Endocrine control of TAG lipase in the fat body of the migratory locust, Locusta migratoria

Aspects of the role and activation of the enzyme triacylglycerol lipase (TAG lipase) in the fat body of the migratory locust Locusta migratoria were investigated. TAG lipase is under the hormonal control of the three endogenous adipokinetic peptides of the migratory locust, Locmi-AKH-I, Locmi-AKH-II and Locmi-AKH-III. Injection of low doses (5-10 pmol) of each peptide causes an increase in lipase activity. The activation of lipase is time dependent: an elevated activity was recorded 15 min after injection of 10 pmol Locmi-AKH-I and maximum activation was reached after 45-60 min. The activation of TAG lipase is also dose-dependent. Doses of 2 pmol of each Locmi-AKH had no effect, whereas 5 pmol caused a significant activation. Maximum activation is reached with a dose of 10 pmol. Analogues of the second messengers cAMP (cpt-cAMP) and IP(3) (F-IP(3)) both activate the enzyme glycogen phosphorylase whereas only cpt-cAMP, but not F-IP(3), activates TAG lipase; cpt-cAMP elevates the lipid levels in the haemolymph. Activation of lipase is specific to the three endogenous AKH peptides: 5 pmol of the endogenous peptide Locmi-HrTH and 10 pmol of corazonin failed to activate lipase. High doses of octopamine did not activate lipase nor did they elevate the lipid concentration in the haemolymph. TAG lipase is stimulated by flight activity but activation is slower than that of glycogen phosphorylase: after 30 min of flight or after 5 min of flight plus 1h of subsequent rest, activity of TAG lipase is increased, but not immediately after 5 min of flight. In contrast, glycogen phosphorylase is activated significantly after 5 min of flight. These activation patterns of the two enzymes mirror-image the concentration of their substrates in the haemolymph: there is a significant decrease in the concentration of carbohydrates after 5 min of flight, whereas no change of the concentration of lipids can be measured after such short time of flight activity; however, a subsequent rest period of 1h is sufficient to increase the lipid concentration.     

Characterization of the peptidases of Calliphora: Many features allow the utilization of small peptides as an amino acid reservoir

Electrophoretic separation of whole flies and of haemolymph indicates the presence of four peptidases, named dipeptidase A, B and C (Dip A, B and C) and leucine amino peptidase (LAP) after enzymes of similar substrate specificities and electrophoretic mobilities found in Drosophila (Laurie-Ahlberg, Biochem. Genet.20, 407–424, 1982; Walker et al., Insect Biochem.10, 535–541, 1980). Prominent in both tissues and haemolymph, dipeptidase A and B together hydrolyse a variety of dipeptides in vitro and probably most of the fly's small peptide component in vivo. Though Dip A and Dip B hydrolyse many of the same substrates, their activities differ in at least several respects. Dip A's K_ms are higher than Dip B's K_ms and hence in vivo the two enzymes together are likely to provide peptide hydrolysis through a wide range of substrate concentration. Dip A's unique hydrolyses are of peptides with biosynthesized amino acids in the N-terminal position and Dip B's unique hydrolyses are of peptides with essential amino acids in the N-terminal position. Dip B, but not Dip A, is inhibited by free amino acid. It is inhibited non-competitively and most strongly by essential amino acids. In cell-free haemolymph Dip B's activity is more stable than Dip A's. The accumulation and maintenance of small peptides in times of dietary sufficiency and the utilization of the small peptides as a source of amino acid in times of dietary scarcity (Collett, Insect Biochem.6, 179–185, 1976a; J. Insect Physiol.22, 1433–1440, 1976b) may be attributed to these features.     

Carbohydrate metabolism in the stick insect, Carausius morosus

Age-related changes in some parameters related to carbohydrate metabolism in the stick insect, Carausius morosus, were investigated during the 6th instar and up to day 37 of adult life. Total haemolymph carbohydrate concentration and the fat body glycogen content are low and may be related to the low activity of this insect. Trehalose constitutes about 75–80% of the total blood carbohydrate pool. During the moult, total blood carbohydrate, fat body glycogen and haemolymph volume, decrease while glycogen phosphorylase activity of the fat body is slightly activated. The effects are brought about mainly by reduced feeding activity, but may also be influenced by the shedding and replacement of the cuticle. During starvation, blood homeostasis is maintained at the expense of fat body glycogen via an activation of phosphorylase. During reproduction, although no dramatic changes in fat body glycogen levels occur, blood carbohydrates are maintained and fat body phosphorylase is slightly activated. The possibility is discussed that during moulting and reproduction, blood sugar homeostasis is maintained by a hormonal mechanism controlling glycogen phosphorylase. No circadian rhythm in any parameter investigated is observed.     

