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Bovine Lens Epithelial Cells Have a Ubiquitin-Dependent Proteolysis System

Bovine Lens Epithelial Cells Have a Ubiquitin-Dependent Proteolysis System

L L Huang, J Jahngen-Hodge, A Taylor

Biochim Biophys Acta. 1993 Jan 17;1175(2):181-7.

PMID: 8380340

Abstract:

Lens cells must remove obsolete or damaged proteins produced during development, maturation and aging to maintain lens transparency. In reticulocytes removal of abnormal or obsolete proteins is thought to involve a ubiquitin-dependent proteolytic pathway. Two hallmarks of ubiquitin (Ub) dependent proteolysis have previously been demonstrated in lens cell or tissue supernatants: (1) the presence of ubiquitin conjugates, and (2) ATP-dependent proteolysis. Nevertheless, conclusive proof was lacking of a requirement for ubiquitination of substrate proteins for proteolysis. Here we show that in bovine lens epithelial cell (BLEC) supernatant, ATP-dependent proteolysis is also ubiquitin-dependent. Ubiquitin-activating enzyme (E1), the first enzyme in the cascade of ubiquitin ligation, was purified over 3000-fold from a rabbit reticulocyte lysate using Ubiquitin-Sepharose, and showed ATP-PPi exchange activity. Antiserum to E1 was prepared in goats and affinity-purified on Protein G-Sepharose. Western blot analysis revealed that both the goat antiserum and purified antibody (anti-E1(IgG)) recognize specifically E1. Anti-E1(IgG) inhibits 86% of the ATP-dependent degradation of labeled histone H2A in reticulocyte lysate and 75% of the ATP-dependent degradation in BLEC. Upon reconstitution with purified E1, 100% and 80% of proteolysis was restored in reticulocytes and BLEC supernatant, respectively. This confirms that there is a ubiquitin-dependent proteolytic system in lens.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42417000	Anti-phospho-Cyclin E1 (pThr395) antibody produced in rabbit			Price

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