Purification and characterization of a neutral zinc endopeptidase secreted by Flavobacterium meningosepticum.
Flavobacterium meningosepticum, Elder strain (ATCC 33958), secretes into the medium a neutral zinc endoprotease as a major component of the extracellular proteins. The enzyme was purified to homogeneity in a simple two-step procedure involving ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular weight of this metalloprotease was determined to be about 27,000 (P27) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P27 was comparable to thermolysin in the relative rates of elastin-orcein, azocasein, and azoalbumin hydrolysis. P27 and thermolysin hydrolyzed equally well 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the same primary sites that are susceptible to cleavage by vertebrate collagenases, Gly-Ile, and Gly-Leu. P27 was also capable of partially hydrolyzing Type I acid-soluble calf skin collagen and slowly hydrolyzing N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala, a bacterial collagenase substrate not cleaved by thermolysin. P27 was further differentiated from thermolysin from the inability of the former to hydrolyze N-[3-(2-furyl)acryloyl]-Gly-Leu-NH2. In addition, a vertebrate elastase substrate succinyl-Ala-Ala-Ala-p-nitroanilide was hydrolyzed by P27 but not by thermolysin. P27 is a newly described and unique enzyme from the standpoint of substrate specificity and from the fact that it is resistant to inhibition by phosphoramidon, an inhibitor of a number of zinc endopeptidases, including thermolysin.
Grimwood
B G
BG
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Plummer
T H
TH
Tarentino
A L
AL
D000595:Amino Acid Sequence; D011232:Chemical Precipitation; D002845:Chromatography; D004492:Edetic Acid / Q000494:pharmacology; D005417:Flavobacterium / Q000201:enzymology*; D006020:Glycopeptides / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015260:Neprilysin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000378:metabolism; D017385:Sequence Homology; D013379:Substrate Specificity; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000032:analysis / Q000494:pharmacology
D006020:Glycopeptides; D008670:Metals; D010446:Peptide Fragments; D010455:Peptides; D004492:Edetic Acid; D015260:Neprilysin; D013820:Thermolysin; D015032:Zinc; C008890:phosphoramidon