The cardiac sarcoplasmic reticulum-glycogenolytic complex A possible effector site for cyclic

Cardiac sarcoplasmic reticulum-glycogenolytic complex, isolated as a single peak on sucrose density gradient, may function as a “compartmented” effector site for cyclic AMP resulting in modulation of both glycogenolysis and calcium transport. The conversion of phosphorylase b to a is stimulated by ATP and inhibited by protein kinase inhibitor. Cyclic AMP alone stimulated neither phosphorylase b to a conversion nor calcium uptake. An inhibitor of adenylate cyclase depressed both calcium uptake and phosphorylase activation and both functions were subsequently stimulated by micromolar concentrations of cyclic AMP. Endogenous phosphorylation of sarcoplasmic reticulum was also inhibited by adenylate cyclase inhibitor and the inhibition was reversed by cyclic AMP. These results suggest that the sarcoplasmic reticulum of cardiac muscle is an internal effector site for cyclic AMP which may regulate both calcium and metabolism. It appears that cyclic AMP formation in vitro is not the rate-controlling step in the activation sequence.     

Regulation of the Crustacean Mandibular Organ

The crustacean mandibular organ (MO) produces methyl farnesoate(MF), a juvenile hormone-related compound thought to have rolesin crustacean reproduction and development. Therefore, the controlof MF production by the MO has been of considerable interest.Current evidence indicates that the MO is negatively regulatedby peptides present in the eyestalk (MO inhibiting factor, MO-IH).Several eyestalk neuropeptides have been identified that inhibitMF synthesis by MO incubated in vitro. The amino acid sequencesof these MO-IH peptides are similar to peptides in the crustaceanhyperglycemic hormone (CHH) family of neuropeptides. In addition,there appears to be a compound in the eyestalk that lowers hemolymphlevels of MF in vivo but does not directly affect the MO invitro. The inhibition of MF synthesis by eyestalk peptides involvesthe inhibition of farnesoic acid O-methyl transferase, the lastenzyme in the MF biosynthetic pathway. The activity of thisenzyme is affected by cyclic nucleotides, suggesting that thesecompounds may be involved in the signal transduction pathwaymediating the effects of MO-IH.     

Proteomics and signal transduction in the crustacean molting gland

Regulation of the molting cycle in decapod crustaceans involves2 endocrine organs: the X-organ/sinus gland (XO/SG) complexlocated in the eyestalk ganglia and the Y-organ (YO) locatedin the cephalothorax. Two neuropeptides [molt-inhibiting hormone(MIH) and crustacean hyperglycemic hormone (CHH)] are producedin the XO/SG complex and inhibit ecdysteroidogenesis in theYO. Thus, YO activation is induced by eyestalk ablation (ESA),which removes the primary source of MIH and CHH. Cyclic nucleotides(cAMP and cGMP) and nitric oxide (NO) appear to mediate neuropeptidesuppression of the YO. Proteomics was used to identify potentialcomponents of signal transduction pathways ("targeted" or cell-mapproteomics) as well as assess the magnitude of protein changesin response to activation ("global" or expression proteomics)in the tropical land crab, Gecarcinus lateralis. Total proteinsin YOs from intact and ES-ablated animals were separated bytwo-dimensional gel electrophoresis and expression profileswere assessed by image analysis and gene clustering software.ESA caused a >3-fold increase in the levels of 170 proteinsand >3-fold decrease in the levels of 89 proteins; a totalof 543 proteins were quantified in total YO extracts. ESA inducedsignificant changes in the levels of 3 groups of proteins elutingfrom a phosphoprotein column and detected with phosphoproteinstaining of two-dimensional gels;     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.     

