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Incubation of rabbit skeletal myosin with 1 to 3 mM D-glucose 6-phosphate over a period of several hours resulted in the inhibition of the K(+)- and actin activated-ATPase activities. Substrate ATP (0.5-3 mM final concentration) protected the myosin against the loss of ATPase activity as induced by glucose 6-phosphate. This was also found for ADP. When the myosin was incubated with 3 mM [3H] labeled glucose 6-phosphate for 28 h. up to one mole of glucose 6-phosphate was incorporated per 4.7 x 10(5) g of myosin. A significant reduction in the labeling occurred in the presence of ATP. The labeling was limited to the heavy chain region as judged by gel electrophoresis which resolved the heavy and light chain components of myosin. The non-enzymatic glycation of myosin by glucose 6-phosphate is probably the primary cause for the observed loss of the ATPase activity of myosin. This effect may also occur physiologically modifying the activity of muscle contractile proteins particularly during prolonged hyperglycemia.

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Incubation of tail tendon from a young rat in solutions containing D-ribose resulted in attachment of the monosaccharide to collagen and subsequent cross-link formation at a rate much faster than found for glucose. The collagen rapidly became resistant to solubilization and showed increasing fluorescence. Ribose bound to all major CNBr peptides of collagen, with some preference for the alpha 2-CB3,5 peptide and the triple-helical region of alpha 1-CB6, and was incorporated into higher molecular weight material. Extensive pepsin digestion permitted isolation of dimers of alpha chains cross-linked in triple-helical regions as a result of incubation with ribose. The dimers were identified as beta 11, beta 12, and beta 22 components, and the limited degree of heterogeneity of these components indicated that cross-linking occurred at several sites, some of which must be intermolecular. Isolated beta components were strongly fluorescent with a spectrum similar to that of collagen in aged tissues. Fluorescent dimers with similar characteristics were found in pepsin digests of tail tendons from older rats.

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Exposure of rat tail tendon to a reducing sugar results in covalent attachment of the sugar to collagen, a process termed glycation, and leads to the formation of stable intermolecular cross-links. We have used X-ray diffraction to study the changes in the crystalline unit cell of rat tail tendon collagen brought about by glycation. Ribose was selected as a model compound for most of the study because its reaction with proteins is faster than that of glucose, and therefore more convenient for laboratory studies, but glucose and glyceraldehyde were used as well. A kinetic model describing the process of glycation by ribose and subsequent cross-link formation has been developed. Glycation resulted in an expansion by more than 12% of the unit cell that describes the three-dimensional structure of rat tail tendon collagen. The expansion was in a direction perpendicular to the axes of the rod-shaped molecules, indicating that the intermolecular spacing of the collagen increased. Thus, the structure of collagen in rat tail tendon is significantly altered by glycation in vitro. The expansion was not isotropic, but was directed parallel to the (120) planes, one of the three major planes of the quasi-hexagonal structure that is densely populated by collagen molecules. It is hypothesized that this expansion is brought about by the formation of one, or at most a few, specific intermolecular cross-links in the overlap zone that act to push the molecules apart. It is likely that similar structural changes in collagenous tissues are caused by glycation in vivo during the natural course of aging, and that these changes are accelerated in chronic hyperglycemia such as that associated with diabetes. Analysis of the structure of glycated rat tail tendon potentially can give us new insight into the detailed molecular structure of collagen.

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The mono- (2) and bis-phosphate (3) derivatives of D-threo-2,5-hexodiulose (1) (5-keto-D-fructose) were synthesized enzymically and purified by anion-exchange chromatography. The proportions, sizes of ring, and anomeric configurations were determined by F.t. 31P- and 13C-n.m.r. spectroscopy. Compound 2 was found to exist preponderantly (70-78%) in the beta-pyranose form with the remainder existing in the 2R,5R-furanose form. Compound 3 assumes two different furanose forms in solution, one (77-84%) being the 2R,5R-furanose form and the other the 2S,5R-furanose form.

