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PubMed1988

Cutaneous leishmaniasis in Jordan: biochemical identification of human and Psammomys obesus isolates as Leishmania major.

As part of a series of epidemiological and ecological studies of leishmaniasis in Jordan, we have made functional studies of four isolates from human lesions and from ear lesions of three field-collected Psammomys obesus. Primary isolates were subcultured, frozen stabilates prepared and BALB/c mouse infectivity experiments initiated. Each mouse was inoculated with 4-8 x 10(4) promastigotes into a hind footpad. Quantitative evaluation of the footpads showed enlargement three to four weeks postinoculation. Amastigotes were readily identified in smears from footpad lesions and promastigotes in culture. At 47 days, liver and spleen samples grew out promastigotes. Biochemical characterization of these seven isolates was made by isozyme analysis using cellulose acetate membrane electrophoresis of fructokinase, phosphoglucose isomerase, phosphoglucomutase, aspartate aminotransferase, malate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Reference isolates used for comparison were Leishmania major, L. tropica minor, L. donovani, L. aethiopica and L. m. mexicana. All seven Jordan isolates showed enzyme electromorphs identical to L. major, confirming our ecological/epidemiological studies that P. obesus is a major reservoir for human cutaneous leishmaniasis in Jordan.

Animals↗
PubMed1994

Rodents as reservoir hosts of cutaneous leishmaniasis in Jordan.

Rodents were collected from endemic foci of cutaneous leishmaniasis in Jordan, either by flooding their burrows with water or using Sherman traps. Of the 170 jirds (Psammomys obesus) collected, 39 (23%) had Leishmania amastigotes in one or both ears. Although cultures of ear biopsies from the infected animals were all positive, cultures made using biopsies from their noses, livers or spleens were all negative. The infected jirds were encountered in seven of the nine areas studied. Biochemical characterization of six isolates from P. obesus, using cellulose acetate electrophoresis of six enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, malic enzyme, phosphoglucomutase, phosphoglucoisomerase and fructokinase) showed that the jird isolates were isoenzymatically identical with two Jordanian human Leishmania isolates and reference isolates of L. major but differed from reference strains of L. tropica. None of the other rodents caught (Meriones libycus, M. crassus, M. tristrami, Allactaga euphratica and Gerbillus spp.) yielded Leishmania parasites, confirming that P. obesus is the major reservoir host of cutaneous leishmaniasis in Jordan.

Animals↗
PubMed1964

QUANTITATIVE STUDIES OF WHITE MATTER. II. ENZYMES INVOLVED IN TRIOSE PHOSPHATE METABOLISM.

Methods for measurement of glyceraldehyde-P dehydrogenase, triose-P isomerase, fructose 1,6-diphosphate aldolase, and the DPN-linked and flavin-linked alpha-glycero-P dehydrogenases in small amounts of tissue have been worked out. These enzymes have been measured in ten tracts in rabbit central nervous system. The activities of all the enzymes measured, except the flavin-linked alpha-glycero-P dehydrogenase, are present in larger amounts in lightly myelinated than in heavily myelinated tracts, but are relatively low in fibrillar layer of olfactory bulb, which is unmyelinated. Aldolase, like P-fructokinase (measured previously), is especially low in fibrillar layer. Taken together with relatively high 6-P-gluconate dehydrogenase activity found earlier this supports the hypothesis that the pentose-P shunt is particularly active in this tract. The activity of DPN-linked alpha-glycero-P dehydrogenase is inversely proportional to the lipid content of the myelinated tracts, which suggests that its primary role is not related to lipid synthesis in adult brain. The activities of flavin-linked alpha-glycero-P dehydrogenase are unrelated to those of the DPN-linked enzyme, which is contrary to expectation if the two enzymes function as partners in the "alpha-glycerophosphate shuttle."

Animals↗
PubMed1997

Sequence analysis of the groESL-cotA region of the Bacillus subtilis genome, containing the restriction/modification system genes.

We have determined a 35-kb sequence of the groESL-gutR-cotA (45 degrees-52 degrees) region of the Bacillus subtilis genome. In addition to the groESL, gutRB and cotA genes reported previously, we have newly identified 24 ORFs including gutA and fruC genes, encoding glucitol permease and fructokinase, respectively. The inherent restriction/modification system genes, hsdMR and hsdMM, were mapped between groESL and gutRB, and we have identified two open reading frames (ORFs) encoding 5-methylcytosine forming DNA methyl transferase and an operon probably encoding a restriction enzyme complex. The unusual genome structure of few ORFs and lower GC content around the restriction/modification genes strongly suggests that the region originated from a bacteriophage integrated during evolution.

Amino Acid Sequence↗
PubMed2001

Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa.

The phosphorylation of glucose and fructose is an important step in regulating the supply of hexose sugars for biosynthesis and metabolism. Changes in leaf hexokinase (EC 2.7.1.1) activity and in vivo metabolite levels were examined during drying in desiccation-tolerant Sporobolus stapfianus and Xerophyta viscosa. Leaf hexokinase activity was significantly induced from 85% to 29% relative water content (RWC) in S. stapfianus and from 89% to 55% RWC in X. viscosa. The increase in hexokinase corresponded to the region of sucrose accumulation in both species, with the highest activity levels coinciding with region of net glucose and fructose removal. The decline of hexose sugars and accumulation of sucrose in both plant species was not associated with a decline in acid and neutral invertase. The increase in hexokinase activity may be important to ensure that the phosphorylation and incorporation of glucose and fructose into metabolism exceeded production from potential hydrolytic activity. Total cellular glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) levels were held constant throughout dehydration. In contrast to hexokinase, fructokinase activity was unchanged during dehydration. Hexokinase activity was not fully induced in leaves of S. stapfianus dried detached from the plant, suggesting that the increase in hexokinase may be associated with the acquisition of desiccation-tolerance.

Carbohydrate Metabolism↗
PubMed2002

In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues.

A powerful technique is described to localize the activities of a range of enzymes in a wide variety of plant tissues. The method is based on the coupling of the enzymatic reaction to the reduction of NAD and subsequent reduction and precipitation of nitroblue tetrazolium. Enzymes that did not reduce NAD could be visualized by coupling their activities to glucose-6-phosphate dehydrogenase activity via one or more intermediary 'coupling' enzymes. The method is shown to be applicable for the detection of the activities of hexokinase, fructokinase, sucrose synthase, uridine 5'-diphospho-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase, phosphoglucomutase, and phosphoglucose isomerase. It could be used for all tissues tested, including green leaves, stems, roots, fruits, and seeds. The method is specific, very sensitive, and has a high spatial resolution, giving information at the cellular and the subcellular level. The localization of sucrose synthase, invertase, and uridine 5'-diphospho-glucose pyrophosphorylase in transgenic potato plants, carrying a cytokinin biosynthesis gene, is studied and compared with wild-type plants.

Alkyl and Aryl Transferases↗
PubMed1990

Genetic relationships among phlebotomine sand flies (Diptera: Psychodidae) in the verrucarum species group.

Isozyme data were used to identify populations of certain Lutzomyia taxa in the verrucarum species group, mostly in the series townsendi. Lutzomyia youngi Feliciangelis and Murillo and L. spinicrassa Morales, Osorno, Osorno, and Hoyos each have diagnostic allomorphs for phosphogluconate dehydrogenase (6PGDH) and fumarate hydratase (FUM). The 6PGDH and FUM data and those from 6-phospho-fructokinase and phosphoglucomutase distinguish Lutzomyia sp., a new species from Columbia to be described and named later. Data from these enzymes and glucose phosphate isomerase will separate L. townsendi (Ortiz) from the others and from L. longiflocosa Osorno, Morales, Osorno, and Hoyos, L. quasitownsendi Morales, Osorno, Osorno, and Hoyos, and L. sauroida Osorno, Morales, and Osorno (three species that are inseparable using enzyme data). Three other species, L. serrana (Damasceno and Arouck) (series serrana) and L. columbiana (Ristorcelli and Van Ty) and L. andina Osorno, Osorno, and Morales (both in series verrucarum), are morphologically distinct using conventional characters and have fixed diagnostic differences at several enzyme loci. Statistical analyses of the enzyme data using genetic identities (I), differences (D), and the amount of genetic variation among these taxa indicated that such statistics can be as useful in the study of sand fly phylogeny and population genetics as they have been for other organisms. I and D values indicated that L. longiflocosa, L. quasitownsendi, and L. sauroida are very similar (I = 0.991 and D = 0.010) and possibly are populations of the same conspecific species. The levels of divergence, based on combined enzyme data for up to 21 gene loci among the taxa, are discussed, and a dendrogram based on genetic distance is presented. The genetic data confirmed established phylogenetic relationships among the sand fly taxa based on structural similarities.