Cyclic AMP stimulation of membrane phosphorylation and Ca2+-activated,Mg2+-dependent ATPase in cardiac sarcoplasmic reticulum

Ca²⁺-activated ATPase (EC 3.6.1.15) in canine cardiac sarcoplasmic reticulum was stimulated 50–80% by cyclic adenosine 3′ : 5′-monophosphate. The relationship of this stimulation to cyclic AMP-dependent membrane phosphorylation with phosphoester bands was studied. Cyclic AMP stimulation of ATPase activity was specific for Ca²⁺-activated ATPase and was half-maximal at about 0.1 μM which is similar to the concentration required for half-maximal stimulation of membrane phosphorylation by endogenous cyclic AMP-stimulated protein kinase (EC 2.7.1.37). Cyclic AMP stimulation of Ca²⁺-activated ATPase was calcium dependent and maximal at calculated Ca²⁺ concentrations of 2.0 μM. Cyclic AMP-dependent Ca²⁺-activated ATPase correlated well with the cyclic AMP-dependent membrane phosphorylation of which 80% was 20 000 molecular weight protein identified by sodium dodecyl sulfate discontinuous polyacrylamide gel electrophoresis. In trypsin-treated microsomes, cyclic AMP did not stimulate Ca²⁺-activated ATPase or phosphorylation of the 20 000 molecular weight membrane protein. An endogenous calcium-stimulated protein kinase (probably phosphorylase b kinase) with an apparent K_m for ATP of 0.21–0.32 mM was present and appeared to be involved in the cyclic AMP-dependent phosphorylation of the 20 000 molecular weight protein which was calcium dependent. Cyclic guanosine 3′ : 5′-monophosphate did not inhibit any of the stimulatory effects of cyclic AMP. These data suggest that the cyclic AMP stimulation of Ca²⁺-activated ATPase in cardiac sarcoplasmic reticulum is mediated by the 20 000 molecular weight phosphoprotein product of a series of kinase reactions similar to those activating phosphorylase b.     

Activation of membrane guanylate cyclase by an invertebrate peptide hormone.

Peptide hormones can stimulate cyclic GMP synthesis through either of two general mechanisms: some peptides activate the cytoplasmic form of guanylate cyclase via a coupling factor called EDRF (endothelium-derived relaxation factor), while others activate the membrane form by interacting directly with an extracellular binding domain of the cyclase molecule itself. We have investigated the mechanism(s) by which crustacean hyperglycemic hormone (CHH), a neuropeptide that regulates energy metabolism in crustaceans, elevates cyclic GMP levels in lobster muscle. Phosphodiesterase inhibitors potentiate the response in intact tissue. This indicates that the primary effect of the peptide is to activate a cyclase rather than inhibit a phosphodiesterase. Methylene blue, a specific inhibitor of the EDRF pathway, does not block the actions of CHH. In addition, nitroprusside, an agent that directly activates the EDRF pathway in vertebrate animals, does not activate guanylate cyclase either in intact or homogenized lobster muscle. This indicates that the EDRF pathway, although prominent in vertebrate muscle, is not found in crustaceans and further suggests that the membrane cyclase is the most likely target of CHH. Membrane and soluble cyclases can be isolated from homogenates of lobster muscle (in a 3.5:1 ratio), and both are stimulated by Mn2+ and inhibited by Ca2+. CHH has no effect on the soluble enzyme. Coupling of CHH receptors to the particulate cyclase, however, remains intact in isolated membranes, thus providing a new model system for the study of receptor/cyclase interactions.     

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine.The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.     

Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti. Functional analysis by double-stranded RNA interference technique

The crustacean hyperglycemic hormone (CHH) plays an important role in the regulation of hemolymph glucose levels, but it is also involved in other functions such as growth, molting and reproduction. In the present study we describe the first CHH family gene isolated from the Atlantic Ocean shrimp Litopenaeus schmitti. Sequence analysis of the amplified cDNA fragment revealed a high nucleotide sequence identity with other CHHs. Northern blot analysis showed that the isolated CHH mRNA from L. schmitti is present in the eyestalk but not in muscle or stomach. We also investigated the ability of dsRNA to inhibit the CHH function in shrimps in vivo. Injection of CHH dsRNA into the abdominal hemolymph sinuses resulted in undetectable CHH mRNA levels within 24 h and a corresponding decrease in hemolymph glucose levels, suggesting that functional gene silencing had occurred. These findings are the first evidence that dsRNA technique is operative in adult shrimps in vivo.     

