Liver microsomes contain two distinct NADPH-Monooxygenases with NH2-terminal segments homologous to the flavin containing NADPH-monooxygenase of Pseudomonas fluorescens

Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as from 1 and from 2. Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40. Affinity chromatography on 2', 5'-ADP Sepharose was used to increase the purity and to concentrate the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000. The NH2- terminus of form 1 is blocked, whereas the NH2-terminus of form 2 is homologous to the NADPH-phydroxybenzoate hydrolase from Pseudomonas fluorescens. The latter and the form 2 enzyme share 11 identical residues in the NH2-terminal segment of 15 residues. Both forms were subjected to tryptic cleavages and peptide mapping. Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins. A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein. This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins. These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.     

The glucagon-like peptide-1 metabolite GLP-1-(9-36) amide reduces postprandial glycemia independently of gastric emptying and insulin secretion in humans

Glucagon-like peptide 1 (GLP-1) lowers glycemia by modulating gastric emptying and endocrine pancreatic secretion. Rapidly after its secretion, GLP-1-(7-36) amide is degraded to the metabolite GLP-1-(9-36) amide. The effects of GLP-1-(9-36) amide in humans are less well characterized. Fourteen healthy volunteers were studied with intravenous infusion of GLP-1-(7-36) amide, GLP-1-(9-36) amide, or placebo over 390 min. After 30 min, a solid test meal was served, and gastric emptying was assessed. Blood was drawn for GLP-1 (total and intact), glucose, insulin, C-peptide, and glucagon measurements. Administration of GLP-1-(7-36) amide and GLP-1-(9-36) amide significantly raised total GLP-1 plasma levels. Plasma concentrations of intact GLP-1 increased to 21 +/- 5 pmol/l during the infusion of GLP-1-(7-36) amide but remained unchanged during GLP-1-(9-36) amide infusion [5 +/- 3 pmol/l; P < 0.001 vs. GLP-1-(7-36) amide administration]. GLP-1-(7-36) amide reduced fasting and postprandial glucose concentrations (P < 0.001) and delayed gastric emptying (P < 0.001). The GLP-1 metabolite had no influence on insulin or C-peptide concentrations. Glucagon levels were lowered by GLP-1-(7-36) amide but not by GLP-1-(9-36) amide. However, the postprandial rise in glycemia was reduced significantly (by approximately 6 mg/dl) by GLP-1-(9-36) amide (P < 0.05). In contrast, gastric emptying was completely unaffected by the GLP-1 metabolite. The GLP-1 metabolite lowers postprandial glycemia independently of changes in insulin and glucagon secretion or in the rate of gastric emptying. Most likely, this is because of direct effects on glucose disposal. However, the glucose-lowering potential of GLP-1-(9-36) amide appears to be small compared with that of intact GLP-1-(7-36) amide.     

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3H]palmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of alpha-tubulin. 3H-labeled palmitoylated alpha-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to peptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of 3H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify 3H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in alpha-tubulin. The palmitoylated residues in alpha-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.     

Influence of continuous magnetic field on the separation of ephedrine enantiomers by molecularly imprinted polymers

A set of polymers was imprinted with (-)-ephedrine using UV initiation, under the influence of a constant external magnetic field with intensities ranging from 0 to 1.55 T. Synthesised materials were characterised by X-ray crystallography, infrared spectroscopy, swelling and surface area. Recognition properties were assessed by the ability to discriminate between (+) and (-)-ephedrine and by Scatchard analyses on chromatographic mode. It was shown that polymer morphology and recognition properties are affected by the magnetic field. This resulted in considerable improvements in the chromatographic resolution of ephedrine enantiomers by materials synthesised under the influence of magnetic field. Apparently the magnetic field improved the ordering of the polymer structure and facilitated the formation of more uniform imprinting sites.     

