Identity of a second type of allatostatin from cockroach brains: an octadecapeptide amide with a tyrosine-rich address sequence.

An octadecapeptide that inhibits juvenile hormone synthesis has been isolated by HPLC from brain-retrocerebral complexes of the cockroach Diploptera punctata. The primary structure of this allatostatin has been elucidated by tandem mass spectrometry: Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu- NH2 (ASB2). The amidated three-residue C terminus of this type B allatostatin is identical to that of four known type A allatostatins, and the preceding three residues show close structural homology. ASB2 has over twice the activity of the type A tridecapeptide Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 (ASA1) in inhibiting juvenile hormone biosynthesis in corpora allata from females in early vitellogenesis (day 2), and its efficacy persists during pregnancy, but it is equally effective as ASA1 on glands from day-10 females (IC50 = 0.31 nM). The octadecapeptide is characterized by a potential dibasic cleavage site, Lys9-Arg10, the integrity of which is needed for high potency. The ASB2-(11-18)-octapeptide amide gives a full response at high concentrations at day 10 (IC50 = 48 nM), but the C-truncated (1-9)-, (1-11)-, and (1-17)-amide fragments of ASB2 are inactive. Thus, the endocrine message is located at the C terminus. N alpha-acetylation of the N-truncated (9-18), (10-18), and (11-18) fragments of ASB2 increases activity relative to the nonacetylated peptides. The site of action of type A and type B allatostatins is located before mevalonate kinase in the biosynthetic pathway for juvenile hormone.

Changes in the sensitivity of adult cockroach corpora allata to a brain allatostatin.

There are major changes in the sensitivity of corpora allata from the cockroach Diploptera punctata toward the allatostatic tridecapeptide APSGAQRLYGFGL-amide (ASAL) during the female reproductive cycle, as revealed by measurement of juvenile hormone (JH) biosynthesis in vitro. Glands from recently molted adult females show only 30-40% inhibition at 10 nM ASAL, falling to a minimum of less than 10% on day 5 at the peak of spontaneous JH synthesis in vitro. The decline in JH synthesis observed in post-vitellogenic females is accompanied by a dramatic increase to ca. 90% ASAL sensitivity at 10 nM by day 6. Then sensitivity slowly wanes during subsequent ovulation and pregnancy to the levels typical of previtellogenic and virgin females. Full dose/response studies reveal a second level of response at ca. 1 microM, which resembles the pattern obtained from whole brain extracts. We conclude that physiological sensitivity to ASAL (IC50 ca. 0.1 nM) is correlated with the preparation for choriogenesis, and we suggest that 1000 times higher doses give a cross-reaction with related allatostatic receptor(s) that confer important sensitivity at other development stages.

Identification of an allatostatin from adult Diploptera punctata.

A peptide (allatostatin) causing strong and rapid inhibition of juvenile hormone synthesis in vitro by corpora allata from reproductively active females has been isolated from brain/retrocerebral complexes of the cockroach Diploptera punctata. The primary structure of this 13-residue peptide has been determined: Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2. Removal of the terminal amide group caused at least a ten thousandfold loss of activity. This neurohormone has no sequence similarity with any other known neuropeptide. Its target in the biosynthetic pathway is located prior to the conversion of farnesol to juvenile hormone.

Isolation and sequence of cDNA encoding a cytochrome P-450 from an insecticide-resistant strain of the house fly, Musca domestica.

A cDNA expression library from phenobarbital-treated house fly (Musca domestica) was screened with rabbit antisera directed against partially purified house fly cytochrome P-450. Two overlapping clones with insert lengths of 1.3 and 1.5 kilobases were isolated. The sequence of a 1629-base-pair (bp) cDNA was obtained, with an open reading frame (nucleotides 81-1610) encoding a P-450 protein of 509 residues (Mr = 58,738). The insect P-450 protein contains a hydrophobic NH2 terminus and a 22-residue cysteine-containing fragment near the COOH terminus that is known to bind the heme; both of these features have been found in the more than five dozen vertebrate P-450 proteins of which the sequences are presently known. Interestingly, the termination codon UAA may be contained in a putative polyadenylylation signal (AAUAAA) of the mRNA. This P-450 protein exhibits the most similarity (27% amino acid positional identity) with mammalian proteins of the P450III family but qualifies as a member of another family, which we propose to designate the P450VI gene family. This cDNA and deduced protein sequence should provide important information in the study of evolution of the P-450 gene superfamily, as well as provide an important probe for studying the regulation of insect P-450 and understanding the molecular genetics of insecticide resistance.

Forced synthesis of trace amounts of juvenile hormone II from propionate by corpora allata of a juvenile hormone III-producing insect.

Corpora allata of the cockroach Diploptera punctata normally synthesize only the isoprenoid juvenile hormone III (JH III). Only under extreme in vitro conditions (absence of carbon sources other than propionate) do they produce trace amounts of the homoisoprenoid JH II in addition to JH III. The specificity of the in vitro synthesis of JH III by D. punctata is thus consistent with the observed lack of homoisoprenoid JHs in this insect.

