Susumu Y. Takahashi

Isolation and some characterization of the prothoracicotropic hormone from Bombyx mori.

The prothoracicotropic hormone (PTTH) was isolated from adult heads of Bombyx mori. Fifty micrograms of pure PTTH was obtained from 648,000 heads through a 15-step purification procedure with a 2 X 10(6)-fold purification and an 8% recovery. Chemical analyses of this PTTH have shown that it is a single-chain peptide consisting of 40-43 amino acid residues (MW, 4330-4740), the N-terminus of which is glycine. As little as 0.1 ng of PTTH elicited adult development in a debrained pupa of Samia cynthia ricini. Five picograms of PTTH directly stimulated the prothoracic glands in vitro so as to enhance ecdysone release. The hemolymph ecdysteroids of brainless Samia pupae that were developed by PTTH injection increased with essentially the same pattern as in developing normal pupae.

Cloning of a Gene Encoding Bombyxin, an Insulin‐Like Brain Secretory Peptide of the Silkmoth Bombyx mori with Prothoracicotropic Activity

A genomic DNA encoding bombyxin, a 5kD brain peptide of the silkmoth Bornbyx mori with prothoracicotropic hormone activity, has been isolated. The nucleotide sequence coding for bombyxin shows high homology with insulin‐gene family members and the overall organization of the preprobombyxin gene is the same as in preproinsulin genes, indicating that bombyxin shares a common ancestral molecule with insulin‐family peptides. The bombyxin gene has no intron contrasting to other members of insulin‐gene family.

Adenylate cyclase system and the hyperglycemic factor in the cockroach, Periplaneta americana

Abstract Adenylate cyclase (EC 4.6.1.1) and cyclic AMP in the fat body of the cockroach, Periplaneta americana , were investigated in relation to the mode of action of the hyperglycemic factor. The adenylate cyclase was activated about 3-fold by the injection of an extract of the corpora cardiaca. Concomitantly with the activation, the cyclic AMP content in fat body was increased nearly twice, preceding the rise of haemolymph trehalose concentration. Injection of theophylline or dibutyryl cyclic AMP induced a sustained hyperglycemia in the cockroach. These results strongly indicate the involvement of adenylate cyclase system in the hyperglycemic action of the corpora cardiaca extract.

Effect of a hyperglycemic factor on haemolymph trehalose and fat body carbohydrates in the American cockroach

Abstract Effects of the hyperglycemic factor on haemolymph trehalose and fat body carbohydrates have been investigated in adult male cockroaches. Injection of an aqueous extract of the corpus cardiacum into intact and beheaded coackroaches caused a rapid and sustained rise in the haemolymph trehalose level. A linear dose-response relation was obtained over a wide range of doses. Concomitant with the increase in haemolymph trehalose, fat body glycogen decreased, accompanied by a temporary increase in fat body trehalose. Injection of distilled water or saline, or anaesthetization itself caused a significant rise in haemolymph trehalose level, but the effect was temporary. These results are discussed in relation to the regulation of carbohydrate metabolism in the insect.

Multiple protein kinases in the American cockroach, Periplaneta americana

Abstract Cyclic nucleotide-dependent protein kinases were extracted and partially purified from the fat body tissues of the American cockroach, Periplaneta americana . Fat body tissues from the adult male contained two cAMP-dependent protein kinases and those from females contained three species of cyclic nucleotide-dependent protein kinases, namely two cAMP-dependent protein kinases and a protein kinase whose cyclic nucleotide-dependency has not yet been determined. Fat body tissues from larvae contained two cAMP-dependent protein kinases and an independent kinase. The preliminary characterization of these kinases is given and their biological roles are discussed.

Phosphorylation of vitellin of the silkworm,Bombyx mori: identification of vitellin phosphorylation and protein kinase in the ovary

Silkworm vitellin was radioactively labeled both in vivo upon injection of [32P]phosphate and in vitro upon incubation of tissue extracts with [γ-32P]ATP. Phosphorylation of vitellin was also demonstrated in the cultured ovary. [32P]phosphate groups were added to serine residues of the peptide, indicating that silkworm vitellin is a phosphoprotein. SDS slab gel electrophoresis revealed three major labeled peptides in cases of in vitro and in vivo labeling methods. Two of them were identified, with specific anti-vitellin serum, as vitellin subunits, whereas another labeled peptide has not yet been characterized. The latter protein was not found in the hemolymph and fat body, suggesting that it is specifically localized in the ovary but immunologically different from vitellin. Protein kinase activities capable of phosphorylating vitellogenin and vitellin were identified in various tissues of the silkworm and the kinases from the ovary were partially characterized.

