Abstract
Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis and partial NH2-terminal sequence analysis indicated that GPIII was a disulfide-linked heterodimer with 37-kDa subunits. Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor. Partial NH2-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has greater than 80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain.
Highlights
Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC)
Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor
This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library
Summary
Glycoprotein; bp, base pairs; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SGPB, rat Sertoli cell glycoprotein. 40,40, human serum protein-40,40; WGA, wheat germ agglutinin; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. The WGA-Sepharose was washed with 0.1 M NaHCO,$, 0.2 M NaCl, and incubated with 1. 5 cm WGA-Sepharose column, previously equilibrated with 0.05 M sodium phosphate, pH 7.0, 0.2 M NaCl (phosphate buffered saline), at 16 ml/h. Bovine adrenal medullary poly(A)+ RNA was translated in vitro using a commercial wheat germ extract (Promega) with [‘l”S]methionine as label. Recommended by Promega The cDNA ends were blunted using T, DNA polymerase and ligated to EcoRI adaptors (Promega) This material was fractionated on a Sepharose CL4B column, and cDNA longer than 500 hp was phosphorylated and ligated to dephosphorylated XgtlO arms (Promega). One compression was resolved using 7-deaza-dGTP in place of dGTP
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