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

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.