The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases ofthe kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the chargerelay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and-to a lesser extent-gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the a-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromo- (6) and was tested as a marker for postcoital detection in rape investigations (7). Although the clinical utility of PA has been shown, its biological function and chemical structure are not well characterized (8). We report here the complete amino acid sequence of PA and describe the characteristics of its enzymatic properties. MATERIALS AND METHODSPA was purified from human seminal plasma as described (1). PA was finally purified on a large-pore Vydac (Hesperia, CA) C4 column and eluted either with a linear gradient between 70% (vol/vol) buffer A (buffer A = 0.1% trifluoroacetic acid in H20) and 37% (vol/vol) buffer B (buffer B = 0.1% trifluoroacetic acid in acetonitrile) in 280 min or 10% (vol/vol) buffer B and 80% (vol/vol) buffer B in 60 min.The enzymatic activity of PA was assessed on commercially purified proteins including insulin A, insulin B, gelatin, myoglobin, ovalbumin, fibrinogen (Sigma), and recombinant interleukin 2 (Ala-IL2, Cetus). The substrates (1 mg/ml) were dissolved in either 0.1 M ammonium bicarbonate or 50 mM Tris HCl, pH 7.8, containing PA at 0.1 mg/ml. In some cases, a mass ratio (enzyme:substrate) of 1:20 was used. After an 18-hr digestion at 37°C, the peptides were separated by HPLC using a 60-min linear gradient of 0-80% (vol/vol) buffer B. To determine the peptide bond specificity of PA, hydrazinolysis was performed on each ofthe PA/substrate digestion mixtures, substrate alone, and intact PA as described (9). The free carboxyl-terminal amino acids were lyophilized and analyzed on a Beckman 121MB amino acid analyzer.The kinetics ofPA hydrolytic activity on synthetic substrates were studied by monitoring the absorbance change at room temperature using a Hewlett-Packard 8450A spectrophotometer. The following substrates were used: N...

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The amino acid sequence of the human fibrinogen alpha-chain reveals a structure that can be divided into three zones of unique amino acid composition. The middle of these contains the two primary alpha-chain cross-linking acceptor sites and consists of a remarkable series of internal duplications.

The complete amino acid sequence of the alpha chain of human fibrinogen has been determined. It contains 610 amino acid residues and has a calculated molecular weight of 66,124. The chain has 10 methionines, and fragmentation with cyanogen bromide yields 11 peptides [Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J., Hucko, J.T., & Takagi, T. (1977) Biochemistry 16, 1703]. The arrangement of the 11 fragments was determined by the isolation of peptide overlaps from plasmic and staphylococcal protease digests of fibrinogen and/or alpha chains. In addition, certain of the cyanogen bromide fragments, preliminary reports of whose sequences have appeared previously, have been reexamined in order to resolve several discrepancies. The alpha chain is homologous with the beta and gamma chains of fibrinogen, although a large repetitive segment of unusual composition is absent from the latter two chains. The existence of this unusual segment divides the sequence of the alpha chain into three zones of about 200 residues each that are readily distinguishable on the basis of amino acid composition alone.

A strain of Saccharomyces cerevisiae capable of simultaneous hydrolysis and fermentation of highly polymerized starch oligosaccharides was constructed. The Aspergillus awamori glucoamylase enzyme, form GAI, was expressed in Saccharomyces cerevisiae by means of the promoter and termination regions from a yeast enolase gene. Yeast transformed with plasmids containing an intron-free recombinant glucoamylase gene efficiently secreted glucoamylase into the medium, permitting growth of the transformants on starch as the sole carbon source. The natural leader sequence of the precursor of glucoamylase (preglucoamylase) was processed correctly by yeast, and the secreted enzyme was glycosylated through both N- and O-linkages at levels comparable to the native Aspergillus enzyme. The data provide evidence for the utility of yeast as an organism for the production, glycosylation, and secretion of heterologous proteins.