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S W Applebaum

Publications and source records attributed to S W Applebaum.

At least 37 records · Page 2Linked to original sources
PubMed1988

Purification and characterization of Locusta migratoria chymotrypsin.

A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24,000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5-9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI) which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site.

Amino Acid Sequence↗
PubMed1987

Diuretic action and immunological cross-reactivity of corticotropin and locust diuretic hormone.

A functional similarity and immunological cross-reactivity between adrenocorticotrophic hormone (ACTH) and a locust diuretic hormone (DH) is reported. The functional similarity is expressed in that ACTH mimics DH by stimulating fluid secretion and cyclic AMP (cAMP) secretion in locust Malpighian tubules. Desacetyl-alpha-melanocyte-stimulating hormone is active to a lesser degree but no other POMC-derived peptide tested was found to follow suit. Immunological cross-reactivity is shown by a positive response of HPLC-purified DH with a specific ACTH radioimmunoassay as well as a significant reduction in DH activity (fluid secretion) after incubations with ACTH antiserum. However, ACTH and DH are different peptides since they do not share common separation characteristics on HPLC and ACTH does not induce a high excess secretion of cAMP by the tubule cell as does DH.

Adrenocorticotropic Hormone↗
PubMed1987

The intrinsic synthesis of juvenile hormone-III diol by locust corpora allata in vitro.

Juvenile hormone (JH)-III 10-11-diol is intrinsically synthesized and released from the corpora allata (CA) of adult locust females in vitro, together with JH-III. JH-III synthesis is preferentially stimulated and diol production only slightly enhanced, by cerebral locust allatotropin. The identification of JH-III diol is based on: similar ratio of incorporation of 14C/3H from radiolabelled [2-14C]acetate and [methyl-3H]methionine, to that of JH-III; similar chromatographic properties to those of synthetic diol on an RP-18 column eluted with acetonitrile, and similar chromatographic properties of acetylated derivatives; mass spectrometric analysis of derivatives and fragmentation products. Exogenous radiolabelled JH-III is not degraded during incubation with locust CA in vitro, corroborating the endogenous production of JH-III diol. Allatal diol formation may be an additional mechanism for the control of JH-III levels in locusts, preceding release into the hemolymph.

Animals↗
PubMed1986

Farnesoic acid and allatotropin stimulation in relation to locust allatal maturation.

Allatal maturation during the first gonotrophic cycle of adult female Locusta migratoria can be measured in vitro by the competence of these glands to produce juvenile hormone III (JH-III) when maximally stimulated by either farnesoic acid (FA) or allatotropin. Maturation precedes the full activation of the corpora allata (CA) during the first cycle of oogenesis. Basal activity of mature active female CA is highly correlated to stimulated activity. This suggests that between-gland variability reflects the inherent competence of each individual CA, and is not a consequence of a mere pulsatile on/off activation. Older glands may be inactive but their competence is equivalent to that of mature active glands. The kinetics of FA and allatotropic stimulation in vitro are equivalent, suggesting that allatotropin affects rate-limiting steps preceding FA biosynthesis.

Animals↗
PubMed1984

The effects of synthetic locust adipokinetic hormone on dispersed locust fat body cell preparations: cAMP induction, lipid mobilization, and inhibition of protein synthesis.

A procedure for the preparation of functional cells from adult locust fat bodies by collagenase treatment has been developed. The high variability of replicates encountered when whole fat bodies are incubated in vitro is greatly reduced in incubation of the dispersed cells. Synthetic locust adipokinetic hormone (AKH) (40 nM) stimulated release of lipids from the dispersed fat body cells at a rate comparable to that observed using whole fat bodies in vitro. Synthetic AKH elevated cAMP levels sixfold in dispersed cells. In addition, AKH inhibited protein synthesis to a maximum of 50-70% in a concentration-dependent manner. None of these actions of AKH required the presence of locust hemolymph components. These results demonstrate the utility of the isolated locust fat body cells for investigating hormonal action in vitro.

