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The N epsilon-glycosylation of lysine and hydroxylysine residues in collagen from streptozotocin-induced-diabetic rats was confirmed and the stability of the complex shown to be due to an Amadori rearrangement. The studies also demonstrate the relative specificities of glucose, galactose and mannose in their reaction with collagen. The glycosylation of lysine in vitro occurs with glucose and galactose, but not with mannose, whereas only gucose reacts with hydroxylysine to any significant extent. Glycosylation of collagen occurs slowly during normal aging, but in contrast with reports suggesting accelerated aging of collagen in diabetic animals, we clearly demonstrated that the apparent increased stability is not due to an acceleration of the normal maturation process involving the reducible cross-links.

Acid-soluble collagen from rat tail tendon was glucosylated in vitro and fibrillogenesis parameters were determined first at 35 degrees C then at 4 degrees C. Increased lag phase and half time were shown to be related to the amount of non-enzymatically bound glucose, probably due to a decrease of hydrophobic interactions at this early stage of fibril formation. The absence of intermolecular cross-links and the partial redissolution of fibrils of 4 degrees C, as investigated both by turbidimetry and electron microscopy, suggests a defect in the maturation process in glucosylated collagen fibrils.

Ubiquitin was extracted from testis of 4 mammals and purified to homogeneity by gel filtration chromatography. Amino acid compositions and NH2-terminal sequences were found to be identical in the 4 species and with calf thymus ubiquitin. Ubiquitin conformation was shown to be very sensitive to oxidation. Improved methods for radioimmunoassay of ubiquitin in tissue extracts are also discussed.

A technique of preparative isotachophoresis is described for the preparation of two rat α-1-macroglobulin varieties. The procedure permits a good and fast separation. Antigenic identity of these components is shown by fused rocket immunoelectrophoresis. Some properties of the two varieties are determined, and causes of heterogeneity are also discussed.

Glomerular basement membranes were isolated from normal and streptozotocin-diabetic rats. after elimination of non collagenous components with 8M urea the extent of ketoamine-linked hexoses and cross-links in the preparation was determined using NaB 3 H 4 reduction and radiochromatography. The observed increase in the level of N-glycosylation appeared to be closely related to a lowered amount of intermolecular cross-links in diabetic rats as compared to normal animals. Such a glycosylation may affect the stability and physicochemical properties of G.B.M. and thereby contribute to the vascular sequelae of diabetes mellitus.

1. 1. The study of the amino acid composition, in particular the study of the hydroxyproline, hydroxylysine and glycine content, of the glycopeptide fractions obtained from a clostridropeptidase A hydrolysate of Wistar rat GBM allowed us to distinguish between two types of structure, one of which is collagen-like. 2. 2. Determination of carbohydrate composition revealed the existence of a heteropolysaccharidic glycane in all of the fractions studied. 3. 3. The study of the chemical composition of the high molecular weight components isolated using a Sepharose 4B column allowed us to distinguish between structural glycoproteins (Subfractions A 1 , A 2 , A 4 ) and collagen-like glycopeptides (Subfraction A 3 ) to which they are linked. 4. 4. The immunological studies revealed the antigenic complexity of the material studied and the existence of precipitin lines of identity for the fractions which were isolated.

Non-enzymatic glycosylation of collagen occurs both in vivo during diabetes and in vitro after incubation with glucose. Glycosylated collagen exhibits altered physicochemical and biological properties which could explain some of the complications of diabetes. To provide a mechanistic explanation of this modification the localization of bound glucose was investigated using NaB[3H]H4 reduction and CNBr cleavage. Glucose fixation is distributed mainly on the alpha 1CB6 peptide after in vitro glycosylation whereas this distribution occurs less specifically during diabetes. It is concluded that fibrillogenesis alteration of in vitro glycosylated collagen is related to glucose fixation on free epsilon NH2 sites normally implied in intermolecular interactions.