Human myeloblastin (leukocyte proteinase 3): reactions with substrates, inactivators and activators in comparison with leukocyte elastase.
Human myeloblastin (leukocyte proteinase 3) shares many biochemical properties with leukocyte elastase, but rapidly loses enzymatic activity when raising the pH and/or the ionic strength of an acidic solution or when handled in glass vessels. This poses limits to kinetic experiments requiring long incubation times. After purification, myeloblastin was conveniently stored in a glycine/HCl buffer at pH 3.2, while assays 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 care to avoid any contact with glass surfaces. The kinetic parameters of leukocyte elastase and myeloblastin with peptide substrates, irreversible inactivators and glycosaminoglycans were compared under these conditions. MeO-succinyl-Lys(2-picolinoyl)Ala-Pro-Val-4-nitroanilide, an excellent substrate for leukocyte elastase, also proved to be a good substrate for myeloblastin (Km = 16 microM, kcat/Km = 30,600 M(-1)s(-1)). Inactivation of myeloblastin by 3,4-dichloroisocoumarin (ki/Ki = 6,389 M(-1)s(-1)) and MeO-Suc-Ala-Ala-Pro-Val-chloromethane (ki/Ki = 579 M(-1) S(-1)) occurred via a two-step, irreversible complexing mechanism with potencies one-half and one-fifth that of leukocyte elastase, respectively. Glycosaminoglycans such as chondroitin sulfate, dermatan sulfate and a chondroitin polysulfate, interacted with myeloblastin as non-essential 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 distinguishes myeloblastin from leukocyte elastase, which is always inhibited by glycosaminoglycans, independently of the substrate.
Früh
H
H
University Hospital, Department of Rheumatology, Zurich, Switzerland.
Kostoulas
G
G
Michel
B A
BA
Baici
A
A
D004789:Enzyme Activation / Q000187:drug effects; D006025:Glycosaminoglycans / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D053146:Myeloblastin; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
D006025:Glycosaminoglycans; D015842:Serine Proteinase Inhibitors; D012965:Sodium Chloride; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D053146:Myeloblastin