Assay, purification, and properties of bovine liver D-xylulokinase.

Convenient specific assay methods using commercially available materials were developed for the measurement of D-xylulokinase and D-ribulokinase activities. Using these assays, D-xylulokinase from bovine liver has been purified to homogeneity. Purification involved column chromatography on DEAE-cellulose, Sephadex G-100, Dyematrex Red A, and Superose 12. The enzyme preparation was obtained with final yields of 15-30% and had activity toward both D-xylulose and D-ribulose. The final specific activities ranged from 2.5 to 7.5 and 1.9 to 5.9 mol/min for the two substrates and Km values of 0.14 and 0.27 mM were obtained, respectively. For ATP, a Km value of 0.080 mM was obtained with either substrate. AMP, ADP, and cAMP inhibited competitively with respect to ATP; Ki values were 0.34, 0.35, and 1.0 mM, respectively. Xylulokinase thus prepared was a monomeric protein of 51,000 Da and had a pH optimum between 7.4 and 8.2. The kinetics of the enzyme do not support any significant regulation of flux through the enzyme by metabolite level changes.

Protein fructosylation: fructose and the Maillard reaction.

Fructose, as is the case for other reducing sugars, undergoes the Maillard reaction with proteins and amino acids. The first stage of the reaction results in one or more substituted amino sugars. These products in turn enter the advanced and final stages of the Maillard reaction, which involve the formation of reactive intermediates, cross-linking of proteins, and the formation of brown and fluorescent polymeric materials. It would appear that the initial stages of the reaction occur more rapidly with fructose than with glucose. The Maillard reaction with any sugar, including fructose, results in a decrease in protein quality due to the loss of amino acid residues and decreased protein digestibility. Maillard products can inhibit the uptake and metabolism of free amino acids and of other nutrients such as zinc and some advanced Maillard products have mutagenic and/or anticarcinogenic properties. In vivo the Maillard reactions between proteins and fructose, glucose, and other reducing sugars may play a role in aging and in some of the clinical complications of diabetes.

Nutritional and physiological consequences of tumour glycolysis.

A frequent characteristic of many malignant tumours is an increase in anaerobic glycolysis, that is the conversion of glucose to lactate, when compared to normal tissues. The causes of this intensification involve changes in enzyme and glucose transporter levels, shifts of the isoenzyme patterns in the cancer cells to those similar to foetal tissues and a breakdown in the normal control mechanisms, most notably the Pasteur effect. The host must adapt, with a corresponding increase in gluconeogenesis. This change, along with other adaptations made by the host, eventually results in the syndrome known as cancer cachexia, which is characterized by anorexia and depletion and redistribution of the host energy stores. In some ways many malignant tumours behave much like parasites, drawing upon the host for nutrients such as glucose and returning waste products such as lactate to the host for recycling or disposal. This cycling of glucose and lactate between host and tumour has been the target for a number of proposed and tested treatments, with regard to the possible inhibition of tumour growth and/or possible prevention of some or all of the cachectic effects. Some of these suggested treatments have reached the point of clinical testing and show promise for continued research.

Sugar alcohols as bulk sweeteners.

The polyols are a family of bulk sweeteners, some of which are currently used in the United States and in other nations. The use of these compounds is likely to increase in the future. The greatest advantage of polyols as sweeteners is their reduced cariogenicity compared with sucrose, fructose, or glucose. This reduced cariogenicity has been observed with all of the polyols considered in this review. Furthermore, evidence suggests that one of these polyols, xylitol, may have cariostatic properties. More research is needed to clarify the mechanism of this cariostatic effect. Evidence suggests that moderate usage of the polyols in human diets over long periods is not likely to produce many toxic effects. This conclusion is supported by the facts that (a) both sorbitol and mannitol have been used as sweeteners for some time without apparent side effects, and (b) extensive long-term studies with dietary xylitol in Europe have not yielded any reports of toxicity. At this point there is no reason to believe that the disaccharide polyols differ significantly in a qualitative sense from sorbitol or mannitol with regard to their effects in humans. There are some research needs with regard to the inclusion of the polyol sweeteners in human diets: 1. All of the polyols can cause osmotic diarrhea in humans if higher levels are consumed. This fact is noted in the labelling of products containing mannitol and sorbitol in the United States (see "Current Regulatory Status"). If the disaccharide polyols are to be used as bulk sweeteners, further studies of the dose levels that can cause diarrhea may be needed. 2. The polyols, like other slowly absorbed carbohydrates, enhance the absorption of certain minerals, particularly divalent cations. More comparative and mechanistic studies of this effect are needed. 3. All of the polyols, lactose, and other slowly absorbed carbohydrates appear to cause adrenal medullary hyperplasia at high doses in laboratory rats. Evidence suggests that these lesions are linked in some way to the lactose or polyol-induced changes in calcium homeostasis. Despite long-term use of lactose, sorbitol, and mannitol in human diets, similar lesions in humans have not been reported and some investigators have concluded that the lesion in rats has no relevance to humans. Nevertheless further studies are needed to elucidate the mechanisms of the dietary lactose and polyol-induced adrenal hyperplasias in rats to ascertain definitively if they also operate in other species.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of rat Sertoli cells with a collagen lattice in vitro.

Sertoli cells from rats aged 15, 20, and 25 d were subcultured onto collagen-coated, plastic dishes. If the collagen was released from the plastic surface by rimming, the floating rafts of collagen showed uniform shrinkage. If the collagen was allowed to remain attached to the plastic, holes appeared in the collagen with cells from rats aged 25 d but not with those of 15 d. Cells from rats aged 20 d caused fewer and smaller holes to appear. The holes were associated with the formation of clumps of spherical cells from which elongated Sertoli cells extended into the surrounding collagen to end near holes. Rhodamine-phalloidin revealed diffusely distributed actin in the spherical cells in contrast to well-developed microfilaments in the peripheral elongated cells. Addition of cytochalasin B (5 micrograms/ml) to the medium prevented contraction of the floating rafts and the development of holes in the attached collagen. In addition, cytochalasin B caused the peripheral cells to become spherical and to separate from the clumps. Moreover, rhodamine-phalloidin revealed that actin in the peripheral cells occurred as clumps without microfilaments when cytochalasin B was present. When Sertoli cells were subcultured onto silicone rubber films, the cells produced wrinkling of the rubber surface within 4 h of plating. These observations were interpreted to mean that Sertoli cells exert local tractional forces on various substrata. These forces require actin, presumably acting by a contractile mechanism. When the collagen is attached to plastic and the cells are organized into clumps with radiating elongated cells (cells from rats aged 25 d), the tractional forces in the elongated cells reorganize the collagen fibers to produce holes. When cells are uniformly distributed (cells from rats aged 15 d), holes are not formed. When the collagen is released from the plastic surface, tractional forces cause the floating rafts to shrink. These interactions of the cells with collagen are likely to be important in determining the shape of the Sertoli cell in vivo, the polarity of the cell, and its biochemical differentiation.

Dietary potentiation of the antifertility effects of 5-thio-D-glucose in male rats.

Male rats of proven fertility were fed the following diets for 28 d either with or without 0.075% 5-thioglucose (5-THG): AIN-76 diet (A76): a diet with 13% casein, 2% glucose and the balance of the calories as free corn oil fatty acids from corn oil (2G); and a similar diet, isocaloric with 2G, with the glucose level increased to 20% (20G). The diets alone without 5-THG had no influence on any of the parameters measured. Body weight gain was lower in rats fed diets containing 5-THG than in those fed diets without 5-THG. In rats fed A76, the only 5-THG effects on male reproductive tract (MRT) tissues was the appearance of testicular multinucleate giant cells (MGC). In rats fed either 2G or 20G, the MRT effects of 5-THG included the appearance of MGC, a lower number of germ cells at most stages of maturation, lower sperm counts and biochemical changes in testis slices and in germ cell preparations compared to rats not fed 5-THG. There were fewer Step 7 spermatids in rats fed 5-THG in 2 G than in those fed 5-THG in 20G. It is concluded that the MRT toxicity of 5-THG is influenced by diet, being potentiated by the low protein diet high in free fatty acids and, to a lesser extent, by low glucose levels within these diets.

Inhibition of gluconeogenesis by 2,5-anhydro-D-mannitol in isolated rat hepatocytes.

2,5-Anhydro-D-mannitol inhibited glucose synthesis, increased the pyruvate/phosphoenolpyruvate ratio and altered adenine nucleotide concentrations in hepatocytes isolated from fasted rats. The accumulations of 2,5-anhydro-D-mannitol 1,6-diphosphate, an allosteric activator of pyruvate kinase, and of ADP in treated hepatocytes can account for the increase in pyruvate/phosphoenolpyruvate ratio and the inhibition of glucose synthesis from lactate.

The influence of diet on the regression of the Walker carcinosarcoma 256 in rats.

Regression of the Walker carcinosarcoma 256 has been observed in rats inoculated with 10(4) viable tumor cells. The regression was found to be affected by dietary composition. Tumors in rats fed a commercial laboratory diet (CLD) regressed after a 9-day initial growth period while tumors in rats fed a purified diet high in free fatty acids continued to grow. Diets with 20% corn oil promoted tumor regression, but rats fed diets containing 20% free fatty acids from corn oil had tumors that continued to grow. The nonsaponifiable fraction (NSF) of corn oil appeared to promote tumor regression when this fraction was added to diets containing corn oil fatty acids. At the end of the experiment (14 days), the tumors of rats fed a free fatty acid diet weighed 13-17 g. The tumors of rats fed CLD, corn oil diet and the corn oil free fatty acid diet plus the NSF of corn oil weighed 1-5 g. These results indicate that something in the NSF of corn oil was associated with the regression of the tumor. However, the nature of the dietary components promoting tumor regression, and the host response to those components, have not been determined.

Effects of diets deficient in glucose and glucose precursors on the growth of the Walker carcinosarcoma 256 in rats.

The effects of carbohydrate-deficient diets on the growth of the Walker carcinosarcoma 256 in rats and on the carcinostatic action of the glucose analogue 2-deoxyglucose were studied. All diets contained 13.0% casein with glucose levels as indicated and the balance of calories present as corn oil free fatty acids. The growth of the tumor was directly related to the glucose level in such diets; after 16 days rats fed 0.0, 1.5 and 4.5% glucose had tumors weighing 7.0, 11.1 and 13.3 g, respectively. The decrease in tumor weight was related to dietary glucose level rather than the anorexia produced by the diets low in glucose, as shown by the fact that tumors in rats fed 4.5% glucose were larger than rats fed 1.5% glucose even when the rats fed 4.5% glucose were pair-fed to the levels consumed by those fed 1.5% glucose. 2-Deoxyglucose (0.2%) also caused a reduction in tumor growth in a manner independent from the anorexia produced by its presence in the diet. This carcinostatic effect was potentiated by low glucose levels in the diets in the rats fed 4.5% glucose plus 0.2% 2-deoxyglucose had proportionally greater reductions in tumor weights due to the glucose analogue than did rats fed 20% glucose plus 0.2% 2-deoxyglucose.

Inhibition of glycogenolysis by 2,5-anhydro-D-mannitol in isolated rat hepatocytes.

2,5-Anhydro-D-mannitol, an analog of D-fructofuranose, inhibited basal and glucagon-stimulated glycogenolysis and glucose production in hepatocytes isolated from fed rats. Glucose formation from galactose was unaffected by the inhibitor. 2,5-Anhydro-D-mannitol-1-phosphate inhibits phosphorylase alpha with a Ki value of 2.4 mM. This same phosphorylated metabolite accumulates to the extent of 9.2 mumol/g wet wt in treated hepatocytes suggesting that phosphorolysis is the locus of the inhibition of glucose production from glycogen. Our results suggest that 2,5-anhydro-D-mannitol can be used to produce a model of hereditary fructose intolerance and that it merits further study as a hypoglycemic agent.

Enzymatic analysis of 2,5-anhydro-D-mannitol and related compounds.

Two enzymatic assay procedures for the measurement of 2,5-anhydrohexitol fructose analogs have been devised. Both procedures are based on the measurement of ADP formed during enzymatic phosphorylation of the analogs either by hexokinase or by fructokinase. The actual measurement makes use of the coupled assay system using pyruvate kinase, PEP, lactate dehydrogenase, and NADH. Both systems can be used to measure fructose and appropriate analogs at cuvette concentrations up to 0.10 mM. The hexokinase procedures allows the measurement of fructose, 2,5-anhydromannitol, and 2,5-anhydromannose. Glucose, which also reacts, can be removed by pretreatment of the samples with glucose oxidase. The fructokinase procedure allows the measurement of fructose, 2,5-anhydromannitol, 2,5-anhydroglucitol, and 2,5-anhydrotalitol.

The involvement of liver fructokinase in the metabolism of D-xylulose and xylitol in isolated rat hepatocytes.

Hepatocytes isolated from fed, male, Sprague-Dawley rats accumulate xylulose-1-phosphate and glycolaldehyde as well as xylulose-5-phosphate when incubated with 2-20 mM D-xylulose. Fructokinase inhibitors (fructose and 1-deoxyfructose) decreased xylulose-1-phosphate and glycolaldehyde (but not xylulose-5-phosphate) levels in xylulose-treated hepatocytes, demonstrating the role of fructokinase in xylulose-1-phosphate and glycolaldehyde formation. As the fructokinase inhibitors had no overall effects on the conversion of D-xylulose to glucose, the overall flux through the pathway involving fructokinase was less than 27% of the total D-xylulose utilized. In hepatocytes from fed or fasted rats there was no detectable accumulation of either xylulose-1-phosphate or glycolaldehyde after treatment with 20 mM xylitol. Other differences between xylitol and D-xylulose metabolism in rat hepatocytes included a slower rate of xylitol metabolism in all preparations and a difference in the relative conversion of xylitol to glucose in hepatocytes from fasted rats. Rats adapted to 20% xylitol (diarrhea-free) had a lower water consumption than those fed a control cornstarch diet; there were no differences in weight gain, food consumption or in rates or metabolite patterns of xylitol metabolism in hepatocytes isolated from these rats. Despite the minor role of fructokinase in the overall metabolism of xylitol and of D-xylulose as shown by these results, it is not possible to exclude the possibility of some flux through the pathway involving xylulose-1-phosphate and glycolaldehyde formation as a possible route for oxalate formation.

Purification and subunit composition of guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung.

The guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung was purified to apparent homogeneity by affinity chromography using 8-2-aminoethylthio-cGMP coupled to Sepharose 4B. The kinase activity was purified approximately 6000-fold with an overall recovery of approximately 20%. The product isolated by affinity chromatography contained both cGMP-binding and cGMP-dependent histone kinase activity, indicating that the enzyme was not dissociated into regulatory and catalytic components by the immobilized cGMP derivative. The enzyme had a molecular weight of approximately 165,000 and a sedimentation coefficient of 7.8 S. The purified kinase displayed several characteristics similar to that of the partially purified enzyme including specificity for cGMP and stimulation by high concentrations of magnesium. On sodium dodecyl sulfate gels, only one major polypeptide chain was present having a molecular weight of approximately 81,000. This subunit bound 1 mol of cGMP and exhibited cGMP-dependent protein kinase activity. It is proposed that the native enzyme consists of two identical subunits (Mr=81,000), each of which binds cGMP and catalyzes protein phosphorylation.