Purification and characterization of endogenous protein activator of human platelet proteasome

Masao Yukawa, Masato Sakon, Jun Ichi Kambayashi, Ei Ichi Shiba, Tomio Kawasaki, Yoshio Uemura, Rohei Murata, Takaharu Tanaka, Toru Nakayama, Hiroshi Shibata, Takesada Mori

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

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°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 μM), while it inhibited the activity at higher substrate concentrations (400-800 μM). It is concluded from these findings that the endogenous protein activator may regulate the intracellular proteasome activity by functioning as a positive allosteric effector.

Original languageEnglish
Pages (from-to)317-323
Number of pages7
JournalJournal of biochemistry
Volume114
Issue number3
DOIs
Publication statusPublished - 1993 Sep
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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