Chemical synthesis, initial conformational studies, and activity of rat steroidogenesis activator peptide and a truncated analog.
A 30-residue peptide corresponding to the amino acid sequence of steroidogenesis activator peptide (SAP) from rat Leydig tumor cells has been synthesized by the solid-phase method using Boc protection. SAP is the putative cycloheximide-sensitive, cAMP-regulated mediator of ACTH-stimulated conversion of cholesterol to pregnenolone in adrenal cortex. N alpha-acetyl-SAP(11-30), an NH2-terminally truncated steroidogenesis activator peptide analog that is missing the most hydrophobic portion of SAP, was also prepared. In addition to these two peptides, N alpha-acetyl-(Cys0)SAP was synthesized in both non-radiolabeled and tritiated forms for coupling to carrier proteins for use as an immunogen to raise anti-SAP antibodies. Chain elongation during synthesis of SAP on PAM resin proceeded with an average coupling yield of 99.3% as determined by quantitative ninhydrin tests. After HF cleavage at -7 degrees, the crude products were purified by semi-preparative HPLC. Peptides were analyzed by analytical HPLC, amino acid analysis, tryptic peptide mapping, and by UV and CD spectroscopy. As determined by CD spectra, SAP showed little evidence of preferred structure either in aqueous solution in the presence of divalent cations or in micelles of reduced Triton X-100 in the absence or presence of either cholesterol or phosphatidylcholine. SAP, in conjunction with GTP, enhanced side chain cleavage activity in isolated adrenal mitochondria.
Glass
D B
DB
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322.
Robertson
D G
DG
Xu
T S
TS
Bowman
E P
EP
Lambeth
J D
JD
D000311:Adrenal Glands / Q000378:metabolism / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006360:Heat-Shock Proteins; D008928:Mitochondria / Q000378:metabolism; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D051381:Rats; D013057:Spectrum Analysis; D000091342:Endoplasmic Reticulum Chaperone BiP
D000596:Amino Acids; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011506:Proteins; C051684:GRP78 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP