Evidence for presence of proinsulin-immunoreactive glycoprotein(s) in fetal bovine pancreatic extracts.

A large-molecular-weight proinsulin-immunoreactive protein fraction was obtained from an extract of fetal bovine pancreases by gel filtration in 6 M guanidine-1 M acetic acid. Concanavalin A-Sepharose-affinity column chromatography of the large-molecular-weight fraction yielded a discrete alpha-methyl-mannoside-displaceable immunoreactive peak that also displayed N-acetylglucosamine-specific binding to wheat germ lectin-Sepharose. Chemically tritiated and radioiodinated lectin-reactive proteins interacted specifically with antibodies to insulin and bovine proinsulin. Immunochemically purified (reaction with antibodies followed by separation of antigen-antibody complexes on protein A-Sepharose) radiolabeled lectin-reactive proteins were analyzed by gel filtration in guanidine-acetic acid and by sodium dodecyl sulfate polyacrylamide gel electrophoresis after disulfide bond-cleavage treatments. Results from these studies suggest the existence of an approximately 67,000-Mr glycoprotein that contains antigenic domains common to proinsulin and insulin.

Beta-endorphin-like immunoreactivity in extracts of the fetal bovine pancreas. Column chromatographic characterizations of a high-molecular-weight immunoreactive species.

Acid-ethanol extracts of fetal bovine pancrease were examined for the presence of beta-endorphin-like immunoreactivity. Gel-filtration analyses revealed the presence of a major large-molecular-weight beta-endorphin immunoreactive species of approximately 20K delta. This molecular form maintained its size upon resubmission to gel filtration in the presence of 6 M guanidine hydrochloride, separated from the bulk of the glucagon immunoreactivity upon ion-exchange chromatography, showed proportional dilution in the beta-endorphin radioimmunoassay, and interacted in a biospecific manner with Concanavalin-A-Sepharose.

Glycoprotein-like large glucagon immunoreactive species in extracts of the fetal bovine pancreas.

Large glucagon immunoreactive substances, extracted from the fetal bovine pancreas and separated by gel filtration in the presence of 6 M guanidinium-hydrochloride, were submitted to lectin-sepharose affinity column chromatograph. Gel-filtered peak I (approximately 45 K delta) and peak II (approximately 10 K delta) interacted biospecifically with concanavalin-A- and wheat-germ-lectin-sepharoses, suggesting glycoproteins as possible constituents of large glucagon immunoreactive substances in extracts of the fetal bovine pancreas. The glucagon-like immunochemical identity of the lectin-sepharose-bound substances was further substantiated by binding to antiglucagon antibodies-sepharose and by characteristic proportional dilutions in the glucagon radioimmunoassay.

Biosynthesis of insulin in bovine fetal pancreatic slices: the incorporation of tritiated leucine into a single-chain proinsulin, a double-chain intermediate, and insulin in subcellular fractions.

This communication reports the biosynthesis of insulin in the bovine fetal pancreatic slices in vitro. Double-chain proinsulin and insulin were found as major components in the mitochondrial-granule fraction of bovine fetal pancreas. Tritiated leucine was incorporated into a single-chain proinsulin, a double-chain proinsulin, and insulin. Subcellular fractionation of the slices incubated with tritiated leucine showed that radioactive single-chain proinsulin was present in the deoxycholate-soluble microsomal fraction, deoxycholate-insoluble microsomal fraction, and mitochondrial-granule fraction. Labeled double-chain proinsulin and insulin were present in the deoxycholate-soluble microsomal and mitochondrial-granule fractions. These results are consistent with the hypothesis that insulin is synthesized as a single-chain polypeptide on the ribosomes, and that intracellular proteolysis in the subcellular membranous organelles and beta-granules converts the single-chain proinsulin to insulin via a double-chain intermediate.

Characterization of a chemically tritiated large molecular weight glucagon immunoreactive protein species by lectin-affinity column chromatography and reaction with anti-glucagon antibodies.