Animals↗
PubMed1989

Sucrose metabolic pathways in sweetgum and pecan seedlings.

Sucrose metabolism and glycolysis were studied in one- to two-year-old seedlings of sweetgum (Liquidambar styraciflua L.) and pecan (Curya illinoinensis (Wangenh.) C. Koch). The sucrose synthase pathway was identified as the dominant sucrose metabolic activity in sucrose sink tissues such as terminal buds and the root cambial zone. The sucrose synthase pathway was completely dependent on uridine diphosphate and pyrophosphate and it was activated by fructose 2,6-bisphosphate. Both acid and neutral invertases were less active than sucrose synthase in sucrose sink tissues. According to the magnitude of seasonal changes in activity, sucrose synthase, the pyrophosphate-dependent phosphofructokinase, and fructokinase were identified as adaptive enzymes, whereas neutral invertase, uridine diphosphate-glucopyrophosphorylase, phosphoglucomutase, and the nonspecific, nucleotide triphosphate-dependent phosphofructokinase were identified as maintenance enzymes. The periodically high activities of pyrophosphate-dependent phosphofructokinase indicate that pyrophosphate can serve as an energy source in trees. The observations support the hypothesis that sucrose glycolysis and gluconeogenesis in plants proceed by a network of alternative enzymes and substrates.

Journal Article↗
PubMed1978

An alternative pathway for the degradation of endogenous fructose during the catabolism of sucrose in Rhodopseudomonas capsulata.

Sucrose catabolism was studied in Rhodopseudomonas capsulata. Sucrose was hydrolysed by the action of a constitutive cytoplasmic sucrase. The use of a glucose-6-phosphate dehydrogenase-deficient mutant and radiorespirometric experiments demonstrated that both the glucose and fructose moieties of sucrose were catabolized via the Entner-Doudoroff pathway. This result was confirmed by enzyme analysis and studies on sugar assimilation. All the enzymes of the Entner-Doudoroff pathway were present in bacteria grown on secrose but fructokinase (EC 1.7.1.4) activity was relatively low. In contrast, phosphoenolpyruvate:fructose phosphotransferase and 1-phosphofructokinase, the key enzymes for the catabolism of exogenous fructose, were only partially induced. Bacteria grown on sucrose and treated with chloramphenicol were, therefore, not able to assimilate exogenous fructose. We conclude that under these conditions endogenous fructose is catabolized via the Entner-Douboroff pathway, while exogenous fructose is degraded via fructose 1-phosphate and the Embden-Meyerhof pathway.

Fructose↗
PubMed2002

Metabolism of sucrose and its five isomers by Fusobacterium mortiferum.

