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A multicatalytic high-molecular-weight neutral endopeptidase from human kidney.
Arch Biochem Biophys. 1987 Oct; 258(1):42-50.AB

Abstract

A multicatalytic endopeptidase (ME) with three distinct activities, chymotrypsin-like, cucumisin-like, and trypsin-like, occurred in all rat tissues examined with highest activities in kidney, testes, liver, and spleen; they were assayed with benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide (Z-Gly-Gly-Leu-pNA), benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Z-Leu-Leu-Glu-2NA), and benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide (Z-Gly-Gly-Arg-2NA), respectively. All three activities were recovered from a single protein band on a polyacrylamide gel after electrophoresis of purified human kidney ME. The native enzyme had a Mr of 650,000, and it consisted of about 5,135 amino acid residues. After denaturation and electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels kidney ME dissociated into several low Mr components ranging from 23,000 to 33,000. Kidney ME had a pH optimum of 7.6-8.1 with Z-Gly-Gly-Leu-pNA, 7.3 with Z-Leu-Leu-Glu-2NA, and 9.8 with Z-Gly-Gly-Arg-2NA. SDS enhanced chymotrypsin- and cucumisin-like activities by two to three times whereas trypsin-like activity was not enhanced. The specificity constant (kappa cat/Km) of human kidney ME for Z-Gly-Gly-Leu-pNA was 6.7 X 10(3) M-1 S-1; Z-Gly-Gly-Leu-2NA was not hydrolyzed. The specificity constant for Z-Leu-Leu-Glu-2NA was similar to, and for Z-Gly-Gly-Arg-2NA was one half of that for Z-Gly-Gly-Leu-pNA. ME cleaves only the Phe5-Ser6 bond of bradykinin (BK); however, all three ME activities were inhibited by BK. Strong inhibition of ME by albumin suggests that ME is involved in cleavage of larger polypeptides. Antipain and leupeptin almost completely inactivated the trypsin-like activity whereas they had no significant effect on the other two activities. ME is not a metal-loenzyme nor is the serine residue essential for its activities; however, thiol groups are involved. Na+ and K+ inhibited all ME activities. Trypsin-like activity was more sensitive to divalent cations than the other two.

Authors+Show Affiliations

Zolfaghari R
Department of Surgery, Texas Tech University School of Medicine, Lubbock 79430.
Baker CR
No affiliation info available
Amirgholami A
No affiliation info available
Canizaro PC
No affiliation info available
Behal FJ
No affiliation info available

MeSH

Amino AcidsCatalysisCationsChymotrypsinElectrophoresis, Polyacrylamide GelEndopeptidasesHumansHydrogen-Ion ConcentrationKidneyKineticsMolecular WeightNeprilysinPeptidesProtease InhibitorsSerine EndopeptidasesSubstrate SpecificityTrypsin

Pub Type(s)

Comparative Study
Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

3310903

Citation

Zolfaghari, R, et al. "A Multicatalytic High-molecular-weight Neutral Endopeptidase From Human Kidney." Archives of Biochemistry and Biophysics, vol. 258, no. 1, 1987, pp. 42-50.
Zolfaghari R, Baker CR, Amirgholami A, et al. A multicatalytic high-molecular-weight neutral endopeptidase from human kidney. Arch Biochem Biophys. 1987;258(1):42-50.
Zolfaghari, R., Baker, C. R., Amirgholami, A., Canizaro, P. C., & Behal, F. J. (1987). A multicatalytic high-molecular-weight neutral endopeptidase from human kidney. Archives of Biochemistry and Biophysics, 258(1), 42-50.
Zolfaghari R, et al. A Multicatalytic High-molecular-weight Neutral Endopeptidase From Human Kidney. Arch Biochem Biophys. 1987;258(1):42-50. PubMed PMID: 3310903.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A multicatalytic high-molecular-weight neutral endopeptidase from human kidney. AU - Zolfaghari,R, AU - Baker,C R, AU - Amirgholami,A, AU - Canizaro,P C, AU - Behal,F J, PY - 1987/10/1/pubmed PY - 1987/10/1/medline PY - 1987/10/1/entrez SP - 42 EP - 50 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 258 IS - 1 N2 - A multicatalytic endopeptidase (ME) with three distinct activities, chymotrypsin-like, cucumisin-like, and trypsin-like, occurred in all rat tissues examined with highest activities in kidney, testes, liver, and spleen; they were assayed with benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide (Z-Gly-Gly-Leu-pNA), benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Z-Leu-Leu-Glu-2NA), and benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide (Z-Gly-Gly-Arg-2NA), respectively. All three activities were recovered from a single protein band on a polyacrylamide gel after electrophoresis of purified human kidney ME. The native enzyme had a Mr of 650,000, and it consisted of about 5,135 amino acid residues. After denaturation and electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels kidney ME dissociated into several low Mr components ranging from 23,000 to 33,000. Kidney ME had a pH optimum of 7.6-8.1 with Z-Gly-Gly-Leu-pNA, 7.3 with Z-Leu-Leu-Glu-2NA, and 9.8 with Z-Gly-Gly-Arg-2NA. SDS enhanced chymotrypsin- and cucumisin-like activities by two to three times whereas trypsin-like activity was not enhanced. The specificity constant (kappa cat/Km) of human kidney ME for Z-Gly-Gly-Leu-pNA was 6.7 X 10(3) M-1 S-1; Z-Gly-Gly-Leu-2NA was not hydrolyzed. The specificity constant for Z-Leu-Leu-Glu-2NA was similar to, and for Z-Gly-Gly-Arg-2NA was one half of that for Z-Gly-Gly-Leu-pNA. ME cleaves only the Phe5-Ser6 bond of bradykinin (BK); however, all three ME activities were inhibited by BK. Strong inhibition of ME by albumin suggests that ME is involved in cleavage of larger polypeptides. Antipain and leupeptin almost completely inactivated the trypsin-like activity whereas they had no significant effect on the other two activities. ME is not a metal-loenzyme nor is the serine residue essential for its activities; however, thiol groups are involved. Na+ and K+ inhibited all ME activities. Trypsin-like activity was more sensitive to divalent cations than the other two. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/3310903/A_multicatalytic_high_molecular_weight_neutral_endopeptidase_from_human_kidney_ DB - PRIME DP - Unbound Medicine ER -