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.

Salicylic acid stimulates secretion of the normally symplastic enzyme mannitol dehydrogenase: a possible defense against mannitol-secreting fungal pathogens

“…As previously reported (e.g., Schnarrenberger 1990;Yamamoto et al 2000), we found that HK was not only associated with mitochondria and chloroplasts, but also had a signiWcant cytosolic presence. In contrast, no HK was detected in the apoplast of either untreated or SA treated tissues (Fig.…”

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

“…Different from studies on cytosolic enzymes, phenyl-Sepharose step was 50 times more efficient in purifying maize NC-HK 1 with similar yields ( Table 1), indicating that this is a suitable step for the purification of NC-HKs. Several studies have been performed with non-cytosolic HKs from different plants (1)(2)(3)(5)(6)(7)(8)(9)(10)(11)(12)(13)28,37). However, since these studies focused on the sub-cellular localization or the determination of the kinetic properties of these enzymes, the current study presents for the first time a procedure for the purification of particulate HKs from plants.…”

Spinach SoHXK1 is a mitochondria-associated hexokinase

“…Biochemical studies suggested that hexokinase isozymes diVer in their location within plant cells. Thus, hexokinases have been reported to be associated with mitochondria (Dry et al 1983;Miernyk and Dennis 1983;Tanner et al 1983;Cosio and Bustamante 1984;Schnarrenberger 1990;Galina et al 1995;Wiese et al 1999;Yamamoto et al 2000;da-Silva et al 2001;Giege et al 2003;Rolland and Sheen 2005), the Golgi complex (da-Silva et al 2001), the chloroplast stroma (Miernyk and Dennis 1983;Singh et al 1993) and the chloroplast membrane (Singh et al 1993). Yet, most of these biochemical studies were carried out without knowledge of the number or sequences of the genes encoding hexokinases and the intracellular localization of the corresponding isozymes.…”