Adipose Tissue↗
PubMed1984

Synthesis of shrimp red pigment-concentrating hormone analogs and their biological activity in locusts.

Two analogs of the red pigment-concentrating hormone (RPCH) have been synthesized by the solid-phase method: [Thr6]-RPCH (I) and [Tyr4, Thr6]-RCPH (II). Analog I has the same amino acid composition as the second adipokinetic hormone (AKH-II) isolated from locust corpora cardiaca. Bioassay for lipid-mobilizing activity in adult male locusts gave the following increases in hemolymph lipid content: AKH-I, 3.5; I, 2.4; II, 2.9. The biological response shown by I lends support to the conclusion that its sequence is that of the presumptive AKH-II. Replacement of Phe in position 4 by Tyr does not reduce the adipokinetic response.

Animals↗
PubMed1984

Preparation of a specifically tritiated locust adipokinetic hormone analog with full biological potency.

A synthetic peptide related to locus adipokinetic hormone ( AKH ) and shrimp red pigment concentrating hormone ( RPCH ) containing a tyrosine residue in place of phenylalanine was iodinated and the 3,5- diiodotyrosyl derivative was isolated by reverse phase HPLC. Catalytic dehalogenation of the diiodo derivative in the presence of tritium yielded the tritiated AKH analog which was isolated by gel filtration on Sephadex LH-20 and reverse phase HPLC. The tritiated peptide was formed to be identical to AKH in its ability to stimulate lipid release into the hemolymph of locusts in vivo where the diiodotryrosyl derivative was inactive. The specific radioactivity of the tritiated peptide was 57.2 Ci/mmol, or 99% of the theoretical value.

Animals↗
PubMed1983

Determination of locust vitellogenin by radioimmunoassay with [3H]Propionyl-vitellogenin.

A simple procedure for radiolabeling of locust vitellogenin is described. This procedure involves coupling of [3H]propionyl succinimidate to purified vitellogenin with high yield and specific activity. Using this radiolabeled analog, a specific and sensitive radioimmunoassay was developed for determining locust vitellogenin content, with a lower detection limit of 1 ng. [3H]Propionyl-vitellogenin binds completely to rabbit anti-vitellogenin (locust) and can be completely competed out by locust vitellogenin. The structural similarity of locust vitellogenin with that of locust egg vitellin, male locust lipophorin (a diglyceride-carrying lipoprotein), Xenopus laevis vitellogenin, and chicken egg yolk lipovitellin was examined with this RIA procedure. Comparable binding competition was obtained with locust vitellin only. Male locust lipophorin, Xenopus vitellogenin, and chicken lipovitellin did not inhibit vitellogenin binding at concentrations 1000-fold greater than that of locust vitellogenin. The use of this RIA in determination of vitellogenin synthesis in vivo and in vitro, using isolated fat body preparations, is described.

Animals↗
PubMed1982

Construction and partial characterization of a recombinant DNA probe for locust vitellogenin messenger RNA.

Double-stranded DNA complementary to poly(A)-containing RNA from the fat body of adult female locusts, Locusta migratoria, was synthesized. Hybrid molecules containing this cDNA was constructed in the PstI site of the plasmid pAT 153 by the technique of dC . dG tailing and amplified in Escherichia coli K-12 strain HB 101. Ten colonies of bacteria were identified as carrying recombinant plasmids containing DNA complementary to locust vitellogenin mRNA by (a) 'Northern' blot hybridization analysis and (b) hybrid selection of vitellogenin mRNA and immunological detection of the products of translation of the mRNA. Of the ten recombinant plasmids, one, termed plasmid 4E, containing a cDNA insert of about 650 nucleotides, was characterized in greater detail and a partial restriction map obtained. Using this hybrid plasmid it was possible to derive a value for the average content of vitellogenin mRNA in the adult female locust fat body as 1.5 x 10(5) molecules/cell, and to establish that the haploid genome of L. migratoria contains only one or two genes coding for vitellogenin.

