Abstract
Proteinase A was purified by an improved large scale procedure and split into fragments by means of trypsin, cyanogen bromide, hydroxylamine, and o-iodosobenzoic acid. On the basis of high degrees of homology with cathepsin D and pepsin its amino acid sequence was determined. Proteinase. A contains 329 amino acid residues, and in addition 8.5% neutral sugar and 1% glucosamine, attached to asparagines in positions 67 and 267. Proteinase A contains two disulfide bonds, as opposed to three in mammalian aspartic proteinases. Comparison with the tertiary structure of pepsin indicates, that the two catalytically essential aspartic acid residues, and the residues corresponding to their surroundings, are conserved. The sequence shows 46% identity with porcine cathepsin D and 40% with porcine pepsin. An aspartic proteinase from Saccharomyces carlsbergensis had the same N-terminal 40 amino acid sequence as proteinase A. Immunological cross-reactivity between proteinase A and calf chymosin was demonstrated by immune blotting assay.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- DAN:
-
diazoacetyl-norleucine methyl ester
- DPCC:
-
diphenyl carbamyl chloride
- EPNP:
-
1,2-epoxy-3-(p-nitrophenoxy) propane
- FPLC:
-
fast protein liquid chromatography
- HPLC:
-
high performance liquid chromatography
- PMSF:
-
phenyl methylsulfonyl fluoride
- SDS-PAGE:
-
sodium dodecylsulphate-polyacrylamide gel electrophoresis
- TEA:
-
triethylamine
- TFA:
-
trifluoroacetic acid
References
Bech, A.-M. &B. Foltmann: Partial primary structure of Mucor miehei protease. Neth. Milk Dairy J. 35, 275–280 (1981)
Bjerrum, O. J., K. P. Larsen &M. Wilken: Some recent developments of the electroimmunochemical analysis of membrane proteins. In: Modern Methods in Protein Chemistry, Tschesche, H., Ed., Walter de Gruyter, Berlin, New York 79–124 (1983)
Bornstein, P. &G. Balian: Cleavage at Asn-Gly bonds with hydroxylamine. Meth. Enzymol. 47, 132–145 (1977)
Dixon, H. B. F. &R. N. Perham: Reversible blocking of amino groups with citraconic anhydride. Biochem. J. 109, 312–314 (1968)
Dreyer, T., K. Biedermann &M. Ottesen: Yeast proteinase in beer. Carlsberg Res. Commun. 48, 249–253 (1983)
Dreyer, T., I. Svendsen &M. Ottesen: Partial primary structure and substrate specificity of proteinase A from Saccharomyces cerevisiae. Biochem. Soc. Transac. 13, 1142–1143 (1985)
Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers &F. Smith: Colorimetric method for determination of sugar and related substances. Anal. Chem. 28, 350–356 (1956)
Dunn, B. M., B. Kammermann &K. R. McCurry: The synthesis, purification and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases. Anal. Biochem. 138, 68–73 (1984)
Edman, P. &A. Begg: A protein sequenator. Eur. J. Biochem. 1, 80–91 (1967)
Edelhoch, H.: Spectroscopic determination of tryptophan and tyrosin in proteins. Biochemistry 6, 1948–1954 (1967)
Erickson, A. H., G. E. Conner &G. Blobel: Biosynthesis of a lysosomal enzyme. J. Biol. Chem. 256, 11224–11231 (1981)
Faust, P. L., S. Kornfeld &J. M. Chirgwin: Cloning and sequence analysis of cDNA for human cathepsin D. Proc. Natl. Acad. Sci. USA 82, 4910–4914 (1985)