Fusobacterium mortiferum utilizes sucrose [glucose-fructose in alpha(1-->2) linkage] and its five isomeric alpha-D-glucosyl-D-fructoses as energy sources for growth. Sucrose-grown cells are induced for both sucrose-6-phosphate hydrolase (S6PH) and fructokinase (FK), but the two enzymes are not expressed above constitutive levels during growth on the isomeric compounds. Extracts of cells grown previously on the sucrose isomers trehalulose alpha(1-->1), turanose alpha(1-->3), maltulose alpha(1-->4), leucrose alpha(1-->5) and palatinose alpha(1-->6) contained high levels of an NAD+ plus metal-dependent phospho-alpha-glucosidase (MalH). The latter enzyme was not induced during growth on sucrose. MalH catalysed the hydrolysis of the 6'-phosphorylated derivatives of the five isomers to yield glucose 6-phosphate and fructose, but sucrose 6-phosphate itself was not a substrate. Unexpectedly, MalH hydrolysed both alpha- and beta-linked stereomers of the chromogenic analogue p-nitrophenyl glucoside 6-phosphate. The gene malH is adjacent to malB and malR, which encode an EII(CB) component of the phosphoenolpyruvate-dependent sugar:phosphotransferase system and a putative regulatory protein, respectively. The authors suggest that for F. mortiferum, the products of malB and malH catalyse the phosphorylative translocation and intracellular hydrolysis of the five isomers of sucrose and of related alpha-linked glucosides. Genes homologous to malB and malH are present in both Klebsiella pneumoniae and the enterohaemorrhagic strain Escherichia coli O157:H7. Both these organisms grew well on sucrose, but only K. pneumoniae exhibited growth on the isomeric compounds.

Bacterial Proteins↗
PubMed1994

Cloning and nucleotide sequencing of a 15 kb region of the Bacillus subtilis genome containing the iol operon.

Within the framework of an international project on the sequencing of the whole Bacillus subtilis genome, a 15 kb chromosome segment, which contains the iol operon involved in inositol utilization, has been cloned and sequenced. This region (14,974 bp) contains 12 complete open reading frames (ORFs; genes) and two partial ones; the seventh gene (E83G) is the idh gene encoding inositol dehydrogenase. All the genes identified are transcribed in the same direction as that of the movement of the replication fork. A homology search for their products deduced from the 12 complete ORFs revealed that eight of them exhibit significant homology to known proteins such as fructokinase, acetolactate synthase, fructose-1,6-bisphosphate aldolase (B. subtilis), and PhoB and FtsE proteins (Escherichia coli). It also implied that two genes (B65D and B65E) might encode a set of two-component regulatory proteins and that the B65F gene might encode a protein belonging to the ATP-binding cassette (ABC) family. Based on the features of the nucleotide sequence determined and the results of the homology search, the primary structure of the iol operon is predicted.

Amino Acid Sequence↗
PubMed1993

Photoregulation of Fructose and Glucose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii F-60 and Spinach.

The photoregulation of chloroplastic respiration was studied by monitoring in darkness and in light the release of 14CO2 from whole chloroplasts of Chlamydomonas reinhardtii F-60 and spinach (Spinacia oleracea L.) supplied externally with [14C] glucose and [14C]-fructose, respectively. CO2 release was inhibited more than 90% in both chloroplasts by a light intensity of 4 W m-2. Oxidants, oxaloacetate in Chlamydomonas, nitrite in spinach, and phenazine methosulfate in both chloroplasts, reversed the inhibition. The onset of the photoinhibitory effect on CO2 release was relatively rapid compared to the restoration of CO2 release following illumination. In both darkened chloroplasts, dithiothreitol inhibited release. Of the four enzymes (fructokinase, phosphoglucose isomerase, glucose-6-P dehydrogenase, and gluconate-6-P dehydrogenase) in the pathway catalyzing the release of CO2 from fructose, only glucose-6-P dehydrogenase was deactivated by light and by dithiothreitol.

Journal Article↗
PubMed1993

Respiration of Sugars in Spinach (Spinacia oleracea), Maize (Zea mays), and Chlamydomonas reinhardtii F-60 Chloroplasts with Emphasis on the Hexose Kinases.

