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Leptin, a recently discovered hormone secreted mainly from adipose tissue, was first described as a regulator of adiposity, food intake and energy metabolism. It is now apparent that leptin physiology is much more complex and is likely to play an important role in many other systems including reproduction, haematopoiesis and immunity. Leptin levels have been shown to be well correlated with body fat in both humans and rodents, with administration of exogenous leptin to rats and mice resulting in loss of body fat. Leptin is, therefore, likely to be an important humoral signal to the central nervous system on body composition and regulation of food consumption. Due to the limited cross-reactivity of leptin from other species in the current assays for leptin, physiological research on leptin has, to a large extent, been restricted to rodents and humans. The aim of this study was to develop a leptin immunoassay suitable for use with sheep, enabling the investigation of the basic physiology of leptin in an animal larger than rats or mice, thus allowing repeated blood sampling. Using this assay we investigated the short-term effects of insulin, adrenaline and glucagon (all modulators of blood glucose) on plasma leptin levels. Antiserum to bovine recombinant leptin (brLeptin) raised in chickens was used to develop a competitive ELISA. Using brLeptin as standard, the assay has a sensitivity of 0. 5 ng/ml with inter- and intra-assay variation of 15% and 7% respectively. The cross-reactivity of human recombinant leptin was 36.5%, while mouse leptin showed no cross-reactivity. Plasma samples from ewes, male castrate animals and rams (n=4-5) diluted in parallel to the standard with mean leptin concentrations of 6.0+/-2. 9, 3.3+/-0.4 and 3.1+/-1.3 ng/ml respectively. Leptin levels in rams were significantly lower than in ewes. The non-significant difference in leptin levels between rams and male castrate animals suggests that testosterone may not be responsible for the lower levels of leptin. Four groups of 3-4 ewes were given intravenous insulin (1 iu/kg), adrenaline (65 microg/kg), glucagon (24 iu/kg) or saline. Blood samples were taken at 1, 3, 5, 10, 20, 30, 60, 90 and 120 min after injection. As expected, glucose levels declined within 10 min of the insulin injection and rose after 3 min following both adrenaline and glucagon injections. Leptin levels, however, remained relatively unchanged for the 2 h following the treatments. Finally, a bolus intravenous dose of glucose (240 mg/kg) was given and sequential blood samples taken. Despite plasma glucose levels rising to over 200 mg/dl, leptin levels did not significantly change over the three hours following treatment. These data indicate that plasma leptin levels in sheep, in contrast to rodents, are not responsive to short-term changes in blood glucose or insulin, as has been shown in humans.

The fluorescence of the cation auramine O was substantially enhanced by the presence of actin monomer. Titrations of this fluorescence enhancement indicated that actin monomer had two auramine O binding sites, each with a dissociation constant of approx. 20 microM. Calcium ions had no effect on the number of actin monomer-bound auramine O molecules or on the dissociation constant for that interaction. However, calcium ions increased the maximum change of fluorescence that occurs when actin monomer was fully saturated with auramine O. This effect of calcium was saturable and yielded a Ca2+ dissociation constant of 1.6 mM. It was concluded that auramine O bound to sites on actin monomer and independently monitored the binding of Ca2+ ion(s) to other site(s) on actin monomer. Further, the magnitude of the Ca2+ dissociation constant suggested that this Ca2+-binding site may be representative of the multiple bivalent cation-binding sites on actin monomer which are thought to be directly involved in actin polymerization. However, the exact relationship between these sites remains unclear.