Foltmann, B., V. B. Pedersen, H. Jacobsen, D. Kauffman &G. Wybrandt: The complete amino acid sequence of prochymosin. Proc. Nat. Acad. Sci. USA 74, 2321–2324 (1977)
Friedman, M., J. C. Zahnley &J. R. Wagner: Estimation of the disulfide content of trypsin inhibitors as S-β-(2-pyridylethyl)-L-cysteine. Anal. Biochem. 106, 27–34 (1980)
Graham, R. J., Jr.U. Lundholm &M. J. Karnovsky: Cytochemical demonstration of peroxidase activity with 3-amino-9-ethyl-carbazole. J. Histochem. Cytochem. 13, 150–153 (1965)
Hapner, K. D. &P. E. Wilcox: Fragmentation of bovine chymotrypsinogen A and chymotrypsin Aα. Specific cleavage at arginine and methionine residues and separation of peptides including B and C chains of chymotrypsin. Biochemistry 9, 4470–4480 (1970)
Harboe, N. &A. Ingild: Immunization, isolation of immunoglobulins, estimation of antibody titre. In: A Manual of Quantitative Immunoelectrophoresis. Methods and Applications, eds., Axelsen, N. H., J. Krøll & B. Weeke. Universitetsforlaget, Oslo, pp. 161–164 (1973)
Hasilik, A. &E. Neufeld: Biosynthesis of lysosomal enzymes in fibroblasts: Phosphorylation of mannose residues. J. Biol. Chem. 255, 4946–4950 (1980)
Hata, T., R. Hayashi &E. Doi: Purification of yeast proteinases I. Fractionation and some properties of these proteinases. Agr. Biol. Chem. 31, 150–159 (1967)
Hirs, C. H. W.: Determination of cystine as cysteic acid. Methods Enzymol. 11, 59–62 (1967)
Hobart, P. M., M. Fogliano, B. A. O'Connor, I. M. Schaefer &J. M. Chirgwin: Human renin gene: Structure and sequence analysis. Proc. Nat. Acad. Sci. USA 81, 5026–5030 (1984)
Hsu, I.-N., L. T. J. Dalbaere, M. N. G. James &T. Hofmann: Penicillopepsin from Penicillium janthinellum. Crystal structure at 2.8 Å and sequence homology with porcine pepsin. Nature 266, 140–145 (1977)
James, M. N. G. &A. R. Sielecki: Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin. Biochemistry 24, 3701–3713 (1985)
Johansen, J. T., K. Breddam &M. Ottesen: Isolation of carboxypeptidase Y by affinity chromatography. Carlsberg Res. Commun. 41, 1–14 (1976)
Johansen, J. T., C. Overballe-Petersen, B. Martin, V. Hasemann &I. Svendsen: The complete amino acid sequence of copper, zinc superoxide dismutase from Saccharomyces cerevisiae. Carlsberg Res. Commun. 44, 201–217 (1979)
Jones, E. W.: Genetic approaches to the study of protease function and proteolysis in Saccharomyces cerevisiae. In: Yeast Genetics, Fundamental and Applied Aspects, Spencer, J. F. T., D. M. Spencer & A. R. W. Smith Eds., Springer Verlag, New York 167–203 (1983)
Keller, H. P., F. Erni, H. R. Linder &R. W. Frei: Dynamic slurry-packing technique for liquid chromatography columns. Anal. Chem. 49, 1958–1963 (1977)
Krøll, J.: Tandem crossed immunoelectrophoresis. In: Handbook of Immunoprecipitation-in-gel-techniques, Axelsen, N. H. Ed., Blackwell, pp. 135–139 (1983)
Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophag T4. Nature 227, 324–331 (1980)
Lenney, J. F. &J. M. Dalbec: Purification and properties of two proteinases from Saccharomyces cerevisiae. Arch. Biochem. Biophys. 120, 42–48 (1967)
Lenney, J. F., P. Matile, A. Wiemken, M. Schellenberg &J. Meyer: Activities and cellular localization of yeast proteases and their inhibitors. Biochem. Biophys. Res. Commun. 60, 1378–1383 (1974)
