Effect of vanadate on the glucose-1,6-bisphosphate synthase and glucose-1,6-bisphosphatase activities of phosphoglucomutase

Phosphoglucomutase, in addition to catalyzing the interconversion of glucose 1-P and glucose 6-P, catalyzes both the synthesis of glucose 1,6-P2 from glucose monophosphate and either fructose 1,6-P2 or glycerate 1,3-P2, and the hydrolysis of glucose 1,6-P2. Vanadate inhibits the mutase activity, activates the synthase activities, and does not affect the phosphatase activity. These effects suggest that the "exchange" step postulated for the phosphoglucomutase pathway is specifically inhibited by vanadate.     

Tunicamycin--an inhibitor of yeast glycoprotein synthesis

Tunicamycin, a glucosamine-containing antibiotic, halted synthesis of the external glycoproteins invertase, acid phosphatase and mannan by yeast protoplasts within 30 min; formation of two intracellular proteins, alpha-glucosidase and alkaline phosphatase, and of glucan continued at the control rate for at least 60–80 min. No accumulation of mannan-free acid phosphatase or invertase was evident in treated cells. Utilization of hexoses and incorporation of ¹⁴C-amino acids into protein were not affected. Incorporation of ³H-glucosamine into trichloroacetic acid-insoluble products was only partially reduced. In yeast tunicamycin acts primarily as an inhibitor of glycoprotein synthesis and not of general glucosamine metabolism.     

Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.     

The uptake of 2-deoxy-d-glucose by Pseudomonas aeruginosa and its regulation

The non-metabolizable glucose analogue 2-deoxy-d-glucose is taken up by Pseudomonas aeruginosa against a concentration gradient, in a predominantly unchanged form. d-Glucose competitively inhibits 2-deoxy-d-glucose uptake and also causes a rapid exit of intracellular 2-deoxy-d-glucose. Thus these two sugars share the same stereospecific carrier system, and glucose transport can be studied reliably with 2-deoxy-d-glucose. The transport system is inducible, and is strongly repressed by a number of organic acids such as acetate, citrate, succinate, fumarate and malate, even in the presence of adequate excess of the inducer (d-glucose). Repression by organic acids can be relieved by transferring cells to a glucose medium, but in the presence of chloramphenicol the cells fail to recover from repression, indicating that the formation of the transport system involves the synthesis of protein. The results demonstrate that the regulation of glucose metabolism effected by citric acid-cycle intermediates in P. aeruginosa is manifest at the level of the glucose-transport system.     

Kinetics of alpha-amylase synthesis from Bacillus amyloliquefaciens

The microbial production of alpha-amylase from Bacillus amyloliquefaciens was investigated. The microorganism was grown using media containing glucose or maltose at 37 degrees C and under aerobic conditions in a 16-L fermentor. The alpha-amylase synthesis from maltose was not found to be inducible but was found to be subject to catabolite repression. The maltose uptake rate was observed to be the rate-limiting step compared to the conversion rate of maltose to glucose by intracellular alpha-glucosidase. The alpha-amylase activity achieved with maltose as a substrate was higher than that achieved with glucose. A slower growth rate and a higher cell density were obtained with maltose. The enzyme production pattern depended upon the nutrient composition of the medium.     

Osmotic regulation of invertase formation and secretion by protoplasts of Saccharomyces

Osmotic regulation of invertase formation and secretion by protoplasts of Saccharomyces was examined using sorbitol, KCl, NaCl, or magnesium sulfate as the osmotic support. The synthesis and secretion of the enzyme is remarkably sensitive to the osmolarity of the supporting medium irrespective of the particular support employed. Invertase formation was inhibited at high osmolarity and was maximal at 0.65 to 0.75 osmolal, even though some leakage of the intracellular enzyme alpha-glucosidase and of ultraviolet (UV)-absorbing materials occurred under these conditions. The reduction of invertase formation and secretion due to high osmolarity was eliminated promptly when protoplasts were transferred into a medium of lower osmolarity. The rate of fructose uptake and of threonine incorporation into protein was decreased by high osmolarity; also reduction of invertase formation could be partially reversed by increasing the level of sugar supplied as energy source. Thus changes in the permeability of the plasma membrane (and presumably also in its structure) are important factors in the response of protoplasts to high osmolarity, though certainly not the complete explanation. Protoplasts suspended in 0.8 m sorbitol, with 10mm fructose as the energy source, increased their invertase level 5- to 10-fold during a 2-hr incubation without substantial release of alpha-glucosidase or UV-absorbing materials. Both the large and small forms of invertase were present in the protoplasts, but only the large form was released into the medium when enzyme was being actively synthesized. Formation and secretion of newly formed invertase and the release of enzyme initially present were inhibited by cycloheximide.     

Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.     

Glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels in different types of rat skeletal muscle

1. The concentration of glycogen, glucose 1,6-P2, fructose 2,6-P2 and the content of glycogen phosphorylase, phosphofructokinase, 6-phosphofructo 2-kinase and glucose 1,6-P2 phosphatase activity, have been determined in rat muscles which differ in their fiber composition: extensor digitorum longus, gastrocnemius, diaphragm and soleus. 2. Glucose 1,6-P2 concentration seems to be related to the glycolytic capacity of the muscle, while fructose 2,6-P2 concentration does not. 3. No significant relationship exists between the fiber type and the content in glucose 1,6-P2 phosphatase and 6-phosphofructo 2-kinase activities.     

Maltose metabolism of Pseudomonas fluorescens.

Pseudomonas fluorescens W uses maltose exclusively by hydrolyzing it to glucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to maltobionic acid was found in this organism. The alpha-glucosidase was totally intracellular and was most active at pH of 7.0. Induction occurred when cells were incubated with maltotriose or maltose. Induction was rapid and easily detectable within the first 5 min after the addition of the inducer. Glucose and its derivatives did not repress induction. Cells growing on DL-alanine or succinate plus maltose exhibited lower levels of alpha-glucosidase than those grown on maltose alone or maltose plus glucose. Induction required both messenger ribonucleic acid and protein synthesis.     

Uptake and phosphorylation of glucose and fructose in Daucus carota cell suspensions are differently regulated

Cell suspensions of Daucus carota L. were grown in batch culture on 50 mM sucrose, 100 mM glucose or 100 mM fructose. Sucrose was rapidly converted extra-cellularly into equimolar amounts of glucose and fructose, and glucose was then taken up preferentially. This impaired uptake of fructose could partially be explained by the eight-fold lower affinity of the hexose carrier in the plasmamembrane for fructose compared to glucose. However, cells grown on fructose as the sole carbon source showed a shorter lag phase and showed more biomass production compared to glucose-grown cells, indicating that conversion of glucose and fructose were also differently regulated. Ninety-five % of the glucose phosphorylating activity was membrane-associated and most probably confined to mitochondria; therefore, it might be present in a respiratory ‘compartment’ making glucose a better substrate for respiration than fructose. The soluble fraction contained the majority of the fructokinase activity. This activity was hypothesized to be more or less randomly distributed through the cytosol; in this soluble ‘compartment’ a pool of fructose-6-phosphate is formed. Concomitantly, via glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P), it is converted into UDPG-glucose, resulting in structural cell components. The observed transient obstruction of the conversion of G-1-P into UDP-glucose in fructose-grown cells, leading to G-1-P accumulation, might be a result of both an altered equilibrium maintained by phosphoglucomutase, interconverting G-6-P and G-1-P and low levels of nucleotide triphosphates. Low nucleotide triphosphate production, connected with a low initial respiration rate, might be caused by the ten-fold lower affinity of the membrane-associated phosphorylating enzymes for fructose compared to glucose. Our results were taken to indicate that two separate pools of glycolytic intermediates exist in D. carota cells: one distributed throughout the cytosol and one surrounding the mitochondria.     

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required. Analysis of label in saccharides provides complementary data to better define fluxes around hexose, pentose, and triose phosphate pools. Here, we propose a gas chromatography-mass spectrometry (GC-MS) method to analyze 13C labeling in glucose and fructose moieties of sucrose, free glucose, fructose, maltose, inositol, and starch. Our results show that saccharide labeling for isotopomer quantification is better analyzed by chemical ionization than by electron ionization. The structure of the generated fragments was simulated and validated using labeled standards. The method is illustrated by analysis of saccharides extracted from developing rapeseed (Brassica napus L.) embryos. It is shown that glucose 6-phosphate isomerase and plastidial glucose 6-phosphate transport reactions are not at equilibrium, and light is shed on the pathways leading to fructose, maltose, and inositol synthesis.     

Inhibition of sucrose phosphatase by sucrose

1. Partially purified sucrose phosphatase from immature stem tissue of sugarcane is inhibited by sucrose. The enzyme was also inhibited by maltose, melezitose and 6-kestose but not by eight other sugars, including glucose and fructose. 2. The relative effectiveness of sucrose, maltose and melezitose as inhibitors is different for sucrose phosphatase from different plants. 3. The inhibition of the sugar-cane enzyme by sucrose was shown to be partially competitive. The K(i) for sucrose is about 10mm. 4. Melezitose is also a partially competitive inhibitor of the enzyme but the inhibition by maltose is probably mixed. 5. The possibility that sucrose controls both the rate of accumulation of sucrose in stems of sugar-cane and sucrose synthesis in leaves by inhibiting sucrose phosphatase is discussed.     

Enzyme activities associated with maize kernel amyloplasts

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.     

Zinc inhibition of hepatic fructose metabolism in rats

The ability of Zn to modulate key metabolic processes was investigated in a study of gluconeogenesis in isolated hepatocytes from fasted rats. Zn (100 μM) inhibited glucose production from fructose by 41%, sorbitol by 28%; glycerol by 17%, and glyceraldehyde by 26%. Maximum inhibition of gluconeogenesis from fructose occurred at 25 μM Zn. Zn inhibited the rate of lactate production from fructose by 24% but not from sorbitol, glycerol, or glyceraldehyde. Fructose uptake by hepatocytes was not affected by Zn. A positive linear relationship (r=0.994) was obtained between inhibition by Zn of glucose and lactate production, indicating that a common step in both pathways is inhibited by Zn. The effect of Zn on fructokinase, aldolase-B, and triokinase activities was determined on semipurified rat liver enzyme preparations. Zn had no affect on triokinase activity but inhibited the two other enzymes in a dose-dependent manner, with the inhibition of aldolase-B being much greater than of fructokinase for concentrations of Zn between 2.5 and 20 μM. Zn increased the intracellular concentration of fructose-1-P in hepatocytes incubated with fructose, indicating a more potent Zn inhibition of aldolase-B than fructokinase. In addition, hepatocytes treated with Zn had decreased ATP and ADP concentrations, but had normal energy charge, suggesting an effect of Zn on adenine nucleotide degradation or synthesis. The demonstration that Zn inhibits two enzymes in fructose metabolism adds to the growing list of metabolic pathways that are catalyzed by enzymes that are sensitive to Zn.     

A kinetic analysis of glucokinase and glucose-6-P phosphatase in dictyostelium

Summary Using a mathematical model of carbohydrate metabolism in Dictyostelium discoideum, the kinetic expressions describing the activities of glucokinase and glucose-6-P phosphatase have been analyzed. The constraints on the kinetic mechanisms and relative activities of these two enzymes were investigated by comparing computer simulations to experimental data. The results indicated that, (1) glucose-6-P is compartmentalized with respect to the enzymes involved in glucose-6-P, trehalose and glycogen metabolism, (2) a differences of approximately 0.6 mm/min in maximum specific activity of glucokinase compared to glucose-6-P phosphatase is required in order for the model to produce end product carbohydrate levels consistent with those observed experimentally, (3) the Km of glucokinase for glucose strongly influences the steady state levels of glucose in the absence of external glucose, and (4) changing the order of product removal in the reaction catalyzed by glucose-6-P phosphatase influences the level of glycogen and trehalose.     

Catabolism of D-fructose and D-ribose by Pseudomonas doudoroffii. I. Physiological studies and mutant analysis.

Pseudomonas doudoroffii, a strict aerobe of marine origin, was able to utilize fructose and ribose but not glucose, gluconate, or other hexoses, pentoses, or sugar alcohols as sole sources of carbon and energy. Evidence was presented indicating that in this organism fructose was utilized via an inducible P-enolpyruvate: fructose phosphotransferase system (FPTS) which catalyzed the phosphorylation of fructose in the 1 position. The resulting fructose-1-P (F-1-P) was converted to fructose-1,6-P2 (FDP) by means of an inducible 1-P-fructokinase (1-PFK). The subsequent conversion of FDP to pyruvate involved enzymes of the Embden-Meyerhof pathway (EMP) which, with the exception of glyceraldehyde-3-P dehydrogenase (G3PDH), were constitutive. Two G3PDH activities were detected, one of which was inducible and NAD-dependent while the other was constitutive and NADP-dependent. Cell-free extracts of P. doudoroffii also contained enzymes of the methylglyoxal pathway (MGP) which converted dihydroxyacetone-P to pyruvate. The low specific activities of enzymes of this pathway as compared to the EMP suggested that the major route of FDP catabolism was via the latter pathway. 2. Ribose catabolism appeared to involve an inducible uptake system and an inducible ribokinase, the resulting ribose-5-P being converted to glyceraldehyde-3-P and fructose-6-P (F-6-P) by means of constitutive activities of the pentose-P pathway. The F-6-P formed as a result of these reactions was converted to FDP by means of a constitutive 6-P-fructokinase (6-PFK). Since no activity converting fructose or F-1-P to F-6-P could be detected in cell-free extracts of P. doudoroffii, the results suggested that fructose and ribose were catabolized via 1-PFK and 6-PFK, respectively, the two pathways converging at the level of FDP. Further evidence for this suggestion was obtained from a mutant which lacked an NAD-dependent G3PDH, accumulated FDP from both fructose and ribose, and was not able to grow on either of these compounds. 3. Ribose grown cells had increased amounts of the fructose uptake system and 1-PFK suggesting that a compound (or compounds) common to the catabolism of both fructose and ribose acted as the inducer(s) of these activities. Evidence was presented suggesting that the probable inducer(s) of 1-PFK and FPTS could be FDP, glyceraldehyde-3-P, or dihydroxyacetone-P. 4. A mutant unable to grow on fructose was characterized and found to lack FPTS while retaining 1-PFK and other enzyme activities of the EMP and MGP, indicating that a functional FPTS was essential for growth on fructose and suggesting that all or most of this sugar was catabolized via F-1-P.     

Effect of exogenous application of sugars on the salt tolerance of perennial ryegrass protoplasts

Various sugars were introduced by electroporation into perennial ryegrass protoplasts, and the involvement of intracellular functional groups of the sugars in salt tolerance was investigated. The protoplasts were prepared from the young leaves of perennial ryegrass, and those into which sugars were introduced were treated with NaCl solution (250 mM, pH 7.0) for 6 h at 10°C. The survival rate of the protoplasts increased when xylitol, cellobiose, 1-kestose, maltose, maltotriose, raffinose and trehalose were introduced, while no changes occurred when fructose, fucose, galactose, glucose, inositol, mannitol, mannose, rhamnose, sorbitol, sorbose, fructobiose, lactose and sucrose were introduced. Cellobiose, 1-kestose, maltose, maltotriose, raffinose and trehalose possess a number of equatorial OH (e-OH) groups that promote the structuration of H₂O. Xylitol, however, structures H₂O even though it does not possess the e-OH groups. Hence, it is suggested that under conditions of NaCl stress, structured H₂O protects the structure of cell membranes and the activity of enzymes, and that e-OH groups are involved in enhancing salt tolerance.     

A Saccharomyces cerevisiae mutant unable to convert glucose to glucose-6-phosphate accumulates excessive glucose in the endoplasmic reticulum due to core oligosaccharide trimming

D-Glucose is the preferred carbon and energy source for most eukaryotic cells. Immediately following its uptake, glucose is rapidly phosphorylated to glucose-6-phosphate (Glc-6-P). The yeast Saccharomyces cerevisiae has three enzymes (Hxk1p, Hxk2p, and Glk1p) that convert glucose to Glc-6-P. In the present study, we found that yeast mutants lacking any two of these enzymes retain the ability to efficiently convert glucose to Glc-6-P and thus maintain a low level of cellular glucose. However, a mutant strain lacking all three glucose-phosphorylating enzymes contained up to 225-fold more intracellular glucose than normal. Drugs that inhibit the synthesis or the trimming of the lipid-linked core oligosaccharide Glu(3)Man(9)GlcNac(2) effectively reduced the accumulation of glucose. Similarly, mutations that block the addition of glucose residues to the core oligosaccharide moiety, such as alg5Delta or alg6Delta, also diminished glucose accumulation. These results indicate that the intracellular glucose accumulation observed in the glucose phosphorylation mutant results primarily from the trimming of glucose residues from core oligosaccharide chains within the endoplasmic reticulum (ER). Consistent with this conclusion, both [(14)C]glucose exchange and subcellular fractionation experiments indicate that much of the accumulated glucose is retained within an intracellular compartment, suggesting that the efficient transport of glucose from the ER to the cytosol in yeast may be coupled to its rephosphorylation to Glc-6-P. The high level of cellular glucose was associated with an increased level of protein glycation and the release of glucose into the culture medium via its transit through the secretory pathway. Finally, we also found that the accumulation of glucose may lead to a subtle alteration in ion homeostasis, particularly Ca(2+) uptake. This suggests that this mutant strain may serve as a useful model to study the consequences of excessive glucose accumulation and protein glycation.     

Enzyme co-localization in pea leaf chloroplasts: glyceraldehyde-3-P dehydrogenase,triose-P isomerase,aldolase and sedoheptulose bisphosphatase

Nearest neighbor analysis of immunocytolocalization experiments indicates that the enzymes glyceraldehyde-3-P dehydrogenase, triose-P isomerase and aldolase are located close to one another in the pea leaf chloroplast stroma, and that aldolase is located close to sedoheptulose bisphosphatase. Direct transfer of the triose phosphates between glyceraldehyde-3-P dehydrogenase and triose-P isomerase, and from glyceraldehyde-3-P dehydrogenase and triose-P isomerase to aldolase, is then a possibility, as is direct transfer of sedoheptulose bisphosphate from aldolase to sedoheptulose bisphosphatase. Spatial organization of these enzymes may be important for efficient CO₂ fixation in photosynthetic organisms. In contrast, there is no indication that fructose bisphosphatase is co-localized with aldolase, and direct transfer of fructose bisphosphate from aldolase to fructose bisphosphatase seems unlikely.     

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.