Chromogranin from normal human adrenal glands: purification by monoclonal antibody affinity chromatography and partial N-terminal amino acid sequence.
A method is presented for the purification of human chromogranin from adrenal glands obtained at autopsy. The procedure involved homogenization of whole glands in aqueous buffer, salt precipitation, affinity chromatography using a highly specific monoclonal antibody (LK2H10) and reverse-phase high-pressure liquid chromatography. Chromogranin purified from autopsy adrenal glands revealed a high degree of polypeptide heterogeneity when analyzed by silver-stained SDS polyacrylamide gels. Greater than 90% of the protein was represented by a cluster of polypeptides with an Mr = 70 000 (i.e. chromogranin A), while the remaining protein was highly disperse in molecular weight. That these various polypeptides were in fact chromogranin was shown by Western blotting using monoclonal antibody LK2H10. About 6 nmol of chromogranin were obtained from 97 g of starting adrenals which was estimated to be a 25% yield and a 250-fold enrichment from adrenal homogenates. Critical to achieving reasonable yields of this protein was the need for particular low pH buffers for resuspension of chromogranin after solvent removal steps. Chromogranin purified from human adrenal glands was similar in amino acid composition, and identical in the N-terminal amino acid sequence (24 residues) to bovine chromogranin A. A secondary sequence representing 25% of the total protein and missing the first three residues of the N-terminus suggested the possibility of N-terminal processing of chromogranin in situ. The conservation of the N-terminal amino acid sequence of human and bovine chromogranin contrasts with the strong sequence variability predicted by antisera cross-reactivity and suggests that the N-terminus of chromogranin may be critical for its biological activity.
B S
BS
Wilson
S H
SH
Phan
R V
RV
Lloyd
D000311:Adrenal Glands / Q000032:analysis*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002864:Chromogranins / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D002864:Chromogranins; D009419:Nerve Tissue Proteins