Glycogen phosphorylase kinase deficiency: a survey of enzymes in phosphorylase activating system

A four year-old Japanese boy with hepatomegaly and hypoglycemia has low activity of hepatic phosphorylase. A survey of enzymes involved in the phosphorylase activating system has revealed that liver phosphorylase kinase is deficient although adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase and total phosphorylase measured in a mixture supplemented by rabbit muscle phosphorylase kinase show normal activities. The hormone receptor as well as adenyl cyclase system appears to be normal since cyclic AMP increases immediately after intravenous injection of glucagon. His muscle phosphorylase activating system is normal.     

Transient changes in cyclic AMP and in the enzymic activity of protein kinase and phosphorylase during the cardiac cycle in the canine myocardium and the effect of propranolol

Summary Oscillation of cyclic AMP and in the activity ratio of cyclic AMP-dependent protein kinase and of glycogen phosphorylase with the cardiac cycle were demonstrated in the canine heart in situ. For tissue sampling an ECG (R-wave)-triggered, automatically working push-freeze-drill apparatus was developed which allows intraventricular cryobiopsies from the left ventricular muscle of anaesthetized open-chest dogs. The nucleotide cyclic AMP oscillated with the cardiac cycle during normal working condition, the higher cyclic AMP level occuring during systole. Cyclic GMP was assayed to be without oscillatory changes during the contraction-relaxation cycle. The rise in the activity ratio of protein kinase was found to coincide with the maximum in the level of cyclic AMP. Propranolol pretreatment prevents the transient in the level of the nucleotide as well as in the activity ratio of the kinase indicating i) a causal relationship between these changes and ii) a neurohumoral, beat-to-beat regulation by catecholamines released from the sympathetic nerve endings within the heart. Contrary the activity ratio of phosphorylase retains its transient changes during the cardiac cycle in the presence of propranolol, indicating a Ca-mediated activation of phosphorlase kinase during the contraction process.     

The possible role of cyclic AMP in the control of genetic tumor induction

Young seedlings of the tumor-prone amphiploid Nicotiana suaveolensx N. langsdorffii, grown aseptically on nutrient medium, weretreated with 1x10^–2 M cyclic adenosine 3': 5'-monophosphate(cyclic AMP). The incidence of tumor formation was scored atregular intervals subsequent to exposure. Cyclic AMP causeda significant reduction in the rate of tumor formation. In addition,untreated plants grown on nutrient medium were harvested atregular intervals after the seeds had been sown. Cyclic AMPwas extracted, partially purified, and assayed radioimmunologically.The endogenous level of cyclic AMP in stem tissue was highestin young seedlings, rapidly fell reaching a low point in 18day-old plants, and eventually leveled off. The presence ofindoleacetic acid (IAA) in the growth medium at a final concentrationof 2x10^–5 M prevented the decline in cyclic AMP that occurredin seedlings grown on unsupplemented medium. (Received May 21, 1976; )     

The cardiac sarcoplasmic reticulum-glycogenolytic complex. A possible effector site for cyclic AMP.

Cardiac sarcoplasmic reticulum-glycogenolytic complex, isolated as a single peak on sucrose density gradient, may function as a "compartmented" effector site for cyclic AMP resulting in modulation of both glycogenolysis and calcium transport. The conversion of phosphorylase b to a is stimulated by ATP and inhibited by protein kinase inhibitor. Cyclic AMP alone stimulated neither phosphorylase b to a conversion nor calcium uptake. An inhibitor of adenylate cyclase depressed both calcium uptake and phosphorylase activation and both functions were subsequently stimulated by micromolar concentrations of cyclic AMP. Endogenous phosphorylation of sarcoplasmic reticulum was also inhibited by adenylate cyclase inhibitor and the inhibition was reversed by cyclic AMP. These results suggest that the sarcoplasmic reticulum of cardiac muscle is an internal effector site for cyclic AMP which may regulate both calcium and metabolism. It appears that cyclic AMP formation in vitro is not the rate-controlling step in the activation sequence.     

