HUMAN MYELOBLASTIN (LEUKOCYTE PROTEINASE-3) - REACTIONS WITH SUBSTRATES, INACTIVATORS AND ACTIVATORS IN COMPARISON WITH LEUKOCYTE ELASTASE

Citation
H. Fruh et al., HUMAN MYELOBLASTIN (LEUKOCYTE PROTEINASE-3) - REACTIONS WITH SUBSTRATES, INACTIVATORS AND ACTIVATORS IN COMPARISON WITH LEUKOCYTE ELASTASE, Biological chemistry, 377(9), 1996, pp. 579-586
Citations number
45
Categorie Soggetti
Biology
Journal title
ISSN journal
14316730
Volume
377
Issue
9
Year of publication
1996
Pages
579 - 586
Database
ISI
SICI code
1431-6730(1996)377:9<579:HM(P-R>2.0.ZU;2-X
Abstract
Human myeloblastin (leukocyte proteinase 3) shares many biochemical pr operties with leukocyte elastase, but rapidly loses enzymatic activity when raising the pH and/or the ionic strength of an acidic solution o r when handled in glass vessels. This poses limits to kinetic experime nts requiring long incubation times. After purification, myeloblastin was conveniently stored in a glycine/HCl buffer at pH 3.2, while assay s were performed in sodium/potassium phosphate buffer at pH 7.0, ionic strength 0.11, in the presence of 0.05% w/v Triton X-100 and taking c are to avoid any contact with glass surfaces. The kinetic parameters o f leukocyte elastase and myeloblastin with peptide substrates, irrever sible inactivators and glycosaminoglycans were compared under these co nditions. cinyl-Lys(2-picolinoyl)Ala-Pro-Val-4-nitroanilide, an excell ent substrate for leukocyte elastase, also proved to be a good substra te for myeloblastin (K-m=16 mu M, k(cat)/K-m=30600 M(-1) s(-1)). Inact ivation of myeloblastin by 3,4-dichloroisocoumarin (k(i)/K-i=6389 M(-1 ) s(-1)) and MeO-Suc-Ala-Ala-Pro-Val-chloromethane (k(i)/K-i=579 M(-1) S-1) occurred via a two-step, irreversible complexing mechanism with potencies one-half and one-fifth that of leukocyte elastase, respectiv ely. Glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and a chondroitin polysulfate, interacted with myeloblastin as non-ess ential activators in the presence of peptide substrates (activation up to a 6.7-fold factor) and as partial inhibitors (about 50% inhibition at saturation) in the presence of elastin. This property distinguishe s myeloblastin from leukocyte elastase, which is always inhibited by g lycosaminoglycans, independently of the substrate.