Elucidation of the primary structures of the cockroach hyperglycaemic hormones I and II using enzymatic techniques and gas-phase sequencing

ABSTRACT. Two peptides, HGHI and HGHII, both inducing hyperglycaemia and activation of fat body glycogen phosphorylase can be isolated from the corpora cardiaca of the American cockroach, Periplaneta americana , using high-performance liquid chromatography. Both peptides are N-terminally blocked by a pyroglutamate residue and are thus not available for sequencing methods using the Edman degradation as this technique requires a free N-terminus. The blocked peptides were treated with pyroglutamate aminopeptidase to cleave the pyroglutamate residue, and the C-terminus of each peptide is also blocked and neither molecule can be cleaved by carboxypeptidase A. The following sequences for hyperglycaemic hormones HGHI and HGHII have been revealed using gas-phase sequencing.
HGHI pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂
HGHII pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂
Both peptides show remarkable similarities to locust adipokinetic hormones I and II and prawn red-pigment concentrating hormone, and are identical to two myotropic peptides MI and MII (O'Shea et al. , 1984; Witten et al. , 1984) and two cardioactive and hyperglycaemic peptides CC-1 and CC-2 (Scarborough et al. , 1984), respectively, also isolated from the corpora cardiaca of P. americana.     

Phosphorylation of the glycogen-binding subunit of protein phosphatase-1G by cyclic-AMP-dependent protein kinase promotes translocation of the phosphatase from glycogen to cytosol in rabbit skeletal muscle

The glycogen-bound form of protein phosphatase-1 (termed protein phosphatase-1G) is composed of the catalytic (C) subunit complexed to a glycogen-binding (G) subunit that anchors the enzyme to glycogen [Str?lfors et al. (1985) Eur. J. Biochem. 149, 295-303]. Incubation of purified protein phosphatase-1G with cyclic-AMP-dependent protein kinase and MgATP, which leads to stoichiometric phosphorylation of the G-subunit [Caudwell et al. (1986) FEBS Lett. 194, 85-90], was found to promote the release of the phosphatase from glycogen; similar observations were made using glycogen-protein particle preparations. An intravenous injection of adrenaline decreased protein phosphatase-1 activity associated with the glycogen-protein particles by 50% with a corresponding increase in the amount present in the cytosol. By contrast, adrenaline did not affect the distribution of glycogen synthase or glycogen phosphorylase which remained entirely bound to glycogen in these experiments. The specific release of protein phosphatase-1 from glycogen may facilitate its inactivation by inhibitor-1 in the cytosol, thereby preventing dephosphorylation of the glycogen metabolising enzymes. Translocation of protein phosphatase-1 may represent a novel mechanism for the activation of glycogenolysis and inhibition of glycogen synthesis by adrenaline.     

Unique features of the structural model of 'hard' cuticle proteins: implications for chitin-protein interactions and cross-linking in cuticle

Cuticular proteins are one of the determinants of the physical properties of cuticle. A common consensus region (extended R&R Consensus) in these proteins binds to chitin, the other major component of cuticle. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Subsequently, we verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). Homology modelling of soft (RR-1) cuticular proteins using bovine plasma retinol binding protein (RBP) as a template revealed an antiparallel beta-sheet half-barrel structure as the basic folding motif (Hamodrakas et al., Insect Biochem. Molec. Biol. 32 (2002) 1577). The RR-2 proteins characteristic of hard cuticle, have a far more conserved consensus and frequently more histidine residues. Extension of modelling to this class of consensus, in this work, reveals in detail several unique features of the proposed structural model to serve as a chitin binding structural motif, thus providing the basis for elucidating cuticle's overall architecture and chitin-protein interactions in cuticle.     

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.     

