of the intrinsic kinase and phosphatase of the pyruvate dehydrogenase complex of lactating rat mammary gland are not explicable by current theories of regulation of the complex.     

Effect of starvation and insulin treatment on glycogen synthase D and synthase D phosphatase activity in rat heart

Summary We have previously shown that synthase phosphatase activity was decreased in starved animals and was rapidly restored by insulin administration (1). In order to determine whether the decreased phosphatase activity was due to a decrease in phosphatase enzyme per se or to a change in the substrate, synthase D, phosphatase activity has been determined using purified synthase D substrate. Using purified heart or liver synthase D, phosphatase activity was lower in extracts from starved animals than in fed animals. Insulin administration rapidly increased phosphatase activity in extracts from the starved animals. The total amount of endogenous synthase D which was convertible to synthase I was lower in extracts from starve animals, but this was rapidly increased within 15 minutes following insulin administration. These data suggest that starvation and insulin have a direct effect on the phosphatase enzyme activity per se and probably on the substrate suitability of synthase D as well.     

Induction of Rat Liver Alkaline Phosphatase by Bile Duct Ligation

Bile duct ligation causes a five- to sevenfold increase in the activity of rat liver alkaline phosphatase within 12 hours after ligation and a similar rise in the activity of alkaline phosphatase in serum. The increased serum activity is due entirely to the appearance of a new isoenzyme that has the properties of rat liver alkaline phosphatase. The increase in both serum and liver alkaline phosphatase is prevented by the prior administration of cycloheximide in a dose that inhibits protein synthesis by 70%. Rat liver alkaline phosphatase was then purified to homogeneity. Antibody was raised to purified rat liver alkaline phosphatase in rabbits. The antibody was coupled to sepharose 4B and affinity columns made. ³-H-leucine was then injected into the portal veins of sham operated rats and rats with bile duct ligation four hours after ligation. One hour after injection and five hours after ligation, animals were sacrificed. Liver alkaline phosphatase was purified by means of affinity chromatography and double immunoprecipitation with rabbit antibody to rat liver alkaline phosphatase and goat anti-rabbit gamma globulin. Bile duct ligation increased the incorporation of ³-H-leucine into liver alkaline phosphatase more than threefold compared with sham operated rats, 164 CPM/mg protein vs. 49 CPM/mg protein (p < .001). The data indicate that the increased activity of rat liver alkaline phosphatase after bile duct ligation is due to enzyme induction rather than to activation of a pre-existing, relatively inactive enzyme.     

Association of inorganic pyrophosphatase activity with normal calcification of rat costal cartilage in vivo

1. Dialysed extracts of rat costal cartilage were shown to possess an enzyme that hydrolyses inorganic pyrophosphate. 2. Inorganic pyrophosphatase activity assayed in the presence of 2mm substrate was maximal at pH6.8. 3. Mg(2+) was essential for activity, which was greatest with 10mm or higher concentrations of Mg(2+). 4. Extracts prepared from cartilage taken from suckling rats (<20g.) showed little or no hydrolytic activity, but as rat weight increased inorganic pyrophosphatase activity was detected, increased to a maximum in tissue from animals weighing about 40g., and then rapidly declined. 5. The increase in inorganic pyrophosphatase activity was associated with an increase in the uptake of (45)Ca by the cartilage in vivo. 6. Accumulation of calcium, inorganic phosphate and magnesium occurred when inorganic pyrophosphatase activity was at its maximum. 7. Alkaline phosphatase activity, measured in the same extracts used to determine pyrophosphatase activity, was highest in the tissues of the animals weighing <20g., and decreased as inorganic pyrophosphatase activity increased to its maximum. 8. There was no direct relationship between alkaline phosphatase activity and the onset of calcification.     

Enzymic activity in rat uterus during early pregnancy.

Total protein, RNA and DNA content and the activity of acid and alkaline phosphatases, 5'-nucleotidase and isocitrate dehydrogenase were studied in rat uterus during the first 8 days of pregnancy. Isocitrate dehydrogenase activity showed marked fluctuations from day to day. Nucleotidase and acid phosphatase activities showed a significant increase on day 8. The most marked change in activity was that of alkaline phosphatase which showed a 10-fold increase between days 6 and 8, due largely to an increase in the activity of this enzyme in the decidual nodule. The rise in alkaline phosphatase activity did not occur in rats ovariectomized on days 1, 2 or 4 of pregnancy and was markedly decreased in those ovariectomized on day 6. [3H]-uridine incorporation into RNA showed a significant increase between days 2 and 6 whereas [3H]-thymidine incorporation into DNA showed a significant increase on day 6.     

Biochemical compartmentation of fish tissues: chronic toxicity of mercuric nitrate on visceral phosphomonoesterases in Channa punctatus (Bloch)

Effect of sub-lethal dose of mercuric nitrate was studied in anterior, middle and posterior regions of kidney, anterior and posterior regions of left and right liver lobes, cephalic, thoracic and caudal regions of muscle and left and right gill tissues of C. punctatus in relation to acid and alkaline phosphatase under chronic studies. Middle region of kidney registered maximum rise and fall of acid and alkaline phosphatase, respectively. Right lobe of liver showed more rise and fall of acid and alkaline phosphatase activity respectively. Similarly, left gill is more pronounced than the right one. The observed enzymatic variations were discussed in relation to the biochemical constituents and physiological coordination rendered by these tissues.     

Simultaneous measurement of atrial natriuretic polypeptide (ANP) messenger RNA and ANP in rat heart--evidence for a preferentially increased synthesis and secretion of ANP in left atrium of spontaneously hypertensive rats (SHR)

Tissue levels of atrial natriuretic polypeptide (ANP) messenger RNA (ANPmRNA) and ANP in the rat heart were measured simultaneously. In Wistar rats, ANPmRNA of the same size (approximately 0.95 kbp) was detected in all four chambers of the rat heart. The ANPmRNA level was the highest in the right atrium, and the left atrial level was slightly lower than the right atrial level. Ventricular levels were more than two orders of magnitude lower than atrial levels. Tissue ANP concentrations of four chambers were roughly parallel to ANPmRNA levels. In spontaneously hypertensive rats (SHR) with the elevated plasma ANP level, the ANPmRNA level in the left atrium was substantially increased. The left/right ratio of atrial ANPmRNA level in SHR (150%) was higher than that in control Wistar Kyoto rats (WKY) (90%). In contrast, the left/right ratio of atrial ANP concentration was decreased in SHR (44%) compared with that in WKY (84%). The ratio of ANP to ANPmRNA levels in the left atrium of SHR was about three times smaller than that in the right atrium of SHR, and those in bilateral atria of WKY. These results indicate that the biosynthesis and secretion of ANP from the left atrium is preferentially increased in SHR. Thus, simultaneous determination of ANPmRNA and ANP levels is a refined strategy of investigation for the biosynthesis, storage and secretion of ANP.     

Molecular aspects of the regulation of rat kidney alkaline phosphatase

The mechanisms by which phosphate regulates the activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) in rat kidney were investigated. Measurements of incorporation of [(14)C]leucine into kidney alkaline phosphatase in rats fed on complete or phosphate-free diet provide evidence of a twofold increase in the rate of synthesis of the enzyme in diet-treated animals. Cycloheximide experiments indicated that control and diet-adapted enzyme decreases in activity according to first-order kinetics with a calculated half-life of 10.3 and 6.5h after complete and phosphate-free diet administration respectively. Basal and diet-adapted enzymes exhibit similar K(m) values for several phosphomonoesters and an identical degree of inhibition is produced by cysteine. In addition, the enzyme from both sources is the same with regard to heat inactivation at 45, 56 or 64 degrees C, to the profile of elution from Sephadex and to electrophoretic properties on polyacrylamide gel. A failure of rat kidney alkaline phosphatase to respond to cortisol (hydrocortisone) was also observed.     

