1. Human intestinal brush border peptidases
N Tobey, W Heizer, R Yeh, T I Huang, C Hoffner Gastroenterology. 1985 Apr;88(4):913-26. doi: 10.1016/s0016-5085(85)80008-1.
Hydrolysis of small peptides, like disaccharide hydrolysis, is an important function of the intestinal brush border, but little is known of the individual human peptidases. The purposes of this study were to detect all human brush border enzymes hydrolyzing dipeptides and tripeptides, identify the most discriminating substrate for each enzyme in order to permit assays in crude mixtures, and begin biochemical characterization of each enzyme. Four brush border peptidases were identified. Enzymes I (aspartate aminopeptidase, E.C. 3.4.11.7) and III (amino-oligopeptidase, E.C. 3.4.11.2) are known brush border enzymes. Enzymes II (membrane Gly-Leu peptidase) and IV (zinc stable Asp-Lys peptidase) have not been identified in human brush border previously. They are distinct from dipeptidyl aminopeptidase IV, carboxypeptidase, and gamma-glutamyl transferase. The substrate most discriminating for each enzyme is alpha-Glu-beta-naphthylamide for I (100% of the brush border activity for this substrate is due to enzyme I), glycylleucine for II (80%), leucyl-beta-naphthylamide for III (91%), and aspartyl-lysine in 5 mM Zn2+ for IV (63%). The enzymes are immunologically distinct and antibodies to each one localize to the brush border on immunohistochemical staining. Purification of 142-, 79-, 158-, and 46-fold was achieved for enzymes I through IV, respectively. Biochemical characteristics include slightly alkaline pH optima, molecular weights of 91,000-190,000, and evidence of metal ion involvement in activity. These studies provide necessary information for determining the role of brush border peptidase deficiencies in human disease.
2. Substrate specificity of an adenohypophyseal endopeptidase capable of hydrolyzing luteinizing hormone-releasing hormone: preferential cleavage of peptide bones involving the carboxyl terminus of hydrophobic and basic amino acids
B Horsthemke, K Bauer Biochemistry. 1982 Mar 2;21(5):1033-6. doi: 10.1021/bi00534a032.
The substrate specificity of a peptidase from anterior pituitaries that is capable of hydrolyzing luteinizing hormone-releasing hormone (LH-RH; less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) at the Tyr5-Gly6 peptide bond has been investigated by using inhibitors and model substrates. While trypsin and chymotrypsin inhibitors from plants and animals are without any effect, many microbial protease inhibitors and synthetic peptides containing hydrophobic and basic amino acids inhibit the degradation of radiolabeled LH-RH by this enzyme. The model substrates N-acetyl-Phe-Gly-Leu-beta-naphthylamide, N-acetyl-Leu-Gly-Leu-beta-naphthylamide, and N alpha-benzoyl-Arg-Gly-Leu-beta-naphthylamide are hydrolyzed at the X-Gly peptide bonds; N-acetyl-Gly-Gly-Leu-beta-naphthylamide is not degraded. Hydrolysis of typical amino- and carboxypeptidase substrates was not observed. Degradation of the general protease substrates insulin B chain and denatured hemoglobin also could not be detected. Thus, the enzyme is not LH-RH specific but may be characterized as an endopeptidase that hydrolyzes peptides preferentially at the carboxyl terminus of hydrophobic and basic amino acids.
3. Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids
M Orlowski, C Cardozo, C Michaud Biochemistry. 1993 Feb 16;32(6):1563-72. doi: 10.1021/bi00057a022.
Initial studies on the specificity of the multicatalytic proteinase complex (MPC; EC 3.4.99.46) led to the identification of three distinct proteolytic components designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing, all sensitive to inactivation by 3,4-dichloroisocoumarin (DCI), a general serine proteinase inhibitor. The three components cleave the peptidyl-arylamide bonds in the model synthetic substrates, Z-(D)-Ala-Leu-Arg-2-naphthylamide, Z-Gly-Gly-Leu-p-nitroanilide, and Z-Leu-Leu-Glu-2-naphthylamide, respectively. We report here evidence for the presence in the MPC of two additional distinct components, neither of them capable of cleaving the three model substrates. One of these components cleaves the Leu-Gly and the Leu-Ala bonds in the substrates Cbz-Gly-Pro-Ala-Leu-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Leu-Ala-p-aminobenzoate, respectively, and is activated by treatment of the MPC with DCI, N-ethylmaleimide, Mg2+, Ca2+, and low concentrations of sodium dodecyl sulfate and fatty acids. This component is apparently identical with the previously identified DCI-resistant component of the MPC that cleaves preferentially bonds on the carboxyl side of branched chain amino acids in natural peptides including neurotensin and proinsulin [Cardozo, C., Vinitsky, A., Hidalgo, M. C., Michaud, C., & Orlowski, M. (1992) Biochemistry 31, 7373-7380]. It is probably also identical with the component proposed to be the main factor responsible for the caseinolytic activity [Pereira, M. E., Nguyen, T., Wagner, B. J., Margolis, J. W., Yu, B., & Wilk, S. (1992a) J. Biol. Chem. 267, 7949-7955]. The designation "branched chain amino acid preferring" (BrAAP) is proposed for this component. The second component cleaves peptide bonds between the small neutral amino acids Ala-Gly and Gly-Gly in the substrates Cbz-Gly-Pro-Ala-Ala-Gly-p-aminobenzoate and Cbz-Gly-Pro-Ala-Gly-Gly-p-aminobenzoate, respectively. This component is sensitive to inactivation by DCI, N-ethylmaleimide, and organic mercurials, but unlike the BrAAP it is significantly activated neither by Mg2+ or Ca2+ nor by fatty acids or sodium dodecyl sulfate. The designation "small neutral amino acid preferring" (SNAAP) is proposed for this component. Both components are sensitive to inhibition by the peptidyl-aldehydes N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO; calpain inhibitor I) and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO; calpain inhibitor II) but are resistant to inhibition by Z-LLF-CHO, a potent inhibitor of the chymotrypsin-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)