Trehalases from the male accessory glands of the American cockroach: Developmental changes and the hormonal regulation of the enzymes

Two types of trehalases, designated CM-I and CM-II, were detected in the male accessory gland of the American cockroach and they could be separated by CM-cellulose chromatography (S. Y. Takahashi, S. Higashi, S. Minoshima, M. Ogiso, and K. Hanaoka, 1980, Int. J. Invert. Reprod. 2, 373-381). Trehalase activity in the gland showed a rapid increase after adult emergence. The relative activities of the two enzymes were followed separately during adult development. The appearance of CM-II preceded that of CM-I during adult development. Allatectomy and decapitation of newly molted adults resulted in inhibition of the increase of enzyme activity, and in the allatectomized cockroach, CM-II, which is the major enzyme activity in the gland, was missing. Implantation of the corpora allata as well as application of JH-III and the JH analogs isopropyl-11-methoxy-3,7,11-trimethyl-2,4,-dodecadienoate (ZR-515) and 6,7-epoxy-1-(p-ethylphenoxy)-3,7-dimethyl-2-octene (R-20458) into the decapitated animals restored the enzyme activities. The data suggested that trehalase in the male accessory gland was under the control of the corpora allata. The regulation of trehalase activity in the male accessory gland was discussed with respect to its function.

Studies on the phosphorylation of ovarian proteins from the silkworm, Bombyx mori: Identification of band 2 protein as egg specific protein

Abstract Following in vivo labeling with [ 32 P]o- phosphate , or in vitro phosphorylation with [γ 32 P ]ATP, ovarian proteins were prepared and analyzed by one and two dimensional polyacrylamide gel electrophoresis and subsequent autoradiography. Several 32 P-labeled proteins were evident in ovarian extracts after labeling in vivo . We had previously identified band 1 and 3 as subunits of vitellin (Takahashi, 1983, 1984, 1985), whereas another phosphoprotein (band 2), which was specifically localized in the ovary had not been identified. In the present communication, we used a specific antiserum and identified the band 2 protein as egg specific protein (ESP). The radioactivity in hydrolysates of ESP appeared to be in the form of serine phosphate (15–25%) and inorganic phosphate (75–85%). The 32 P-labelling of ESP in the cultured ovary was selectively enhanced by dibutyryl cAMP. Analysis of proteolytic phosphopeptides by one-dimensional peptide mapping by the procedure of Cleveland et al. (1977) indicated that ESP is phosphorylated at multiple sites in the ovary. In vitro phosphorylation with [γ 32 P]ATP showed that the protein is predominantly phosphorylated in a cAMP-dependent manner in the late developmental stages of the ovary. Protein kinase inhibitor protein from rabbit skeletal muscle was shown to inhibit the phosphorylation, indicating the involvement of cAMP-dependent protein kinases in phosphorylation of ESP. The functional role of the phosphorylation of ESP, however, remains unknown.

Characterization of the guanosine 3′∶5′-monophosphate-dependent protein kinase from silkworm eggs and analysis of the endogenous protein substrates

SummaryIn a previous paper, two types of cyclic nucleotide-dependent protein kinases, namely cGMP dependent “G-kinase” and cAMP dependent “A-kinase”, in silkworm eggs has been reported (Takahashi et al. 1975; Takahashi 1976). One of these, G-kinase, has now been purified 2400-fold by means of ammonium sulfate fractionation, chromatography on hydroxylapatite, DEAE cellulose, and gel filtration.Some of the properties of the enzyme are described. The enzyme is highly dependent on cGMP; it is strongly inhibited by GTP in a noncompetitive manner not only for ATP but also for cGMP. GTP was found to be highly inhibitory on G-kinases from various tissues of the silkworm, but did not inhibit the A-kinase.Incubation of the egg extract with [γ-32P]ATP and Mg2+ led to the formation of three major32P-labelled proteins, with molecular weights of 42.000, 70.000 and 180.000 as analyzed by SDS polyacrylamide gel electrophoresis. Two of them corresponded to the subunits of vitellin.The silkworm vitellin was effectively phosphorylated both by the highly purified G-kinase and by the A-kinase. It is concluded that the G-kinase is involved in the phosphorylation of vitellin in developing silkworm eggs.

Polyol dehydrogenases from silkworm eggs: Purification and properties

Abstract Extract from silkworm eggs contains two kinds of nicotinamide adenine dinucleotide phosphate (NADP)-specific polyol dehydrogenases, which catalyse the dehydrogenation of various polyols as well as the reduction of a number of sugars to the corresponding polyol. These are designated as polyol dehydrogenase-I and -II. The purified polyol dehydrogenase-I catalyses the reduction of glucose, xylose, erythrose and d,l -glyceraldehyde but does not catalyse the reduction of the phosphate esters of sugars (except ribose-5-phosphate, R-5-p). Polyol dehydrogenase-II catalyses the reduction of glucose-6-phosphate (G-6-P) as well as xylose, erythrose and d,l -glyceraldehyde, but does not catalyse the reduction of hexoses (except galactose). The molecular weights of polyol dehydrogenase-I and -II were determined to be 56,000 and 76,000 respectively by gel filtration. The roles of these dehydrogenases are discussed in relation to the metabolic pathway which leads to the accumulation of sorbitol and glycerol in the diapausing silkworm eggs.