The rate of calcium uptake into sarcoplasmic reticulum of cardiac muscle and skeletal muscle. Effects of cyclic AMP-dependent protein kinase and phosphorylase b kinase.

Calcium transport into sarcoplasmic reticulum fragments isolated from dog cardiac and mixed skeletal muscle (quadriceps) and from mixed fast (tibialis), pure fast (caudofemoralis) and pure slow (soleus) skeletal muscles from the cat was studied. Cyclic AMP-dependent protein kinase and phosphorylase b kinase stimulated the rate of calcium transport although some variability was observed. A specific protein kinase inhibitor prevented the effect of protein kinase but not of phosphorylase b kinase. The addition of cyclic AMP to the sarcoplasmic reticulum preparations in the absence of protein kinase had only a slight stimulatory effect despite the presence of endogenous protein kinase. Cyclic AMP-dependent protein kinase catalyzed the phosphorylation of several components present in the sarcoplasmic reticulum fragments; a 19000 to 21 000 dalton peak was phosphorylated with high specific activity in sarcoplasmic reticulum preparations isolated from heart and from slow skeletal muscle, but not from fast skeletal muscle. Phosphorylase b kinase phosphorylated a peak of molecular weight 95000 in all of the preparations. Cyclic AMP-dependent protein kinase-stimulated phosphorylation was optimum at pH 6.8; phosphorylase b kinase phosphorylation had a biphasic curve in cardiac and slow skeletal muscle with optima at pH 6.8 and 8.0. The addition of exogenous phosphorylase b kinase or protein kinase increased the endogenous level of phosphorylation 25-100%. All sarcoplasmic reticulum preparations contained varying amounts of adenylate cyclase, phosphorylase b and a (b:a = 30.1), "debrancher" enzyme and glycogen (0.3 mg/mg protein), as well as varying amounts of protein kinase and phosphorylase b kinase which were responsible for a significant endogenous phosphorylation. Thus, the two phosphorylating enzymes stimulated calcium uptake in the sarcoplasmic reticulum of a variety of muscles possessing different physiologic characteristics and different responses to drugs. In addition, the phosphorylation catalyzed by these enzymes occurred at two different protein moieties which make physiologic interpretation of the role of phosphorylation difficult. While the role phosphorylation in these mechanisms is complex, the presence of a glycogenolytic enzyme system may be an important link in this phenomenon. The sarcoplasmic reticulum represents a new substrate for phosphorylase b kinase.     

Phosphorylation of synthetic peptides by (32P)ATP and cyclic GMP-stimulated protein kinase.

Cyclic GMP-stimulated protein kinase from pig lung has been shown to phosphorylate synthetic peptides. The rate of phosphorylation was about one order of magnitude higher than that for mixed histones at a comparable concentration, $\text{i.e.}$ 0.1 mM. The peptides represented sites, phosphorylatable by cyclic AMP-stimulated protein kinase, in pyruvate kinase type L from rat liver, calf thymus histone H2B and the α-subunit of rabbit muscle phosphorylase b kinase. The shortest pyruvate kinase peptide that could be phosphorylated at a significant rate by cyclic GMP-stimulated protein kinase was Arg-Arg-Ala-Ser-Val-Ala, which is one amino acid residue longer than the minimal substrate of cyclic AMP-stimulated protein kinase. The apparent K_m was 0.3 mM which is about 10 times higher than that with cyclic AMP-stimulated protein kinase. The K_m was only slightly decreased upon successive extension of the peptide in the N-terminal direction to Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala. Modification of the sequence showed the importance of two adjacent arginyl residues, and substitution of arginine for the C-terminal alanine abolished the measurable activity. Thus, it has been demonstrated that there are both differences and similarities in substrate specificity of the two protein kinases.     

The autosomal form of phosphorylase kinase defficiency in man: Reduced activity of the muscle enzyme

A muscle biopsy from a boy with the autosomal form of phosphorylase kinase deficiency has been analysed. The glycogen content was higher than normal; phosphorylase was mostly in the b form, and the activity of phosphorylase kinase was undetectable at pH 6.8 and reached about 15 % of the mean control value at pH 8.3. The residual activity could be enhanced by trypsin and inhibited by EGTA. Cyclic AMP-dependent and independent protein kinases were normally active.     