Analysis of the direct and indirect effects of adenosine on atrial and ventricular cardiac muscle

The effects of adenosine were examined upon the tension developed by isolated paced left atria, left ventricular papillary muscles, and right ventricular strips, and upon the spontaneous rate of contraction of right atria of guinea pigs. Three aspects of the direct and indirect actions were examined: the direct effects upon resting developed tension and rate, the indirect activity when added to tissues prestimulated by isoprenaline, and the indirect activity upon isoprenaline concentration--response curves when added prior to exposure to isoprenaline. The direct effects on the atria were decreases in left atrial tension and right atrial rate. These responses were antagonized by 8-phenyltheophylline (8-PT) and therefore were due to stimulation of cell surface P1 purinoceptors. This antagonism was greater in the left atria than the right. The direct ventricular effects were, in contrast, increases in force of contraction, which were not antagonized by 8-PT. This positive inotropy was also unaffected by reserpine pretreatment of the guinea pigs and therefore not due to release of endogenous catecholamines. A possible intracellular effect of adenosine is discussed. Adenosine reduced the isoprenaline-prestimulated tension or rate in both atrial and ventricular tissues, and this indirect effect was susceptible to antagonism by 8-PT. In the presence of adenosine, concentration-response curves of the left and right atria for isoprenaline were displaced to the right, and the maxima were reduced. In contrast, there was no antagonism of the papillary muscle curves to isoprenaline, but the maximum developed tension was elevated. The minimal inhibitory effects of adenosine in ventricular muscles and the high concentrations required are discussed in the context of a physiological role for endogenous adenosine in attenuating cardiac overstimulation by the sympathetic nervous system or endogenously released catecholamines.     

Histochemical observations of alkaline phosphatase activity of the lingual epithelium after the suppression of salivation

Summary The influence of salivation on the location of gustatory alkaline phosphatase has been examined. In untreated rats, taste buds at the ends of fungiform papillae showed almost no activity. However, if salivation was suppressed for 12 hours in fasted animals, alkaline phosphatase activity could be clearly demonstrated in association with these taste buds. The results indicated that alkaline phosphatase may be removed from its site of secretion by saliva and that the enzyme is secreted from fungiform as well as circumvallate and foliate papillae.     

Status of phosphatase activities in the liver and kidney of rats treated with isosaline leaf and stem-bark extracts of Harungana madagascariensis (L)

The activities of phosphatases and other biochemical parameters were examined in rats treated with isosaline leaf and stem-bark extracts of Harungana madagascariensis (L). Results show that both the leaf and stem-bark extracts significantly increased the activities of serum and liver alkaline phosphatase, liver acid phosphatase, liver and kidney glucose-6-phosphatase, fructose-1,6-diphosphatase and glycogen in the treated rats. While the stem-bark extract significantly elevated the activities of fructose-1,6-diphosphatase and glycogen in the kidney, these biochemical parameters were not affected by treatment with the leaf extract. The activity of serum acid phosphatase was unaffected by the two extracts. The results obtained clearly show that these extracts caused a marked increase in gluconeogenesis in the liver and kidney of the treated rats. While the stem-bark extract increased gluconeogenesis in both liver and kidney, the leaf extract caused an increase in gluconeogenesis only in the liver. The increased serum alkaline phosphatase activity caused by these extracts may, aside from having other tissues contributing to it, be due to damage caused by these extracts to the hepatocytes. The extent of pathological changes as well as the implications of these findings to folklore medicine requires further investigation.     

Investigation of alkaline phosphatase activity in the serum of pregnant rats

An investigation was undertaken to determine if the placental alkaline phosphatase of the rat enters the maternal circulation and to study some of its characteristics. Unlike human, rat placental alkaline phosphatase was found to be heat labile and the alkaline phosphatase activity in the serum of both pregnant and non-pregnant rats was also found to be heat labile. Also unlike the human, the alkaline phosphatase activity in rat serum does not increase as pregnancy progresses to term. In an endeavour to establish if the rat placental enzyme is present in the serum of the pregnant rat, the characteristics of the enzyme in both placental extracts and serum of non-pregnant and 1-, 2- and 3-week pregnant rats were studied using the techniques of heat stability at 56°, gel filtration through Sephadex columns, disc gel electrophoresis, and L-phenylalanine inhibition. The presence of rat placental alkaline phosphatase in maternal serum could not be positively demonstrated by any of these procedures, suggesting that rat placental alkaline phosphatase does not enter the maternal serum.