Development of imaginal competence to adipokinetic hormone in Locusta: lipid and carbohydrate levels, and glycogen phosphorylase activity in precocene-induced adultiforms

Abstract. Adipokinetic responses to injection of synthetic adipokinetic hormone I (sAKH) were investigated in precocene-induced fifth-instar adultiforms and in chemically allatectomized but morphogenetically normal adults of Locusta migratoria (L.). The results were compared with data on normal fifth (=last)-instar nymphs and normal adults. Chemical allatectomy did not affect the resting level of lipid in the haemolymph, nor the slight decrease of haemolymph carbohydrate concentration induced by sAKH. The effects of chemical allatectomy on sAKH-induced hyperlipaemia, the resting level of haemolymph carbohydrate, total glycogen phosphorylase specific activity, as well as sAKH-induced phosphorylase activation in the fat body, were mostly minor and probably indirect. The concentrations of lipid and carbohydrate in the haemolymph, and sAKH-induced activation of fat body glycogen phosphorylase were more or less similar in normal fifth-instar nymphs, normal adults and fifth-instar adultiforms. Thus, precocious metamorphosis did not affect markedly those parameters which show no or slight changes during normal metamorphosis. In contrast, parameters which change substantially during normal metamorphosis (sAKH-induced hyperlipaemia, sAKH-induced changes in haemolymph carbohydrate level and total glycogen phosphorylase activity) showed similar changes in the course of precocious metamorphosis; the values for fifth-instar adultiforms were similar to those obtained for normal adults, but differed markedly from those found in normal fifth-instar nymphs. Thus, accelerated (precocious) morphological adult development is strongly correlated with accelerated imaginal target competence to AKH and with relevant physiological development.     

Control of phosphorylase kinase in the isolated glycogen particle by Ca2+-Mg2+ synergistic activation and cAMP-dependent phosphorylation

The isolated glycogen particle provides a means to examine the regulation of glycogen metabolism with the components organized in a functional cellular complex. With this system, we have studied the control of phosphorylase kinase activation by Ca2+ and cAMP. Contrary to a previous report (Heilmeyer, L. M. G., Jr., Meyer, F., Haschke, R. H., and Fisher, E. H. (1980) J. Biol. Chem. 245, 6649-6656), phosphorylase kinase became activated during incubation of the glycogen particle with MgATP2- and Ca2+. Part of this activation could be attributed to the action of the cAMP-dependent protein kinase; however, it was not possible to quantitatively correlate activation with phosphorylation in the presence of Ca2+ and Mg2+ due to a large, but uncertain, contribution of synergistic activation caused by these ions. This latter activation had properties similar to those described by King and Carlson (King, M. M., and Carlson, G. M. (1980) Arch. Biochem. Biophys. 209, 517-523) with the purified enzyme, and its occurrence also explains why phosphorylase kinase activation in the glycogen particle was not observed previously. The cAMP-dependent activation of phosphorylase kinase in the glycogen particle has been characterized. It occurred in a similar manner when either the cAMP-dependent protein kinase or cAMP was added, thus indicating that the phosphorylation sites of phosphorylase kinase complexed in the glycogen particle were accessible to endogenous or exogenous enzyme. In the glycogen particle, both the alpha and beta subunits were phosphorylated by the cAMP-dependent protein kinase, but the alpha subunit dephosphorylation appeared to be preferentially regulated by Ca2+. The activity of phosphorylase kinase in the glycogen particle is regulated by the phosphorylation of both the alpha and beta subunits.     

Beetles' choice--proline for energy output: control by AKHs

Many beetle species use proline and carbohydrates in a varying ratio to power flight. The degree of contribution of either fuel varies widely between species. In contrast, dung beetle species investigated, thus far, do not have any carbohydrate reserves and rely completely on proline to power energy-costly activities such as flight and, probably, walking and ball-rolling. While the fruit beetle, Pachnoda sinuata, uses proline and carbohydrates equally during flight, proline is solely oxidised during endothermic pre-flight warm-up, as well as during flight after prolonged starvation. Thus, proline seems to be the essential fuel for activity in beetles, even in flightless ones and in those that use proline in combination with carbohydrates; the latter can be completely substituted by proline in certain circumstances. It is apparent from the rapid decline of energy substrates in flight muscles and haemolymph after the onset of flight that mobilisation of stored fuels of the fat body is necessary for prolonged flight periods. This task is performed by AKH-type neuropeptides. In beetles, like in other insects, these peptides mobilise glycogen via activation of glycogen phosphorylase. They also stimulate proline synthesis from alanine and acetyl-CoA in the fat body. Acetyl-CoA is derived from the beta-oxidation of fatty acids and we propose that the neuropeptides activate triacylglycerol lipase.     