Animals↗
PubMed1982

Evolutionary conservation of vitellogenin genes.

Homologous and heterologous hybridizations in solution were performed between sheared genomic DNA and DNA complementary to vitellogenin mRNA of Xenopus, chicken, and migratory locust. The kinetics of hybridization and the thermal stability of the hybrids formed suggested a high degree of conservation of coding sequences of insect, amphibian, and avian vitellogenin genes. These cDNA probes hybridized to calf thymus DNA to a slight, but significant, extent, and not at all to Micrococcus lysodektikus DNA. DNA complementary to Xenopus albumin mRNA did not cross-hybridize significantly with locust or chicken DNA. Further evidence for the evolutionary conservation of vitellogenin genes was obtained from Southern blot analysis of restriction endonuclease-digested genomic DNA from a variety of vertebrate and invertebrate oviparous animals (Xenopus, chicken, migratory and desert locusts, yellow meal worm, carab moth, and Mediterranean fruitfly). When probed with cloned vitellogenin cDNAs from Xenopus and migratory locust, the DNA of these organisms showed varying degrees of homology of parts of the vitellogenin coding sequences. Southern blot analysis also showed that a part of the sequence specified in the cloned Xenopus vitellogenin cDNA was represented as repetitive DNA in the locust genome. However, cloned locust vitellogenin cDNA hybridized to discrete fragments of the restricted vertebrate DNA. These studies demonstrate a remarkably high degree of conservation of insect, amphibian, and avian vitellogenin genes.

Animals↗
PubMed1981

The preparation and characterization of locust vitellogenin messenger RNA and the synthesis of its complementary DNA.

Poly(A)+ (polyadenylated) RNA was isolated from vitellogenic female-locus fat-body by LiCl/urea extraction and poly(U)-Sepharose 4B affinity chromatography. Agarose-gel electrophoresis of this poly(A)+ RNA under denaturing conditions shows the presence of a high-molecular-weight species (greater than 31 S, 7100 nucleotides) as the major species, which is absent from the RNA prepared from male-locust fat-body. Inclusion of this poly(A)+ RNA in a mRNA-dependent reticulocyte-lysate system directs the synthesis of polypeptides that could be immunoprecipitated with monospecific antibodies against locust egg vitellin. DNA complementary (cDNA) to the poly(A)+ RNA was synthesized, and back-hybridization of the cDNA to its template reveals a major abundant species comprising about 45% of the total poly(A)+ RNA hybridizing with R0t 1/2 of 2 x 10(-2) mol . litre-1 . s. Abundant cDNA isolated from the total cDNA hybridizes to poly(A)+ RNA with a R0t 1/2 of 9 x 10(-3) mol . litre-1 . s. There are 9.1 x 10(3) copies of vitellogenin mRNA per cell of vitellogenic female-locust fat-body, comprising 55% of the poly(A)+ RNA and equivalent to 0.7% of total cellular RNA.

Animals↗
PubMed1981

In vitro incorporation of L-canavanine into vitellogenin of the fat body of the migratory locust Locusta migratoria migratorioides.

L-Canavanine competes with L-arginine for incorporation into vitellogenin secreted in vitro by the fat body of the female locust Locusta migratoria migratorioides. Incorporation of L-[guanidinooxy-14C]canavanine into vitellogenin has been established unequivocally by combined arginase and urease hydrolyses of the acid hydrolysate of antibody-precipitated canavanyl vitellogenin. Continued exposure of the fat body to canavanine decreases in vitro protein secretion but the proportion of canavanyl vitellogenin to native vitellogenin increases. Canavanine-mediated inhibition of fat body protein secretion is dependent on both the canavanine concentration and the arginine retention by the fat body. Canavanine replaces about 10% of the arginyl residues of canavanyl vitellogenin. The electrophoretic mobility of canavanyl vitellogenin is greater than that of native vitellogenin but the ability of this aberrant protein to react with vitellogenin antibody is unimpaired.

Adipose Tissue↗