Biomaterial Database

A pepstatin-insensitive aspartic proteinase from a thermophilic Bacillus sp. 1995

H S Toogood, and M Prescott, and R M Daniel

Thermophile Research Unit, University of Waikato, Hamilton, New Zealand.

Bacillus sp. strain Wp22.A1 produced a cell-associated aspartic proteinase which was purified to homogeneity using phenyl-Sepharose (hydrophobic and affinity chromatography) and Mono Q. The proteinase has a molecular mass of 45 kDa by SDS/PAGE and a pI of 3.8. It is insensitive to pepstatin, but is sensitive to the other aspartic proteinase-specific inhibitors diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)propane. Inactivation by DAN was only partial, suggesting that it had non-specifically modified an aspartate residue at a site other than the active site. The enzyme was not inhibited by any of the serine or cysteine proteinase inhibitors tested. Maximum proteolytic activity was observed at pH 3.5. The proteinase had a higher activity with haemoglobin, but was more specific (Vmax./Km) for cytochrome c. Substrate inhibition was observed with both these substrates. The cleavage of oxidized insulin B chain tended to occur at sites where the P1 amino acid was bulky and non-polar, and the P1' amino acid was bulky and polar, such as its primary cleavage site of Val2-Asn3. The proteinase was stable in the pH range 2.5-5.5. Thermostability was increased in the presence of Ca2+, although to a lesser extent at higher temperatures. The thermostabilities at 60, 70, 80 and 90 degrees C were 45 h, 102, 21 and 3 min respectively in the presence of Ca2+.

UI	MeSH Term	Description	Entries
D007328	Insulin	A 51-amino acid pancreatic hormone that plays a major role in the regulation of glucose metabolism, directly by suppressing endogenous glucose production (GLYCOGENOLYSIS; GLUCONEOGENESIS) and indirectly by suppressing GLUCAGON secretion and LIPOLYSIS. Native insulin is a globular protein comprised of a zinc-coordinated hexamer. Each insulin monomer containing two chains, A (21 residues) and B (30 residues), linked by two disulfide bonds. Insulin is used as a drug to control insulin-dependent diabetes mellitus (DIABETES MELLITUS, TYPE 1).	Iletin,Insulin A Chain,Insulin B Chain,Insulin, Regular,Novolin,Sodium Insulin,Soluble Insulin,Chain, Insulin B,Insulin, Sodium,Insulin, Soluble,Regular Insulin
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008670	Metals	Electropositive chemical elements characterized by ductility, malleability, luster, and conductance of heat and electricity. They can replace the hydrogen of an acid and form bases with hydroxyl radicals. (Grant & Hackh's Chemical Dictionary, 5th ed)	Metal
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010084	Oxidation-Reduction	A chemical reaction in which an electron is transferred from one molecule to another. The electron-donating molecule is the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or redox pairs (Lehninger, Principles of Biochemistry, 1982, p471).	Redox,Oxidation Reduction
D010436	Pepstatins	N-acylated oligopeptides isolated from culture filtrates of Actinomycetes, which act specifically to inhibit acid proteases such as pepsin and renin.
D010455	Peptides	Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS.	Peptide,Polypeptide,Polypeptides
D011480	Protease Inhibitors	Compounds which inhibit or antagonize biosynthesis or actions of proteases (ENDOPEPTIDASES).	Antiprotease,Endopeptidase Inhibitor,Endopeptidase Inhibitors,Peptidase Inhibitor,Peptidase Inhibitors,Peptide Hydrolase Inhibitor,Peptide Hydrolase Inhibitors,Peptide Peptidohydrolase Inhibitor,Peptide Peptidohydrolase Inhibitors,Protease Antagonist,Protease Antagonists,Antiproteases,Protease Inhibitor,Antagonist, Protease,Antagonists, Protease,Hydrolase Inhibitor, Peptide,Hydrolase Inhibitors, Peptide,Inhibitor, Endopeptidase,Inhibitor, Peptidase,Inhibitor, Peptide Hydrolase,Inhibitor, Peptide Peptidohydrolase,Inhibitor, Protease,Inhibitors, Endopeptidase,Inhibitors, Peptidase,Inhibitors, Peptide Hydrolase,Inhibitors, Peptide Peptidohydrolase,Inhibitors, Protease,Peptidohydrolase Inhibitor, Peptide,Peptidohydrolase Inhibitors, Peptide
D011489	Protein Denaturation	Disruption of the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein.	Denaturation, Protein,Denaturations, Protein,Protein Denaturations
D004795	Enzyme Stability	The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat.	Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme