Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A

Abstract

Phosphorylation of glucose by hexokinase is the key step in glucose and energy metabolism of the cell. In the Morris hepatoma 3924A, hexokinase II is the predominant hexokinase isoenzyme and occurs in the cytosol as well as bound to membranes. Hexokinase II was isolated by DEAE-cellulose chromatography from both the cytosolic and the mitochondria-enriched fractions and further resolved by hydrophobic-interaction chromatography on phenyl-Sepharose into two components designated hexokinase IIa and IIb. In both the soluble and the mitochondria-enriched fractions, type IIb was the predominant form, but the IIb/IIa ratio was higher in the particulate (6-8) as compared with the cytosolic fraction (1.5-2.0). Binding of the isolated forms of the enzyme to rat liver mitochondria resulted in a 2-10-fold activation of both subtypes. Biochemical characterization showed that both subtypes are closely related to the isoenzyme commonly referred to as hexokinase II, and that the microheterogeneity was not a consequence of contamination with hexokinase I or III. Both subtypes had a molecular mass of 110 kDa, they were inhibited by Pi at concentrations higher than 5 mM, and activated by the detergent CHAPS. The two subtypes differed in electrophoretic mobility (IIa > IIb), in Km values for glucose (IIa, 0.109 mM; IIb, 0.216 mM), in Ki values for glucose 6-phosphate (IIa, 25 microM; IIb, 0.106 mM), and in Ki values for glucose 1,6-biphosphate (IIa, 12.2 microM; IIb, 5.5 microM). An artificial proteolytic cleavage as cause of the hexokinase II microheterogeneity can be excluded, since both subtypes show the same molecular mass and the ability to bind to mitochondria and phenyl-Sepharose. In addition, the relative proportions of the two subtypes did not vary markedly between several enzyme preparations. Northern-blot analysis with a hexokinase II-specific cDNA probe revealed two distinct mRNA transcripts of 5.2 and 6.3 kb in length, which offers the possibility that hexokinase II microheterogeneity is due to differential RNA transcription and/or processing.

Hexokinase expression in liver preneoplasia and neoplasia

“…The observation that cultured MH 3924A cells showed a higher amount of bound enzyme than the solid tumour is in line with the finding that the cell line exhibits a less differentiated phenotype than the solid tumour [5]. Only two HK isoforms corresponding to the low-K, HKI and HKII could be separated chromatographically on DEAE-cellulose from the cytosolic as well as from the mitochondrial fraction of all cell lines and tumours studied [5,6]. In normal rat liver, all four isoforms were resolved by this method.…”

“…The yield of HKI+HKII is related to I00000 g supernatant; the yield of HKlla+llb is related to total HKll activity obtained after DEAE-cellulose chromatography. Reprinted from [5] and it co-migrated with brain HK (HKI) on native PAGE. The increase in cytosolic HKI activity was 2.4-fold in the solid tumour and between 12.1-and 13.5-fold in the cell lines (Table 1).…”

Expression of Two Type II-Like Tumor Hexokinase RNA Transcripts in Cancer Cell Lines

“…However, identification of two HK isoforms in hepatomas [12,13] and the recent report of high-Km glucose phosphorylation activities in a range of tumor cells [11] prompted us to examine the exact pattern of HK gene expression in cancer cells. Employing highly sensitive RT-PCR experiments and Northern blot analyses in this study, we characterized the isozyme pattern of HK gene expression in a range of cancer cell lines of different tissue origin.…”

“…The smaller form is reported to be more active than the larger one [12]. Chromatographic separation of two forms of HK II from Morris hepatoma has also been described [13,14] and a post-translational modification has been suggested as the mechanism which generates these tumor HK isoforms [12]. Characterization of a HK cDNA sequence isolated from a highly glycolytic c37 mouse hepatoma cell line is now identified as that of type I HK isozyme [15].…”

The pivotal roles of mitochondria in cancer: Warburg and beyond and encouraging prospects for effective therapies

“…In contrast, with type II hexokinase retaining the catalytic capacity in both domains [29], the isoform effectively retains the capacity to double the rate of formation of glucose-6-phosphate, relative to isoforms I and III. Therefore, it is not surprising that type II hexokinase is the isoform predominantly expressed in highly malignant tumors [30, 31]. …”