The newly discovered insect order Mantophasmatodea contains a novel member of the adipokinetic hormone family of peptides

A novel member of the AKH/RPCH family of peptides has been identified from the corpus cardiacum of an, as yet, unidentified species of the newly discovered insect order Mantophasmatodea from Namibia. The primary sequence of the peptide, which is denoted Manto-CC, was deduced from multiple MS(N) electrospray mass data to be an octapeptide: pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp amide. Synthetic Manto-CC co-elutes on reversed-phase HPLC with the natural peptide from the gland of the insect. Interestingly, Manto-CC is structurally very closely related (only one point mutation) to the AKH/RPCH peptides previously identified in mostly more basal insect taxa (Odonata, Blattodea, and Ensifera) and in Crustacea, the sister group of insects, whereas larger structural differences occur with peptides from Mantodea and Phasmatodea, which are thought to be close relatives of Mantophasmatodea. Functionally, Manto-CC may be employed to activate glycogen phosphorylase to mobilize carbohydrates.     

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.     

The amino acid sequence of the alpha chain of the major haemoglobin of the rat (Rattus norvegicus).

1. A partial amino acid sequence of the alpha chain from the rat (Wistar, Rattus norvegicus) major haemoglobin is reported. The soluble tryptic peptides prepared from aminoethylated alpha-globin were separated by peptide 'mapping'. Sequencing of the tryptic peptides was carried out by the dansyl-Edman method and by the overlapping of smaller peptide fragments derived from secondary enzymic digestion. The insoluble 'core' peptides were further digested with chymotrypsin, thermolysin and pepsin to give smaller soluble peptides for sequencing. The tryptic peptides were ordered on the basis of their homology with the corresponding peptides of human alpha chain. 2. The proposed sequence is compared with that obtained by using an automated sequencer [Garrick et al. (1975) Biochem. J. 149, 245-258]. The differences in sequence resulting from the two methods are discussed. 3. It is suggested that the externally situated cysteine (residue 13) is responsible for the observed inhibition of crystallization of rat haemoglobin at alkaline pH. 4. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50047 (9 pages) at the British Library (Linding Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from which copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.     

Isolation and characterization of cyclic AMP-independent glycogen synthase kinase from rat skeletal muscle

Glycogen synthase kinase was isolated from rat skeletal muscle. This kinase, which is cyclic nucleotide-independent and calcium-independent, was separated from phosphorylase kinase, cyclic AMP-dependent protein kinase and phosvitin kinase by phosphocellulose chromatography. Gel filtration on Sephadex G-100 resolved the glycogen synthase kinase into two fractions with apparent molecular weights of 68 000 (peak I) and 52 000 (peak II). This step also separated glycogen synthase kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase, which had an apparent molecular weight of 39 000. Peak II glycogen synthase kinase activity was not affected by the addition of calcium, EGTA or a number of cyclic nucleotides. In addition to ATP, dATP would serve as the phosphate donor. Other trinucleotides tested were either poor or ineffective substrates. Activity was about 5-fold greater with Mg2+ than with Mn2+. Glycogen stimulated activity about 25%. Modifications of the methods of Soderling et al. ((1970) J. Biol. Chem. 245, 6317--6328) and Nimmo et al. ((1976) Eur. J. Biochem. 68, 21--30) were developed for purification of glycogen synthease (UDPglucose:glycogen 4-alpha D-glucosyltransferase, EC 2.4.1.11) to specific activity of 35 units/mg of protein. Using this preparation of glycogen synthase as substrate, the phosphorylation and inactivation catalyzed by glycogen synthase kinase was compared to that catalyzed by cyclic AMP-dependent protein kinase or phosphorylase kinase. Each of the kinases had different specificities for phosphorylation sites on glycogen synthase.     

