Formation of a hexokinase complex is associated with changes in energy utilization in celery organs and cells

Abstract

We previously presented evidence that the hexose-regulated repression of the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) may be mediated by hexokinase (EC 2.7.1.1) (HK) [Prata et al. (1997) Plant Physiol 114: 307–314]. To see if differential regulation of HK forms might be involved in the sugar-regulated repression of MTD we characterized two forms of HK with respect to their expression in various plant organs as well as in celery suspension cell cultures. We found that the vast majority of HK activity was membrane-associated, whereas fructokinase (EC 2.7.1.4) was found largely in the soluble cell fraction. Gel filtration chromatography further revealed the differential expression of two molecular size classes of HK. One HK (HK-L) chromatographed at 68 kDa, a typical size for a plant HK, while the second (HK-H) chromatographed at 280 kDa. This unique 280 kDa HK was shown to be composed of a 50 kDa HK protein, possibly complexed with other, as yet unidentified, components. The HK-L was present in all cells and organs analyzed, and thus may be a likely candidate for mediation of sugar repression. In contrast, the presence of the HK-H complex was specific to certain organs and cells grown under certain conditions. Our analyses here showed no correlation between the presence of the HK-H and MTD repression or derepression in celery cells. Instead, the HK-H complex was present exclusively in rapidly growing organs and cells, but not in non-growing celery storage tissues or in carbon-depleted celery suspension-cultured cells. Furthermore, the HK-H complex was present when Glc in the growth media was replaced with 2-deoxy Glc, a HK substrate that does not provide energy for growth and metabolism. These results imply that the HK-H complex may have a potentially unique role in the metabolism of rapidly growing celery cells, in particular, in hexose phosphorylation. We also found that mitochondria prepared from Glc-grown celery suspension-cultured cells contained substantial HK activity, and that oxygen uptake of these mitochondria was stimulated by Glc. These results are consistent with the hypothesis that mitochondrial localization of celery HK may play a role in rapid recycling of adenylate.