High molecular weight glucagon immunoreactive material, obtained by gel-filtration (in the presence of 6 M guanidine hydrochloride) of fetal bovine pancreatic extracts, was tritiated by reductive methylation. Concanavalin-A-Sepharose column chromatography of the radiolabeled preparation yielded a discrete Concanavalin-A-reactive, alpha-methyl-mannoside-displaceable radioactive peak, coinciding with the glucagon immunoreactive peak. Submission of the Con-A-reactive material to wheat germ agglutinin-Sepharose column chromatography yielded a lectin-reactive, N-acetyl-glucosamine-displaceable radioactive peak, coinciding with the glucagon immunoreactive peak. The tritiated Con-A-reactive component interacted specifically with anti-glucagon antibodies. Sephacryl S-200 gel-filtration (in the presence of guanidine hydrochloride) dissociated a approximately 40 kDa radioactive species from the antibody-antigen complex. These data provide direct evidence for the existence of a large molecular weight glycosylated glucagon-related protein species from the fetal bovine pancreas.

Glucagon from the bovine fetal pancreas: chromatographic and electrophoretic characterizations of high molecular weight immunoreactive species.

Fetal bovine pancreas was extracted for glucagon using (A) ethanol-Hcl after trichloroacetic acid (TCA) treatment of the pancreas, (B) ethanol-HCl and (C) urea-acetic acid. Fractionation of the acetic acid soluble proteins vi Sephadex G-50 columns yielded glucagon immunoreactivity in the void volume, high molecular weight glucagon immunoreactivities (HMW-IRGs), "proglucagon" (approximately equal to 9 K delta), and true glucagon (3.5 K delta) regions. HMW-IRGs were obtainable using all three methods of extraction. The material obtained from the ethanol-HCl-TCA method appeared stable on Sephadex G-100 (1 M acetic acid) rechromatography. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis analysis showed immunoreactive species corresponding to approximately 40 K delta and approximately 12 K delta. HMW-IRGs did not bind to concanavalin A (Con A)-agarose. SDS-polyacrylamide gel electrophoresis of the Con A-agarose filtered IRG again showed a major immunoreactive peak of approximately 40 K delta. Dose-response RIA studies indicated that the HMW-IRGs from both the gel filtration and SDS-polyacrylamide gel experiments were immunochemically indistinguishable from glucagon. HMW-IRGs bind to antiglucagon antibody agarose, further indicating their reactivity towards glucagon antibodies. When HMW-IRGs are incubated with guanidinium hydrochloride and gel filtered in the same system, a significant fraction of HMW-IRG (representing up to 25% of the total IRG analysed) was found to resist disruption. Our data support the contention that a significant portion of the HMW-IRGs (molecular weight greater than 20 K delta) extracted from fetal bovine pancreas are composed of glucagon covalently linked to larger protein unit(s).

Glucagon from avian pancreatic islets: radioreceptor studies.

Glucagon extracted from isolated islets of the pigeon was studied by means of Sephadex gel filtration. Radioreceptor assay, using rat liver plasma membranes and radioiodinated porcine glucagon, showed that the bulk of the activity eluted with glucagon (molecular weight 3500). Avian glucagon appeared to be less effective than porcine glucagon in inhibiting the binding of labeled porcine glucagon to rat plasma membranes.

Lamivudine is effective in suppressing hepatitis B virus DNA in Chinese hepatitis B surface antigen carriers: a placebo-controlled trial.

Lamivudine is a novel 2',3'-dideoxy cytosine analogue that has potent inhibitory effects on hepatitis B virus replication in vitro and in vivo. We performed a single-blind, placebo-controlled study to assess its effectiveness and safety in Chinese hepatitis B surface antigen (HBsAg) carriers. Forty-two Chinese HBsAg carriers were randomized to receive placebo (6 patients) or lamivudine orally in dosages of 25 mg, 100 mg, or 300 mg daily (12 patients for each dosage). The drug was given for 4 weeks. The patients were closely monitored clinically, biochemically, and serologically up to 4 weeks after drug treatment. All 36 patients receiving lamivudine had a decrease in hepatitis B virus (HBV) DNA values of >90% (P < .001 compared with placebo). Although 25 mg of lamivudine was slightly less effective than 100 mg (P = .011) and 300 mg (P = .005), it still induced 94% suppression of HBV DNA after the fourth week of therapy. HBV DNA values returned to pretreatment levels within 4 weeks of cessation of therapy. There was no change in the hepatitis B e antigen status or in aminotransferase levels. No serious adverse events were observed. In conclusion, a 4-week course of lamivudine was safe and effective in suppression of HBV DNA in Chinese HBsAg carriers. The suppression was >90% but reversible. Studies with long-term lamivudine administration should be performed to determine if prolonged suppression of HBV DNA can be achieved.