Carbohydrate metabolism in Manduca sexta during late larval development

The carbohydrate metabolism in Manduca sexta underwent significant changes during late larval development. Approximately 10% of fat body glycogen phosphorylase was active during the feeding period of the 5th instar, pharate-pupal development and after the pupal moult; it is concluded that glycogen synthesis prevailed. During the last larval and the pupal moult, as well as the wandering stage the percentage of active phosphorylase was significantly increased indicating that fat body glycogen stores were broken down to supply substrates to meet the demands of carbohydrate metabolism. In the course of the last larval moult and the wandering stage the fat body glycogen content decreased significantly from about 300 to about 200 μg mg⁻¹ dry mass substantiating that carbohydrates were released from the fat body. Prior to phosphorylase activation, the concentrations of total haemolymph sugars decreased significantly from about 12 to about 6 mg trehalose equivalents ml⁻¹ (last larval moult) and from about 18 to about 12 mg ml⁻¹ (wandering stage), and increased again slightly when phosphorylase was activated. The haemolymph glucose concentration decreased significantly from about 1.1 to 0.3 mg ml⁻¹ (last larval moult) and in the course of the 5th-instar feeding period from about 1.1 to 0.2 mg ml⁻¹, and remained at this level until the beginning of adult development. The amount of chitosan present in the cuticle increased steadily during the feeding period of the 5th instar from about 10 to 110 mg. It appears that fat body glycogen might be broken down during the last larval moult and the wandering period to provide substrates for chitin synthesis. A dramatic decrease in the amount of chitosan was observed prior to the pupal moult.     

Effects of part sequences of human growth hormone on in vivo hepatic glycogen metabolism in the rat.

Acute effects of two part sequences of human growth hormone on the in vivo activity levels of hepatic glycogen synthase and glycogen phosphorylase were examined. The peptide corresponding to residues 6 to 13 of the hormone (hGH 6--13) decreased the percentage of phosphorylase in the active form without affecting synthase activity. This action was indirect and dependent upon insulin. The peptide hGH 177--191 decreased the level of the active form of synthase without affecting phosphorylase activity. This effect was also observed with analogous peptides containing the sequence hGH 178--191 (i.e., hGH 172--191 and hGH 178--191), whereas the peptide hGH 179--191 was inert. The onset of these effects was rapid, and maximum changes in activity were produced in 5 min by both peptides. The effect for hGH 177--191 was short-lived, and synthase activity had returned to normal levels by 15 min, whereas the action of hGH 6--13 was of longer duration and was still quite marked at 60 min. Both peptides showed a linear dependence of response to the log dose of peptide injected over the range 0.1--250 microgram hGH 6--13 per kg body weight and 0.05--25 microgram hGH 177--191 per kg body weight. Hepatic 3',5'-cyclicadenylic acid levels were not affected by either peptide. Incorporation of glycerol carbon into liver glycogen was increased by hGH 6--13 and decreased by hGH carbon into liver glycogen was increased by hGH 6--13 and decreased by hGH 177--191. This is discussed in terms of a futile cycle between glycogen and hexose phosphate in the liver, as the basis for a control mechanism for hepatic glycogen metabolism. The present observations are consistent with other in vivo and in vitro actions of these and related peptides.     

Comparison of calcium-activated, cyclic nucleotide-independent protein kinase and adenosine 3':5'-monophosphate-dependent protein kinase as regards the ability to stimulate glycogen breakdown in vitro.

A cyclic nucleotide-independent protein kinase, which was produced from its proenzyme upon limited proteolysis by a Ca2+-dependent protease (Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., & Nishizuka , Y. (1977) Biochem. Biophys. Res. Commun. 77, 542-550), showed an ability to phosphorylate not only muscle glycogen phosphorylase kinase but also glycogen synthase, resulting in activation and inactivation of the respective enzymes, although the protein kinase was less active than adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase toward glycogen synthase. Available evidence indicates that this new protein kinase shows pleiotropic functions apparently similar to those described for cyclic AMP-dependent protein kinase. Nevertheless, these protein kinases were clearly distinguishable from each other in their response to cyclic nucleotides and susceptibility to protein inhibitor.     

Allatostatins from the retrocerebral complex and antennal pulsatile organ of the American cockroach: structural elucidation aided by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