The role of hexokinase in carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of 14CO2 from darkened spinach (Spinacia oleracea), maize (Zea mays) mesophyll, and Chlamydomonas reinhardtii chloroplasts externally supplied with 14C-labeled fructose, glucose, mannose, galactose, maltose, and ribose. Glucose and ribose were the preferred substrates with the Chlamydomonas and maize chloroplasts, respectively. The rate of CO2 release from fructose was about twice that from glucose in the spinach chloroplast. Externally supplied ATP stimulated the rate of CO2 release. The pH optimum for CO2 release was 7.5 with ribose and fructose and 8.5 with glucose as substrates. Probing the outer membrane polypeptides of the intact spinach chloroplast with two proteases, trypsin and thermolysin, decreased 14CO2 release from glucose about 50% but had little effect when fructose was the substrate. Tryptic digestion decreased CO2 release from glucose in the Chlamydomonas chloroplast about 70%. 14CO2 evolution from [1-14C]-glucose-6-phosphate in both chloroplasts was unaffected by treatment with trypsin. Enzymic analysis of the supernatant (stroma) of the lysed spinach chloroplast indicated a hexokinase active primarily with fructose but with some affinity for glucose. The pellet (membranal fraction) contained a hexokinase utilizing both glucose and fructose but with considerably less total activity than the stromal enzyme. Treatment with trypsin and thermolysin eliminated more than 50% of the glucokinase activity but had little effect on fructokinase activity in the spinach chloroplast. Tryptic digestion of the Chlamydomonas chloroplast resulted in a loss of about 90% of glucokinase activity.

Journal Article↗
PubMed2003

A bypass of sucrose synthase leads to low internal oxygen and impaired metabolic performance in growing potato tubers.

Plants possess two alternative biochemical pathways for sucrose (Suc) degradation. One involves hydrolysis by invertase followed by phosphorylation via hexokinase and fructokinase, and the other route-which is unique to plants-involves a UDP-dependent cleavage of Suc that is catalyzed by Suc synthase (SuSy). In the present work, we tested directly whether a bypass of the endogenous SuSy route by ectopic overexpression of invertase or Suc phosphorylase affects internal oxygen levels in growing tubers and whether this is responsible for their decreased starch content. (a) Oxygen tensions were lower within transgenic tubers than in wild-type tubers. Oxygen tensions decreased within the first 10 mm of tuber tissue, and this gradient was steeper in transgenic tubers. (b) Invertase-overexpressing tubers had higher activities of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase, and (c) higher levels of lactate. (d) Expression of a low-oxygen-sensitive Adh1-beta-glucuronidase reporter gene construct was more strongly induced in the invertase-overexpressing background compared with wild-type background. (e) Intact transgenic tubers had lower ATP to ADP ratios than the wild type. ATP to ADP ratio was restored to wild type, when discs of transgenic tubers were incubated at 21% (v/v) oxygen. (f) Starch decreased from the periphery to the center of the tuber. This decrease was much steeper in the transgenic lines, leading to lower starch content especially near the center of the tuber. (g) Metabolic fluxes (based on redistribution of (14)C-glucose) and ATP to ADP ratios were analyzed in more detail, comparing discs incubated at various external oxygen tensions (0%, 1%, 4%, 8%, 12%, and 21% [v/v]) with intact tubers. Discs of Suc phosphorylase-expressing lines had similar ATP to ADP ratios and made starch as fast as wild type in high oxygen but had lower ATP to ADP ratios and lower rates of starch synthesis than wild type at low-oxygen tensions typical to those found inside an intact tuber. (h) In discs of wild-type tubers, subambient oxygen concentrations led to a selective increase in the mRNA levels of specific SuSy genes, whereas the mRNA levels of genes encoding vacuolar and apoplastic invertases decreased. (i) These results imply that repression of invertase and mobilization of Suc via the energetically less costly route provided by SuSy is important in growing tubers because it conserves oxygen and allows higher internal oxygen tensions to be maintained than would otherwise be possible.

Adenosine Diphosphate↗
PubMed1997

Sucrose-to-Starch Metabolism in Tomato Fruit Undergoing Transient Starch Accumulation.