Glucagon-like peptide 1 and its derivatives in the treatment of diabetes

Glucagon-like peptide 1 (GLP-1) was discovered as an insulinotropic gut hormone, suggesting a physiological role as an incretin hormone, i.e., being responsible, in part, for the higher insulin secretory response after oral as compared to intravenous glucose administration. This difference, the incretin effect, is partially lost in patients with Type 2 diabetes. The actions of GLP-1 include (a) a stimulation of insulin secretion in a glucose-dependent manner, (b) a suppression of glucagon, (c) a reduction in appetite and food intake, (d) a deceleration of gastric emptying, (e) a stimulation of beta-cell neogenesis, growth and differentiation in animal and tissue culture experiments, and (f) an in vitro inhibition of beta-cell apoptosis induced by different toxins. Intravenous GLP-1 can normalize and subcutaneous GLP-1 can significantly lower plasma glucose in the majority of patients with Type 2 diabetes. GLP-1 itself, however, is inactivated rapidly in vivo and thus does not appear to be useful as a therapeutic agent in the long-term treatment of Type 2 diabetes. Other agents acting on GLP-1 receptors have been found (like exendin-4) or developed as GLP-1 derivatives (like liraglutide or GLP-1/CJC-1131). Clinical trials with exenatide (two injections per day) and liraglutide (one injection per day) have shown reductions in glucose concentrations and HbA1c by more than 1%, associated with moderate weight loss (2-3 kg), but also some nausea and, rarely, vomiting. It is hoped that this new class of drugs interacting with the GLP-1 or other incretin receptors, the so-called "incretin mimetics", will broaden our armamentarium of antidiabetic medications in the nearest future.     

Structural characteristics of prostaglandin synthetase from sheep vesicular gland

Prostaglandin synthetase contains both oxygenase and peroxidase activity and catalyzes the first step of prostaglandin synthesis. Aspirin (acetylsalicylic acid) inhibits oxygenase activity by acetylating a serine residue of the enzyme. In the current study, we have investigated the subunit structure of this complex enzyme and the stoichiometry of aspirin-mediated acetylation of the enzyme. The enzyme was purified to near homogeneity in both active and aspirin-acetylated forms. The purified protein was analyzed for enzymatic activity, [3H]acetate content following treatment with [acetyl-3H]aspirin, NH2-terminal sequence, and amino acid composition. The results show first, that the enzyme can be purified to near homogeneity in an active form; second, that the enzyme consists of a single polypeptide chain (molecular weight 72,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis) with a unique NH2-terminal sequence (Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-Met-Gly-); and third, that aspirin inhibits the enzyme by transfer of one acetate per enzyme monomer. Therefore, the two distinct enzymatic activities, oxygenation and peroxidation, are present in a single polypeptide chain. Experiments with a cross-linking agent indicate that in nonionic detergent the enzyme is a dimer of two identical subunits.     

Immunohistochemical demonstration of cytochrome b5 and hemopexin in rat liver parenchymal cells using horseradish peroxidase

The membrane-bound hemoprotein, cytochrome b₅, and the serum protein, hemopexin, were demonstrated in rat liver parenchymal cells using an immunohistochemical approach. Other liver cells contained neither protein. The immunological “sandwich” technique employs a complex of horseradish peroxidase with anti-horseradish peroxidase sequentially coupled to other antibodies including those to rat cytochrome b₅ and hemopexin.     

Xenopus lipovitellin 1 is a Zn2+- and Cd2+-binding protein

This report discusses the identification of a Zn²⁺- and Cd²⁺-binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with ⁶⁵Zn²⁺ or ¹⁰⁹Cd²⁺. The resulting autoradiograms showed binding of both radio-nuclides to a protein, designated pCdZn. Freon extraction of oocyte and embryo homogenates showed pCdZn to be a yolk protein. When pCdZn was isolated from oocyte homogenates by ammonium sulfate precipitation, delipidation, and chromatography, it co-purified with lipovitellin 1. The amino acid composition of pCdZn closely resembled the reported composition of lipovitellin 1 and the molecular weight of purified pCdZn (～115 kD) corresponded to reported values for lipovitellin 1 (111–121 kD). Amino acid sequence analyses of five peptides derived from pCdZn yielded 94% identity to the reported sequence of lipovitellin 1, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Based on these findings, pCdZn was identified as lipovitellin 1. This study suggests that lipovitellin 1 is the major storage protein for zinc in mature oocytes and developing embryos of Xenopus laevis. © 1995 wiley-Liss, Inc.     

Large dense-core vesicle exocytosis in pancreatic β-cells monitored by capacitance measurements

This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic β-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.