Stimulation of gibberellin-induced germination in lettuce seeds by cyclic 3',5'-adenosine monophosphate

Cyclic 3',5'-adenosine monophosphate and sodium dibutyryl cyclic3',5'-adenosine monophosphate had no effect on dark-germinationof light-sensitive lettuce {Lacluca sativa L., var. Grand Rapids)seeds when given alone. Cyclic 3',5'-adenosine monophosphate,however, synergistically enhanced the 3 µ 10^–5 Mgibberellic acid-induced germination, but showed no such effecton kinetin-stimulated germination. 5'-AMP, in contrast to cyclic3',5'-adenosine monophosphate, had no effect on gibberellicacidinduced germination. (Received August 8, 1971; )     

Bioengineering and Application of Novel Glucose Polymers

Abstract

Glucan phosphorylase, branching enzyme, and 4-α-glucanotransferase were employed to produce glucose polymers with controlled molecular size and structures. Linear or branched glucan was produced from glucose-1-phosphate by glucan phosphorylase alone or together with bracnhing enzyme, where the molecular weight of linear glucan was strictly controlled by the glucose-1-phosphate/primer molar ratio, and the branching pattern by the relative branching enzyme/glucan phosphorylase activity ratio. Cyclic glucans were produced by the cyclization reaction of 5-αglucanotransferases and branching enzyme on amylose and amylopectin. Molecular size and structure of cyclic glucan was controlled by the type of enyzyme and substrate chosen and by the reaction conditions. This in vitro approach can be used to manufacture novel glucose polymers with applicable value.     

Activation of glycogen synthetase and inactivation of phosphorylase kinase by the same phosphoprotein phosphatase

Purified rabbit muscle glycogen synthetase D phosphatase inactivates phosphorylase kinase. The inactivation is reversed by cyclic AMP-dependent protein kinase. It is postulated that the synthetase D phosphatase is a general phosphoprotein phosphatase which dephosphorylates proteins that are phosphorylated $\text{in}$$\text{vivo}$ by the cyclic AMP-dependent kinase.     

Significance of Cyclic Nucleotides in Amaranthin Synthesis by Amaranthus caudatus Seedlings

Explants of 72–76 h old Amaranthus caudatus seedlingssynthesize the betalain pigment amaranthin in response to light.Light can be replaced with a cytokinin or a cyclic nucleotidewith an N⁶-substituent. Cyclic 3'5'-AMP shows only weak activityand that only at high unphysiological concentrations. Even cyclic2'3'-AMP, which docs not act as a ‘second messenger’,induces amaranthin synthesis to a greater degree than cyclic3',5'-AMP. But N⁶-monobutyryl-cyclic 3',5'-AMP and N⁶-2'-O-dibutyryl-cyclicAMPshow high activity, higher even than kinetin at its optimumconcentration of 10^–5 M. 2'-O-Monobutyryl-cyclicAMP, onthe other hand, is considerably less active, suggesting thatN⁶-substitution of the adenine ring is responsible for the enhancedactivity. N⁶-Propionyl, butyryl and valeryladenines are allhighly active, indicating that the cyclic monophosphate moietyis unnecessary for this response. All the compounds tested,including cyclic 3',5'-AMP, show additive effects, but thereis no amplification of the response, typical of second messengeraction. Inhibition of amaranthin synthesis imposed by hadacidin, isrelieved by kinetin, DBc AMP, N⁶-monobutyryl-cAMP and N⁶-butyryladenine. Cyclic 3',5'-AMP is weakly active in this regard. Asnatural cytokinins are N⁶-substituted adenine compounds, andas only N⁶-substituted cyclic nucleotides are able to mimicthe effect of cytokinin, it is concluded that these cyclic nucleotidesfunction as cytokinin analogues and not as ‘second messengers’'. Amaranthus caudatus, amaranthin, cytokinins, cyclic nucleotides