A modified colorimetric method for the estimation of n-acetylamino sugars

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b5 reductase and cytochrome b5.

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An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study.

Publisher Summary This chapter discusses the procedures employed for the analysis of the sugar components of glycoproteins. Most of the methods used for the estimation of the monosaccharides present in glycoproteins are colorimetric in nature. Because of the relatively low specificity of some of these color reactions, it is often important to apply them only after appropriate separation of the constituents. In many cases complete identification, preferably by chromatographic means, of all the sugar and amino acid constituents of a glycoprotein should precede any colorimetric analysis so that potentially interfering substances may be taken into account. The hydrolytic release of the monosaccharides is also considered in the chapter. The optimal conditions of acid hydrolysis for the monosaccharides vary substantially because of differences both in the stability of their glycosidic bonds and in their susceptibility to destruction during the hydrolysis. One of the prerequisites for an accurate analysis of the monosaccharide components of a polymer is finding hydrolysis conditions under which all of a given sugar is released and yet is not significantly destroyed.

[1] Analysis of sugars found in glycoproteins

O-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.

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Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.

Hyaluronan fragments: an information-rich system.

N-Acetylation of the Hexosamines

A micromethod for the colorimetric determination of N-acetyl groups in acid mucopolysaccharides

Chemistry of β-Heparin (Chondroitinsulfuric Acid-B)

The metabolism of mucopolysaccharides in animals. I. Isolation from skin.

http://www.jbc.org/content/236/2/333.full.pdf

Julio Ludowieg

Papers

N-Acetylation of the Hexosamines

A micromethod for the colorimetric determination of N-acetyl groups in acid mucopolysaccharides

Chemistry of β-Heparin (Chondroitinsulfuric Acid-B)

The metabolism of mucopolysaccharides in animals. I. Isolation from skin.

The Mechanism of Action of Hyaluronidases