Poly(ethylene glycol) 6000 affected many of the properties of skeletal-muscle actin. It accelerated the rate and increased the extent of actin polymerization as measured by light-scattering and sedimentation studies respectively. Moreover, intrinsic-fluorescence measurements showed that addition of poly(ethylene glycol) 6000 decreased the rate of EDTA-induced denaturation of actin monomer and increased the temperature at which irreversible conformational changes occur in actin monomer. These effects occurred without any apparent direct binding interaction and are postulated to be a consequence of the effect of excluded volume on the thermodynamic activity of actin. A relationship based on spherical geometry was formulated which described the co-volume increment that occurs upon addition of a monomer to a long linear polymer in the presence of a space-filling macromolecule. The application of this relationship to the poly(ethylene glycol) 6000-actin system was not without assumption, but it permitted quantitative estimation of the co-volume increment which proved to be of the sign and magnitude required to explain the increased extent of actin polymerization found experimentally in the presence of various concentrations of poly(ethylene glycol) 6000. It is suggested that, in vivo, excluded volume may play a role in actin-filament formation and in the maintenance of the native G-actin structure.

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

Bacterial alpha-amylase was shown by equilibrium and velocity-sedimentation studies to be a monomer-dimer equilibrium system in 0.10M-NaCl/0.015M-calcium acetate/0.010M-EDTA, pH7.0; an association constant of 2.4 × 10(3)M-1 is obtained. Studies of the binding of Zn2+ to alpha-amylase in 0.10M-NaCl/0.005M-calcium acetate, pH7.0, yielded binding curves that exhibit dependence on the concentration of alpha-amylase (Zn2+-free) used in the equilibrium-dialysis experiments. Results are described very satisfactorily by a reaction scheme in which Zn2+ binds exclusively to the dimer of the above monomer–dimer system with an association constant of 1.0 × 10(6)M-1. The present results refute the earlier scheme for dimer stabilization by Zn2+ in which the metal ion formed a cross-link between two non polymerizing monomer units.

The self-association of alpha-chymotrypsin and its di-isopropyl phosphoryl derivative in in I0.03 sodium phophate buffer, pH7,9, was investigated by velocity sedimentation, equilibrium sedimentation and difference gel chromatography. No differences between the native and chemically modified enzyme were observed in the ultracentrifuge studies, and only a marginal (0.6%) difference in weight-average elution volume was detected by difference gel chromatography of 5g/litre solutions on Sephadex G-75. From quantitative analyses of sedimentation velocity and sedimentation-equilibrium distributions obtained with iPr2P (di-isopropylphosphoryl)-chymotrypsin, the polymerizing system is postulated to involve an indefinite association of dimer (with an isodesmic association constant of 0.68 litre/g) that is formed by a discrete dimerization step with equilibrium constant 0.25 litre/g. In addition to providing the best fit of the experimental results, this model of chymotrypsin polymerization at low ionic strength is also consistent with an earlier observation that dimer formation is a symmetrical head-to-head phenomenon under conditions of higher ionic strength (I0.29, pH7.9) where association is restricted to a monomer-dimer equilibrium. It is proposed that the dimerization process is essentially unchanged by variation in ionic strength at pH7.9, and that higher polymers are formed by an entirely different mechanism involving largely electrostatic interactions between dimeric species.

Glycoproteins located on the luminal surface of the plasma membrane of tick gut epithelial cells, when used to vaccinate cattle, are capable of stimulating an immune response that protects cattle against subsequent tick infestation. One such tick gut glycoprotein, designated Bm86, has been purified to homogeneity and the amino acid sequences of peptide fragments generated by endoproteinase Lys-C digestion have been determined. We report here the isolation and characterization of a cDNA that encodes Bm86. The nucleotide sequence of the cDNA contains a 1982-base-pair open reading frame and predicts that Bm86 contains 650 amino acids including a 19-amino acid signal sequence and a 23-amino acid hydrophobic region adjacent to the carboxyl terminus. The main feature of the deduced protein sequence is the repeated pattern of 6 cysteine residues, suggesting the presence of several epidermal growth factor-like domains. A fusion protein consisting of 599 amino acids of Bm86 and 651 amino acids of beta-galactosidase was expressed in Escherichia coli as inclusion bodies. Ticks engorging on cattle vaccinated with these inclusion bodies were significantly damaged as a result of the immune response against the cloned antigen.