Carbohydrate analysis.

The analysis of the carbohydrate chains attached to proteins is becoming increasingly important as appreciation of the role of glycosylation in the structural and functional properties of biologically significant glycoproteins grows. Over the past year, a number of developments have been made that may improve and promote the analysis of the glycosylation of proteins.     

Carbohydrate in pancreatic ribonucleases.

A survey of the presence and compositions of carbohydrate chains attached to pancreatic ribonucleases is given. Carbohydrate chains may occur at asparagine residues in Asn-X-Ser/Thr sequences at four exposed sites of the molecule (positions 21, 34, 62 and 76). These sites form part of highly variant sequences in pancreatic ribonucleases with the consequence that the enzymes from very closely related species may differ in the presence or absence of carbohydrate. In a number of ribonucleases Asn-X-Ser/Thr sequences occur which carry carbohydrate in only a part of the molecules. The occurrence of Asn-X-Ser/Thr sequences entirely without any carbohydrate also has been demonstrated. Compositions of the carbohydrate moieties of ribonucleases from cow, sheep, pig, whales, giraffe, okapi, moose, horse, coypu, chinchilla, and guinea-pig are presented. Striking differences in complexity have been found, both between chains attached to the same site in different species (cow and giraffe), between chains attached to different sites of the same enzyme in one-species (pig) and even between chains attached to the same site in a single species (chinchilla).     

Carbohydrate preferences of mammalian cells.

Carbohydrate preferences of mammalian cells can be utilized to biochemically distinguish between different cell lines. Ninety-three carbohydrates were examined of which (a) 15 supported cell proliferation and (b) 42 were toxic or growth inhibitory. The present investigation has employed an enzymatic system to eliminate trace glucose levels from reagents and glucose generated by serum enzymes.     

Carbohydrate composition of bovine rhodopsin.

The carbohydrate content of bovine rhodopsin was investigated and found to be different from previously reported values. Rod outer segments were isolated from dark-adapted bovine retinas by sucrose flotation and purified by sucrose density contrifugation. Rhodopsin was extracted with detergents and purified by chromatographic procedures involving calcium phosphate/celite chromatography followed by affinity chromatograpy on concanavalin A-Sepharose (or in some cases, gel filtration on agarose). Purified preparations of rhodopsin had A278/A498 ratios of 1.6 to 2.0. After treatment of the rhodopsin with chloroform/methanol (2/1) to remove lipids and detergents, the carbohydrate content was measured by gas-liquid chromatography, colorimetric and enzymatic analyses, paper chromatography, and electrophoresis. Rhodopsin was found to have about 9 mol of mannose and 5 mol of glucosamine per mol of visual pigment. A molar ratio of mannose/glucosamine of about 2 was also found in samples of rhodopsin obtained from two other laboratories. The amino acid analysis was similar to previously published values.     

Carbohydrate determinants of Rhizobium-legume symbioses.

Rhizobium is a genus of symbiotic nitrogen-fixing soil bacteria that induces the formation of root nodules on leguminous plants and, as such, has been the subject of considerable research attention. Much of this work was initiated in response to the question 'how does recognition occur between free living rhizobial bacteria in the soil and potential host legumes?' The answer to this question has been shown to involve both cell-surface carbohydrates on the external face of the bacteria and secreted extracellular signal oligosaccharides. This review will focus on the structure, function, and biosynthesis of two of these components--the host-specific nodule-promoting signals known as Nod(ulation) factors and the rhizobial lipopolysaccharides.     

Carbohydrate feedings before, during, or in combination improve cycling endurance performance.

This study examined the effects of no carbohydrate (PP), preexercise carbohydrate feeding (CP), carbohydrate feedings during exercise (PC), and the combination of carbohydrate feedings before and during exercise (CC) on the metabolic responses during exercise and on exercise performance. Nine well-trained cyclists exercised at 70% of maximal O2 uptake until exhaustion. Blood glucose peaked 30 min after the preexercise carbohydrate feeding and at the start of exercise was 25% below the prefeeding concentration (4.76 mM). At exhaustion, glucose had declined to 3.8 (PP), 4.0 (CP), 4.6 (PC), and 5.0 mM (CC). Insulin was 300% above basal (7 microU/ml) at the start of exercise for CC and CP and returned to baseline by 120 min of exercise. When carbohydrates were consumed, the rate of carbohydrate oxidation was significantly higher throughout exercise than during PP. Total work produced during exercise was 19-46% (P less than 0.05) higher when carbohydrates were consumed. Time to exhaustion was 44% (CC), 32% (PC), and 18% (CP) greater than PP (201 min; P less than 0.05). Performance was improved by ingestion of carbohydrates before and/or during exercise; performance was further improved by their combination. This is probably the result of enhanced carbohydrate oxidation, especially during the later stages of exercise.     