The occurrence of allatostatins in retrocerebral complexes and antennal pulsatile organs of the American cockroach, Periplaneta americana, was investigated. Previously, molecular cloning of the P. americana allatostatin gene had predicted 14 peptides of this family [Ding et al., Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata. Eur J Biochem 1997;234:737-746], however, only two forms had been identified by peptide isolation procedures [Weaver et al., Identification of two allatostatins from the CNS of the cockroach Periplaneta americana: novel members of a family of neuropeptide inhibitors of insect juvenile hormone biosynthesis. Comp Biochem Physiol 1994;107(C):119-127]. Using an extract of only 200 corpora cardiaca/corpora allata, we have found that at least 11 allatostatins occur in the retrocerebral complex. These peptides were already separated from other substances of the crude extract in the first HPLC step with heptafluorobutyric acid as organic modifier, and subsequently identified by MALDI-TOF mass spectrometry. Moreover, we have demonstrated the occurrence of nearly all allatostatins, including the cleavage product of Pea-AST-2 (LPVYNFGL-NH2), in antennal pulsatile organs of males and females. Allatostatins are predominant neuropeptides in these organs. Additionally, only two other known peptides could be identified in these organs by mass screening: proctolin and leucomyosuppressin. The function of allatostatins in antennal pulsatile organs remains unclear. We assume a release into the hemolymph via the ampullac, which could act as neurohemal release sites. The method described for the identification of allatostatins is a very fast method for neuropeptide screening in neurohemal tissues.     

Role of protein kinase C in the regulation of rat liver glycogen synthase

Rat liver glycogen synthase was phosphorylated by purified protein kinase C in a Ca2+- and phospholipid-dependent fashion to 1-1.4 mol PO4/subunit. Analysis of the 32P-labeled tryptic peptides derived from the phosphorylated synthase by isoelectric focusing and two-dimensional peptide mapping revealed the presence of a major radioactive peptide. The sites in liver synthase phosphorylated by protein kinase C appears to be different from those phosphorylated by other kinases. Prior phosphorylation of the synthase by protein kinase C has no significant effect on the subsequent phosphorylation by glycogen synthase (casein) kinase-1 or kinase Fa, but prevents the synthase from further phosphorylation by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, phosphorylase kinase, or casein kinase-2. Additive phosphorylation of liver glycogen synthase can be observed by the combination of protein kinase C with the former set of kinases but not with the latter. Phosphorylation of liver synthase by protein kinase C alone did not cause an inactivation nor did the combination of this kinase with glycogen synthase (casein) kinase-1 or kinase Fa produce a synergistic effect on the inactivation of the synthase. Based on these findings we conclude that the phorbol ester-induced inactivation of glycogen synthase previously observed in hepatocytes cannot be accounted for entirely by the activation of protein kinase C.     

High and intermediate affinity calmodulin binding domains of the alpha and beta subunits of phosphorylase kinase and their potential role in phosphorylation-dependent activation of the holoenzyme.

Phosphorylase kinase is a calcium-regulated multimeric enzyme of composition (alpha beta gamma delta)4, which contains calmodulin as the integral delta subunit and also is activated further by addition of extrinsic calmodulin. Previous studies by Dasgupta, M., Honeycutt, T., and Blumenthal, D.K. ((1989) J. Biol. Chem. 264, 17156-17163) have identified gamma 302-326 and gamma 342-366 as two calmodulin binding regions. Using peptides that were synthesized based on alpha and beta primary structure and that were predicted to contain the basic amphiphilic alpha-helix motif thought important for calmodulin binding, four additional potential calmodulin binding domains have now been identified: one of high affinity, beta 770-794; two of intermediate affinity, beta 5-28 and beta 920-946; and one with marginally low affinity, alpha 1070-1093. Peptide beta 770-794 was of higher calmodulin affinity than either gamma 302-326 or gamma 342-366; it was of higher affinity than the model synthetic peptide IV defined by O'Neil, K.T., and DeGrado, W.F. ((1990) Trends Biochem. Sci. 15, 59-64); and it is currently the most potent calmodulin-binding peptide so far described. Correlated with their affinity for calmodulin, all six phosphorylase kinase-derived peptides and several other established calmodulin-binding peptides inhibited phosphorylase kinase previously activated by cAMP-dependent phosphorylation, reducing its activity to the level of the nonactivated enzyme. However, these peptides did not inhibit (and some peptides slightly activated) the nonphosphorylated enzyme. Even in the presence of these peptides both activated and nonactivated enzyme remained fully Ca(2+)-dependent. The beta 770-794 peptide has at least a 5-fold greater calmodulin binding affinity than the holo-phosphorylase kinase. This, and its higher affinity for calmodulin than either of the sites on the gamma subunit, raises the possibility that in the native enzyme it may be involved in binding the intrinsic delta subunit. Further, inhibition of activated but not nonactivated enzyme by calmodulin-binding peptides would suggest that the phosphorylation-dependent activation of phosphorylase kinase may be mediated by changes in the binding interactions of the intrinsic calmodulin delta subunit.