Isolation and sequence of cDNA encoding the soybean protease inhibitors PI IV and C-II

Two full-length (or nearly so) cDNA clones containing information for the protease inhibitors PI IV and C-II from soybean seeds were identified by means of a synthetic probe. DNA sequencing revealed that the two protease inhibitors are synthesized as precursors with a short peptide leader. The coding regions of the two clones show 80% homology, wheraes the 5 non-coding regions are 90% homologous. Homology of 75% is found in the region extending beyond the stop codons.     

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells

Summary The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH₂-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH₂-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.     

Purification of an acid protease from Mucor rouxii that inactivates chitin synthetase

An acid protease was purified from the mycelial form of Mucor rouxii by a method which involved salt and acid precipitation, gel filtration and anion-exchange chromatography. The enzyme had a molecular mass of 16,000 Da. Its optimum pH was 4.0, maximal activity was obtained at 50°C, and it was inactivated at 70°C. It was not affected by leupeptin or N -p-tosyl-L-lysine chloromethyl ketone (TLCK) but diazoacetyl-DL-norleucine methyl ester (DNME) in the presence of Cu²⁺ and more noticeably pepstatin A, strongly inhibited the activity. This acid protease did not activate zymogenic chitin synthetase from the fungus, but brought about its inactivation even at low concentrations and after short periods of incubation time.Abbreviations TLCK N -p-tosyl-L-lysine chloromethyl ketone - DNME diazoacetyl-DL-norleucine methyl ester - TCA trichloroacetic acid - SDS sodium dodecyl sulfate     

Analysis of the biomacromolecular architecture of eukaryotic and prokaryotic serine proteases

Summary We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered on the serine proteases and their natural inhibitors and substrates. It is essential that these approaches emphasize the comparison of these macromolecules at the separate levels of secondary, tertiary and quaternary structure. We assume in our analysis that in functionally related macromolecules (i.e., a family of evolutionarily related enzymes), regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of observed specificity. It is the intent of our research to encapsulate such knowledge in a form which is capable of observing patterns which may serve as generalizable rules for macrostructural analysis (Liebman, M.N. 1986. Enzyme 36: 150–163), and to serve as the essential tools for the rational design of modified serine proteases and/or their natural inhibitors by the methods available through genetic engineering.     

Michaelis complexes of porcine pancreatic elastase with 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins: translational sampling of inhibitor position and kinetic measurements.

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed.     

Cloning and sequencing of the cDNA encoding the major albumin of Theobroma cacao

The major albumin, a polypeptide of 21 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), has been identified and partially purified by preparative gel electrophoresis. Some N-terminal sequence was obtained, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 221-amino-acid polypeptide of M_r 24003. The existence of a precursor to the 21-kDa polypeptide of this size was confirmed by immunoprecipitation from total poly(A)⁺ RNA translation products. The polypeptide has a hydrophobic signal sequence of 26 amino acids before the mature start, and the mature polypeptide would have an M_r of 21223. The protein sequence is homologous with sequences of the Kunitz protease and -amylase inhibitor family, and the protein probably functions to defend the seed's protein reserves from the digestive enzymes of invading pests. However because the protein comprises 25–30% of the total seed protein it may itself also function as a storage protein. Electron micrographs of immunogold-labelled embryo sections show that the protein is located in membrane-enclosed organelles.Abbreviations cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies, and to Dr G. Cope, of the Biological Sciences Electron Microscopy Unit, Sheffield University, for taking the electron micrographs.To whom correspondence should be addressed.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

Identification of the peptide bond cleaved during activation of human Clr

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ M_τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly     

Crystal structure of subtilisin complexed with its trapped substrateStreptomyces subtilisin inhibitor—Protein-protein interaction and evolution of serine proteinases and their proteinaceous inhibitors

The complex of a bacterial alkaline serine proteinase, subtilisin BPN’, with its proteinaceous inhibitorStreptomyces subtilisin inhibitor is unique in several respects, compared with other similar complexes containing serine proteinases of trypsin family. In addition to the usual antiparallelβ-sheet involving P₁-P₃ residues of the inhibitor, P₄-P₆ residues form antiparallelβ-sheet with a previously unnoticed chain segment (the ‘S4-6 site’) of subtilisin. The ‘S4-6 site’ does not exist in serine proteinases of trypsin family, whether of mammalian or microbial origin. Global induced-fit movement seems to occur on the ‘trapped substrate’Streptomyces subtilisin inhibitor: a channel-like structure in SSI remote from the contact region becomes about 2 Å wider upon complexing with subtilisin. Main role of the secondary contact region ofStreptomyces subtilisin inhibitor seems to support the reactive site loop (primary contact region). Steric homology for the two contact regions is so high between the inhibitors ofStreptomyces subtilisin inhibitor family and those of pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family that it seems to favour a divergent evolution and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forwarded by Doolittle(Nature (London),272, 581, 1978).     

Protease activities in non-embryogenic and embryogenic carrot cell strains during callus growth and embryo formation

Embryogenic and non-embryogenic cell strains of Daucus carota L. were examined for their protease activity using a wide range of chromogenic synthetic peptides as substrates. High arginine-specific activity was present in all strains, but no protease activity "specific" for embryogenic or non-embryogenic strains could be detected with the substrates tested. The specific protease activity was 5–10 times higher in the non-embryogenic as compared to the embryogenic strain for most tested substrates, and this difference was not due to release of proteases in the latter. All strains showed a decrease in protease activity when cultured in media without 2,4-dichlorophenoxyacetic acid, but the embryos had high protease activity in comparison with the nondifferentiated cell aggregates. In the latter aggregates, hydrolyzing activity towards three of the substrates (H-D-Phe-Pip-Arg- p -nitroanilide, Suc-Ala-Pro-Phe- p -nitro-anilide and Bz-Phe-Val-Arg- p -nitroanilide) was absent, whereas the embryos were able to hydrolyze them.