Lundblad, R. L. &W. H. Stein: On the reaction of diazoacetyl compounds with pepsin. J. Biol. Chem. 244, 154–160 (1969)
Mahoney, W. C., P. K. Smith &M. A. Hermodson: Fragmentation of proteins with o-iodosobenzoic acid and a reactive contaminant that modify tyrosyl residues. Biochemistry 20, 443–448, (1981)
Mechler, B., M. Müller, F. Meussdoerefer &D. H. Wolf: In vivo biosynthesis of the vacuolar proteinases A and B in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 257, 11203–11206 (1982)
Meussdoerffer, F., P. Tortora &H. Holzer: Purification and properties of proteinase A from yeast. J. Biol. Chem. 255, 12087–12093 (1980)
Misono, K. S., J.-J. Chang &T. Inagami: Amino acid sequence of mouse submaxillary gland renin. Proc. Natl. Acad. Sci. USA 79, 4858–4862 (1982)
Pearl, L. &T. Blundell: The active site of aspartic proteinases. FEBS Lett. 174, 96–101 (1984)
Pedersen, V. B.: (Personal communication)
Saheki, T. &H. Holzer: Comparisons of the tryptophan synthase inactivating enzymes with proteinases from yeast. Eur. J. Biochem. 42, 621–626 (1974)
Saheki, T. &H. Holzer: Proteolytic activities in yeast. Biochim. Biophys. Acta 384, 203–214 (1975)
Schwaiger, H., A. Hasilik, K. von Figura, A. Wiemken &W. Tanner: Carbohydrate-free carboxypeptidase Y is transferred into the lysosome-like yeast vacuole. Biochem. Biophys. Res. Commun. 104, 950–956 (1982)
Sepulveda, P., J. Marciniszyn, D. Liu &J. Tang: Primary structure of porcine pepsin. J. Biol. Chem. 250, 5082–5088 (1975)
Shewale, J. G. &J. Tang: Amino acid sequence of porcine spleen cathepsin D. Proc. Nat. Acad. Sci. USA 81, 3703–3707 (1984)
Sibanda, B. L., T. Blundell, P. M. Hobrart, M. Fogliano, J. S. Bindra, B. W. Dominy &J. M. Chirgwin: Computer graphics modelling of human renin. FEBS Lett. 174, 102–111 (1984)
Stevens, T. H.: (Personal communication)
Subramanian, E., I. D. A. Swan, M. Liu, D. R. Davies, J. A. Jenkins, I. J. Tickle &T. L. Blundell: Homology among acid proteases: Comparison of crystal structures at 3 Å resolution of acid proteases from Rhizopus chinensis and Endothia parasitica. Proc. Natl. Acad. Sci. USA 74, 556–559 (1977)
Svendsen, I., B. Martin &I. Jonassen: Characteristics of hiproly barley III. Amino acid sequence of two-lysine rich proteins. Carlsberg Res. Commun. 45, 79–85 (1980)
Tang, J.: Specific and irreversible inactivation of pepsin by substrate like epoxides. J. Biol. Chem. 246, 4510–4517 (1971)
Twobin, H., T. Staehelin &J. Gordon: Electrophoretic transfer for proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354 (1979)
Wolf, D. H. & H. Holzer: Proteolysis in yeast. In: Microorganisms and Nitrogen Sources, Payne, J. W. Ed., John Wiley and Sons Ltd., 431–458 (1980)
Woolford, C. A., L. B. Daniels, F. J. Park, E. W. Jones, J. V. Arsdell & M. A. Innis: Vacuolar hydrolase muturase of Saccharomyces cerevisiae is an aspartyl protease encoded by the PEP4 gene. (in preparation)
Author information
Authors and Affiliations
Additional information
Accepted byH. Klenow
Rights and permissions
About this article
Cite this article
Dreyer, T., Halkier, B., Svendsen, I. et al. Primary structure of the aspartic proteinase A from Saccharomyces cerevisiae. Carlsberg Res. Commun. 51, 27 (1986). https://doi.org/10.1007/BF02907993
DOI: https://doi.org/10.1007/BF02907993