Immature green tomato (Lycopersicon esculentum) fruits undergo a period of transient starch accumulation characterized by developmental changes in the activities of key enzymes in the sucrose (Suc)-to-starch metabolic pathway. Activities of Suc synthase, fructokinase, ADP-glucose (Glc) pyrophosphorylase, and soluble and insoluble starch synthases decline dramatically in parallel to the decrease in starch levels in the developing fruit. Comparison of "maximal" in vitro activities of the enzymes in the Suc-to-starch pathway suggests that these same enzymes are limiting to the rate of starch accumulation. In contrast, activities of invertase, UDP-Glc pyrophosphorylase, nucleoside diphosphate kinase, phosphoglucoisomerase, and phosphoglucomutase do not exhibit dramatic decreases in activity and appear to be in excess of starch accumulation rates. Starch accumulation is spatially localized in the inner and radial pericarp and columella, whereas the outer pericarp and seed locule contain little starch. The seed locule is characterized by lower activities of Suc synthase, UDP-Glc pyrophosphorylase, phosphoglucomutase, ADP-Glc pyrophosphorylase, and soluble and insoluble starch synthases. The outer pericarp exhibits comparatively lower activities of ADP-Glc pyrophosphorylase and insoluble starch synthase only. These data are discussed in terms of the developmental and tissue-specific coordinated control of Suc-to-starch metabolism.

Journal Article↗
PubMed2000

Compression wood-responsive proteins in developing xylem of maritime pine (Pinus pinaster ait.).

When a conifer shoot is displaced from its vertical position, compression wood (CW) is formed on the under side and can eventually return the shoot to its original position. Changes in cell wall structure and chemistry associated with CW are likely to result from differential gene/protein expression. Two-dimensional polyacrylamide gel electrophoresis of differentiating xylem proteins was combined with the physical characterization of wooden samples to identify and characterize CW-responsive proteins. Differentiating xylem was harvested from a 22-year-old crooked maritime pine (Pinus pinaster Ait.) tree. Protein extracted from different samples were revealed by high-resolution silver stained two-dimensional polyacrylamide gel electrophoresis and analyzed with a computer-assisted system for single spot quantification. Growth strain (GS) measurements allowed xylem samples to be classified quantitatively from normal wood to CW. Regression of lignin and cellulose content on GS showed that an increase in the percentage of lignin and a decrease of the percentage of cellulose corresponded to increasing GS values, i.e. CW. Of the 137 studied spots, 19% were significantly associated with GS effect. Up-regulated proteins included 1-aminocyclopropane-1-carboxylate oxidase (an ethylene forming enzyme), a putative transcription factor, two lignification genes (caffeic O-methyltransferase and caffeoyl CoA-O-methyltransferase), members of the S-adenosyl-L-methionine-synthase gene family, and enzymes involved in nitrogen and carbon assimilation (glutamine synthetase and fructokinase). A clustered correlation analysis was performed to study simultaneously protein expression along a gradient of gravistimulated stressed xylem tissue. Proteins were found to form "expression clusters" that could identify: (a) Gene product under similar control mechanisms, (b) partner proteins, or (c) functional groups corresponding to specialized pathways. The possibility of obtaining regulatory correlations and anticorrelations between proteins provide us with a new category of homology (regulatory homology) in tracing functional relationships.

Cellulose↗
PubMed1983

Subcellular localization of hexose kinases in pea stems: mitochondrial hexokinase.

The subcellular localization of hexose phosphorylating activity in extracts of pea stems has been studied by differential centrifugation and sucrose density gradient centrifugation. The hexokinase (EC 2.7.1.1) was associated with the mitochondria, whereas fructokinase (EC 2.7.1.4) was in the cytosolic fraction. Some properties of the mitochondrial hexokinase were studied. The enzyme had a high affinity for glucose (K(m) 76 micromolar) and mannose (K(m) 71 micromolar) and a relatively low affinity for fructose (K(m) 15.7 millimolar). The K(m) for MgATP was 180 micromolar. The addition of salts stimulated the activity of the hexokinase. Al(3+) was a strong inhibitor at pH 7 but not at the optimum pH (8.2). The enzyme was not readily solubilized but, in experiments with intact mitochondria, was susceptible to proteolysis. A location on the outer mitochondrial membrane is suggested for the hexokinase of pea stems.

Journal Article↗
PubMed1988

Carbohydrate Metabolism and Activity of Pyrophosphate: Fructose-6-Phosphate Phosphotransferase in Photosynthetic Soybean (Glycine max, Merr.) Suspension Cells.

Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDP-glucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K(a)) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate.

Journal Article↗