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Patterns of oxidation of dilute solutions of desialylated fetuin and submaxillary mucin by galactose oxidase have been examined. A significant portion (20-40%) of the terminal galactosyls exposed on the glycoproteins, which theoretically were expected to be accessible to the enzyme, was not oxidized. In comparison, galactosyls in oligosaccharides released from completely desialylated glycoproteins were oxidized more effectively with an apparently lower degree of crypticity to the enzyme. Partial desialylation usually resulted in a reduction of both the rate and the final level of substrate oxidation. A second cycle of oxidation of a desialylated substrate earlier oxidized by galactose oxidase and then reduced by NaB3H4 revealed a selectivity in the pattern of galactosyl oxidation. The same galactosyl residues oxidized in the first cycle were again the most susceptible to oxidation in the second cycle, leaving unmodified the same fraction of galactosyls throughout both cycles. The relevance of these results to the application of the galactose oxidase-NaBH4 procedure for detecting and measuring desialylated glycoconjugates in solution and in biological membranes is discussed.

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A simple, rapid, and sensitive spectrophotometric assay procedure for the determination of as low as 2 microM solutions of formaldehyde and acetaldehyde using an alkaline 4-amino-5-hydrazino-3-mercapto-1,2,4-triazole reagent is described. The method is particularly useful for determination of these aldehydes (0.5-50 nmol) when produced by the periodate oxidation of various glycols and can be applied to the assay of dilute solutions of sugars or polyols.

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5-Keto-D-fructose (5KF) is isolated from cultures of Gluconobacter cerinus growing on D-fructose as the sole carbon source. 5KF is a substrate for hexokinase, fructokinase, and several polyol dehydrogenases. 1H and 13C nuclear magnetic resonance studies show that 5KF exists in different forms in anhydrous dimethyl-d6 sulfoxide and D2O. In dimethyl-d6 sulfoxide, 5KF exists as a spirane dimer with linked furanose and pyranose rings, similar to the structure reported for crystalline 5KF [Hassen, L., Hordvik, A., & Hove, R. (1976) J. Chem. Soc., Chem. Commun., 572-. In D2O, 5KF exists predominantly (greater than 95%) in a beta-pyranose form with the 5-keto group hydrated to form a gem-diol. 13C--1H coupling patterns, 13C relaxation measurements, and 13C deuterium-induced differential isotope shifts confirm this structure of 5KF. The phosphorylation of 5KF by fructokinase can be accounted for by an approximately 2% proportion of the beta-furanose form in solution at 25 degrees C. Both the beta-pyranose and beta-furanose forms of 5KF are proposed to be substrates for yeast hexokinase.

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We describe a modified enzymic reagent for determination of cholesterol and cholesterol ester in serum and in high-density lipoprotein. In this new procedure, the hydrogen peroxide produced by the action of cholesterol oxidase is used as a substrate for NAD+ peroxidase. Spectrophotometric determination of the NADH consumed in this coupled reaction provides a direct and absolute measure of the cholesterol originally present in free form and that liberated by the action of cholesterol ester hydrolase.

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The relatively slow reduction of NAD+ and NADP+ by sodium cyanoborohydride leads to formation of the enzymically active form of NADH and NADPH. This reaction could be useful as a simple procedure to enzymically introduce a specific label into substrates when tritiated or deuterated cynanoborohydride is used for obtaining the reduced nicotinamide adenine dinucleotide.

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Reducing sugars, particularly aldoses, react readily with dansyl hydrazine. The fluorescent hydrazones produced can be separated by thin-layer chromatography and determined quantitatively by spectrofluorimetry after elution from the chromatograms.

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Aerolysin, a hemolytic and lethal exotoxin of Aeromonas hydrophila, was analyzed for amino acids. Assuming 8 histidine residues/mol, the purified toxic protein has, by summation, a molecular weight of 49,000, a value in agreement with earlier estimates by other methods. Erythrocytes from different animal species differ greatly in sensitivity to aerolysin's lytic action. There is some correlation between sensitivity and phosphatidyl choline content. Erythrocyte membranes of different species bind the toxin, and the efficiency of binding is a function of sensitivity to lysis. Binding is temperature independent, is not dependent upon membrane sialic acid, and is decreased by prior treatment with phospholipase C and proteases. Preparations of aerolysin convert substantial amounts of membrane phosphorus to water-soluble form; the conversion is concentration and temperature dependent. Most of the conversion is attributable to contaminating phospholipase(s) that is separable from the toxin. Aerolysin purified by electrophoresis in polyacrylamide gel retains some phospholipase activity, and this activity may or may not be a contaminant.

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