Koko Murakami

Gene Expression and Biological Significance of Hexokinase in Erythroid Cells

Red blood cells (RBCs) express two hexokinase (HK) isoforms, HK-I and HK-R. Both isozymes are generated from the HK-I gene by use of an alternate promoter. Gene structure and exon-intron organization of the HK-I gene have been elucidated from a sequence of three contiguous genomic clones localized at human chromosome 10. The sequence spans about 131 kb, and consists of 25 exons, which include 6 testis- and 1 erythroid-specific exons. HK-R has been shown as an erythroid-specific isozyme whose expression is turned on in the early erythroid-progenitors and is significantly induced during their differentiation. HK-R unfolds major HK activity in immature RBCs and is rapidly degraded during the maturation process. HK-I has a porin-binding domain in its N-terminus. Recent studies have shown that HK isozymes with a porin-binding domain play a role in mitochondrial integrity, suggesting that HK-I-deficient erythroid cells might be eliminated by apoptosis. It is most likely that RBCs are most labile as a result of HK-I/R deficiency since the HK-I gene but not the other isozyme genes are expressed in fetal and adult RBCs.

Decay of mammalian hexokinase: characterization of the specific proteolytic activity.

Hexokinase-degrading activity (HKDA) is associated with hexokinase throughout purification. It is not ubiquitin-dependent. With glucose present, HKDA requires MgATP. It is triggered by removal (or ATP-dependent consumption) of glucose, and masked when glucose is restored. PCMB, TLCK and TPCK suppress hexokinase and activate HKDA. Glucose removal and active-site blockage appear to induce HKDA.

Properties of the ubiquitin conjugation system from bovine eye lens.

A post-translational protein modification system involving the polypeptide ubiquitin results in ubiquitin-protein conjugates of various functions. A ubiquitin-conjugating enzyme system was isolated from the epithelial tissue of bovine eye lens by DEAE-Sepharose and Bio-Gel A-1.5m column chromatography. The lens system shows similar enzymatic properties to the one from rabbit reticulocytes: requirement for ATP and sensitivity to thiol reagents. Two sets of prominent ubiquitin conjugates were formed with endogenous ubiquitin-acceptor proteins from fractions of the Bio-Gel column: a pair of ubiquitin conjugates of approximately 130 kDa and others with very high molecular mass. Extreme specificity is indicated by the ability of the lens system to catalyze conjugation of ubiquitin to the few endogenous acceptor proteins, or to histone H2B, but not to lysozyme, S-carboxymethylated bovine serum albumin, or native or heat-denatured lens alpha crystallin.

The Molecular Basis of Different Hexokinase (HK) Defects; Two Isozymes (HKr and HK1) Produced from a Single Gene by Alternate Promoters ♦ 787

The Molecular Basis of Different Hexokinase (HK) Defects; Two Isozymes (HKr and HK1) Produced from a Single Gene by Alternate Promoters ♦ 787

The Isoenzymes of Mammalian Hexokinase: Tissue Specificity and In Vivo Decline

The reticulocyte loses most of its intracellular organelles during maturation into the red blood cell (RBC). The RBC remains dependent on the preformed enzymatic machinery throughout its 120 day life-span, as it is incapable of protein synthesis. This provides an excellent model of molecular aging.