Carbohydrate moieties in human secretory component.

Human secretory component has seven putative sites for N-linked glycosylation. From tryptic and Glu-C digests we have isolated peptides encompassing asparagines 65, 72, 117, 168, 403, 451 and 481. Analysis by on line HPLC-electrospray mass spectrometry indicated that these residues were fully glycosylated and that the major carbohydrate moieties were far less diversified in composition than expected. Fast atom bombardment mass spectrometry performed on oligosaccharides released by peptide-N-glycosidase F treatment of fractionated and unfractionated SC digests showed the following glycan compositions: Fuc(2)Hex(5)HexNAc(4), Fuc(3)Hex(5)HexNAc(4), NeuAcFucHex(5)HexNAc(4), NeuAcFuc(2)Hex(5)HexNAc(4), NeuAc(2)Hex(5)HexNAc4 and NeuAc(2)FucHex(5)HexNAc(4). Three of these oligosaccharides are the major carbohydrate moieties in human lactoferrin. A possible biological role of the secretory component glycans in the protection of mucosal surfaces is discussed.     

Carbohydrate structure of Marburg virus glycoprotein.

Marburg virus was propagated in E6 cells, a cloned cell line of Vero cells, in the presence of [6-3H]glucosamine. Radiolabelled viral glycoprotein was digested with trypsin, and oligosaccharides were liberated by sequential treatment with endo-beta-N-acetylglucosaminidase H, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and O-glycosidase, by beta-elimination, and by alkaline hydrolysis. After fractionation by HPLC and gel filtration, glycans were characterized chromatographically, by digestion with exoglycosidases and, in part, by methylation analysis and liquid secondary ion mass spectrometry. The oligosaccharide structures thus established include oligomannosidic and hybrid-type N-glycans, as well as neutral fucosylated bi-, tri- and tetraantennary species, most of which carry an additional bisecting N-acetylglucosamine. In addition, high amounts of neutral mucin-type O-glycans with type-1 and type-2 core structures were detected. None of the glycans present in this viral glycoprotein carried sialic acid residues.     

Carbohydrate composition of horse spleen ferritin.

The carbohydrate composition of horse spleen ferritin was studied. 1 mol of the apoferritin, the protein moiety of ferritin, contains 25 mol of hexose, 3 mol of hexosamine and 10 mol of fucose. Same carbohydrate composition was detected in the apoferritin from iron rich ferritins. These results indicate that horse spleen ferritin is composed of non-identical subunits as regards its carbohydrate composition.     

Carbohydrate patterns ofCandida,Cryptococcus andRhodotorula species

Within the genusCandida three distinct groups are recognized on the basis of carbohydrate patterns of intact whole cell hydrolyzates. In the first, ascomycetous, group mannose is dominant, while rhamnose, fucose and xylose are absent; this is indicative of an affinity with endomycetous families. Among the basidiomycetous representatives, two groups can be recognized. One group is usually characterized by the presence of xylose and has a low mannose content. The pattern is typical for Cryptococcales and Tremellales (e.g.,Cryptococcus, Trichosporon, Bullera andTremella). The other basidiomycetous group is characterized by the presence of fucose and/or rhamnose with significant amounts of mannose. This pattern is characteristic for Sporobolomycetaceae.     

Carbohydrate nutrition before, during, and after exercise

The role of dietary carbohydrates (CHO) in the resynthesis of muscle and liver glycogen after prolonged, exhaustive exercise has been clearly demonstrated. The mechanisms responsible for optimal glycogen storage are linked to the activation of glycogen synthetase by depletion of glycogen and the subsequent intake of CHO. Although diets rich in CHO may increase the muscle glycogen stores and enhance endurance exercise performance when consumed in the days before the activity, they also increase the rate of CHO oxidation and the use of muscle glycogen. When consumed in the last hour before exercise, the insulin stimulated-uptake of glucose from blood often results in hypoglycemia, greater dependence on muscle glycogen, and an earlier onset of exhaustion than when no CHO is fed. Ingesting CHO during exercise appears to be of minimal value to performance except in events lasting 2 h or longer. The form of CHO (i.e., glucose, fructose, sucrose) ingested may produce different blood glucose and insulin responses, but the rate of muscle glycogen resynthesis is about the same regardless of the structure.