Isolation of renin-rich rat kidney cells.

Enzymatic dispersion and density gradient (Percoll) sedimentation were used to isolate a population of renin-containing, granule-laden cells (density 1.067 g/ml) from rat kidney cortex. Using immunohistochemistry (light microscopy) and electron microscopy, we defined the presence and ability of these cells to store renin protein(s). A 1000 base pair rat renin complementary DNA was used to show that these cells express the renin gene. The reverse hemolytic plaque assay defined the functional properties of the renin-containing cell. The data are consistent with the postulated inverse relationship between calcium concentration and release of renin. Thus, we have isolated a population of functional rat kidney cells that synthesize, store, and release renin.

Inhibition of insect juvenile hormone synthesis by phorbol 12-myristate 13-acetate.

The synthesis of insect juvenile hormone III (JH III) by isolated corpora allata of the cockroach Diploptera punctata incubated in vitro is inhibited by phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate and 1-oleyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate and diolein are inactive. The inhibitory effect of phorbol 12-myristate 13-acetate is fully reversed by 2E,6E-farnesol or by 2E,6E-farnesoic acid. It is highest in corpora allata that are past their peak in secretory activity or that have been inhibited by injections of 20-hydroxyecdysone. This effect of phorbol esters implicates protein kinase C in the regulation of insect corpus allatum activity.

Characterization and regulation of HMG-CoA reductase during a cycle of juvenile hormone synthesis.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was characterized in cockroach corpora allata which produce insect juvenile hormone III (methyl-(10R)10,11-epoxy-3,7,11-tri-methyl-2E,6E-dodecadienoate ). HMG-CoA reductase is a microsomal enzyme dependent on NADPH and dithiothreitol (or glutathione) for activity. The enzyme selectively reduced (3S)-HMG-CoA to (3R)-mevalonate with an apparent KM of 7.6 microM. Mevinolin was a competitive inhibitor of HMG-CoA reductase with a KI of 2.4 nM. No evidence for a modulation of enzyme activity by phosphorylation was obtained. Levels of HMG-CoA reductase were not altered after incubation of the corpora allata with either mevinolin (to decrease isoprenoid flux) or with mevalonate or farnesol (to increase isoprenoid flux). Split pairs of corpora allata were used to compare JH III synthetic activity with HMG-CoA reductase activity during the cycle of JH III synthesis that controls vitellogenesis and oocyte growth in adult females. Both activities changed over 10-fold and peaked on day 5 after emergence/mating, but JH III synthesis did not parallel HMG-CoA reductase activity precisely thereafter. The half-life of HMG-CoA reductase measured in the presence of cycloheximide was significantly different between low and high activity glands and was not related to the half-life of JH III synthesis. The results suggest that HMG-CoA reductase should not be considered 'the rate-limiting enzyme' in juvenile hormone synthesis by Diploptera punctata corpora allata.

Precursor supply for insect juvenile hormone III biosynthesis in a cockroach.

The biosynthesis of the sesquiterpenoid juvenile hormone III (JH III) was studied using corpora allata of the cockroach Diploptera punctata incubated in vitro and a radiochemical assay for the hormone produced. The influence of several exogenous precursors such as glucose, trehalose, acetate, amino acids, and mevalonate on JH synthetic rates was studied. Glucose or trehalose were needed for an optimal rate of JH synthesis. Highest rates were achieved at trehalose concentrations below the normal hemolymph levels (35-40 mM). About one-third of the glucose utilized for the biosynthesis of JH III was metabolized through a pentose pathway, but acetyl-CoA derived from glucose was significantly diluted by acetyl-CoA from other sources. Amino acids provided both a source of carbon for JH III synthesis and a source of energy that allowed JH III synthesis from acetate and stimulated JH III synthesis from glucose. Acetate was a poor substrate, because it could not support JH III synthesis in long term incubations. The incorporation of exogenous mevalonate into JH III was dependent on the physiological state of the glands, but there was a significant dilution with endogenous mevalonate. This dilution reflected in part the poor penetration of mevalonate into the corpora allata cells, because JH synthesis in mevinolin-treated cells was not fully rescued by mevalonate.

A rapid method for preparing insect microsomes.

The 1000 g supernatant of a tissue homogenate is layered on top of a small (less than 5 ml) sucrose density gradient and centrifuged for 20 min at very high centrifugal forces in a vertical rotor. Microsomes can be recovered rapidly in suspended form from the middle of the gradient, well separated from mitochondria and soluble (cytosolic) components. Applications to cockroach midgut microsomes and mosquito abdominal tissue microsomes are described, and the method is compared to the classical differential centrifugation method. Cytochrome P-450 monooxygenase activities can be measured on microsomes prepared from midgut tissue of 2-3 Diploptera punctata using this method.