Isolation and characterization of highly purified mosquito oostatic hormone

Oostatic hormone, the hormone that inhibits vitellogenesis in mosquitoes, was purified 7,000-fold with a recovery of 70% from the ovaries of the mosquito Aedes aegypti. The purification procedure included heat treatment and chromatography on ion exchange and gel filtration columns. The hormone is a small peptidelike molecule of molecular weight 2,200 at pH 4.5, which aggregates into larger molecular species of trimer and octamer at pH 7.0 as determined by gel filtration. The hormone is positively charged at pH 7.8 and has a low Rf at pH 9.4 on disc gel electrophoresis. Injection of purified oostatic hormone (9 ng) into female mosquitoes inhibited yolk deposition and vitellogenin synthesis. Activity of the oostatic hormone in the mosquito ovary increased rapidly following blood feeding and reached a maximum after 48 h. Oostatic hormone of A. aegypti injected into autogenous Aedes taeniorhynchus inhibited egg development. Repeated injections of dilute oostatic hormone at 24 h intervals partially arrested egg development, resulting in 60% reduction in the number of eggs laid. This hormone does not block release of egg development neurosecretory hormone (EDNH) from the mosquito brain but rather appears to act on the ovary.     

Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, -A2 and -A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

Opioid receptor antagonist affinity ligands: 6β-bromoacetamido-6-desoxynaltrexone and 6β-thioglycolamido-6-desoxynaltrexone

The present study, utilizing thioglycolamido as the reactive group, describes the synthesis and pharmacology of a new opioid antagonist affinity ligand, 6-thioglycolamido-6-desoxynaltrexone (TAN) and compares TAN with a related known compound, 6-bromoacetamido-6-desoxynaltrexone (BAN). Both compounds were tested for their reversible and irreversible inhibition of [³H]naloxone binding to calf brain membranes. Reversible binding of BAN and TAN had K_i values of 1×10^–9 and 1×10^–10 M, respectively as determined by log probit plots. Irreversible binding was determined after extensive washing to remove all non-covalently bound ligand. At a concentration of 5×10^–8 and 1×10^–8 M for BAN and TAN irreversible binding was inhibited 50% of the maximum value. A study of the time course of irreversible inhibition of [³H]naloxone binding revealed that maximal inhibition occurred within 5 min with a concentration of 1×10^–7 M of either agent. TAN but not BAN when administered systematically to mice produced an antinociceptive effect as measured by the writhing test. When administered intracerebraventricularly BAN did not block morphine-induced analgesia for more than 2 hr; whereas, with a single ED₅₀ dose of 20 nmoles of TAN i.c.v. morphine-induced analgesia was almost completely blocked for a period of over 24 hr, as determined by the tail flick test. Although the SH group of TAN were required for the covalent interaction with opioid receptors, the site of TAN's interaction appears to involve other than protein SH groups.     

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Pharmacological and biochemical characteristics of partially purified GABAB receptor

Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)_B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [³H]GABA binding to the purified GABA_B receptor showed a linear relationship and the K_D and B_max values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg²⁺ did not affect on the GABA_B receptor binding, Ca²⁺ significantly increased [³H]GABA binding to the purified GABA_B receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca²⁺ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [³H]GABA, but phospholipase A₂ had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [³H]GABA to the purified GABA_B receptor. These results suggest that purification of the solubilized GABA_B receptor by the affinity column chromatography may result in the functional uncoupling of GABA_B receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABA_B receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A₂ treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts.     

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.     

High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase

Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants were calculated from chromatographic data and found to correspond well with literature values. The dissociation constants for a number of nucleotide derivatives with potential application in affinity chromatography were also determined. The spaces were found to affect the binding strength of the nucleotides in a qualitatively predictable way. Theoretical plate heights were calculated and found to be in the range 0.01 to 0.1 cm. Attempts to correlate peak widths with the rate constants for the binary complexes involved were only partially successful.     

Isolation of the vitellogenin-binding protein from locust ovaries

A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-β-D-glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 μg (intact ovarian membranes) to 170.9 μg (affinity purified binding protein) per mg membrane protein, which corresponds to a purification factor of 35.