Purification and characterization of endogenous protein activator of human platelet proteasome.
An endogenous activator of 20S proteasome was purified from human platelets and its effect on three peptidase activities of proteasome was studied. This activator had a molecular weight of 170 kDa, and was composed of 32 kDa polypeptides as determined by SDS-PAGE. It was highly labile upon heat treatment (56 degrees C, 20 s) and proteinase (pronase CB) digestion. Suc-LLVY-MCA degrading activity of the platelet proteasome showed positive cooperativity between two or more catalytic sites because the coefficient was 1.54 when analyzed by use of the Hill plot. The endogenous activator increased Vmax and caused a loss of cooperativity. The plot of reaction velocity as a function of activator concentration yielded a saturation curve, implying the binding of the activator to proteasome. Boc-LTR-MCA degrading activity followed Michaelis-Menten kinetics. The activator enhanced the activity by increasing Vmax and decreasing Km. In contrast, CBz-LLE-2NA degrading activity could not be analyzed according to any kinetic scheme reported so far. The activator stimulated this activity at lower substrate concentrations (below 200 microM), while it inhibited the activity at higher substrate concentrations (400-800 microM). It is concluded from these findings that the endogenous protein activator may regulate the intracellular proteasome activity by functioning as a positive allosteric effector.
Yukawa
M
M
Department of Surgery II, Osaka University Medical School.
Sakon
M
M
Kambayashi
J
J
Shiba
E
E
Kawasaki
T
T
Uemura
Y
Y
Murata
K
K
Tanaka
T
T
Nakayama
T
T
Shibata
H
H
D001792:Blood Platelets / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000097:blood*; D010450:Endopeptidases / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000097:blood*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011402:Pronase; D046988:Proteasome Endopeptidase Complex
D009097:Multienzyme Complexes; D010455:Peptides; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D011402:Pronase; D046988:Proteasome Endopeptidase Complex