Leon W. Cunningham

Variations in human urinary O-hydroxylysyl glycoside levels and their relationship to collagen metabolism

Two O-hydroxylysyl glycosides, Hyl-Gal-Glc and Hyl-Gal, have been isolated from normal human urine and shown to be identical to two glycosides isolated from alkaline hydrolysates of collagen. A relatively sample and reproducible analytical procedure has been devised to measure the levels of these glycosides in human urine. By the use of this procedure it was shown that a normal diet has only a small effect on 24-hr urinary excretion levels of these glycosides indicating an endogenous origin. Urinary glycoside levels appear to be highest in children, roughly paralleling collagen turnover as indicated by urinary hydroxyproline levels. Collagen turnover equivalents calculated from urinary hydroxylysyl glycoside levels were found to be significantly larger than collagen turnover equivalents calculated from urinary hydroxyproline levels. This suggests that urinary glycosides are more quantitative indicators of collagen metabolism than urinary hydroxyproline. The ratio of Hyl-Gal-Glc to Hyl-Gal was measured in urines of diseased as well as normal individuals and a bimodal distribution was found. Alkaline hydrolysates of different human connective tissue collagens showed that only bone collagen, of the collagens examined, had a low ratio of Hyl-Gal-Glc to Hyl-Gal compared to human urine. Other collagens examined had higher ratios than found in human urine. On the basis of these results it is postulated that the bimodal distribution of glycoside ratios represents two populations of collagen turnover, the lower ratio population having a high bone collagen turnover, the lower ratio population having a high bone collagen turnover relative to the second population. Examination of the types of subjects making up the two populations supports this hypothesis. These data suggest that urinary O-hydroxylysyl glycoside excretion, in addition to providing a more quantitative estimate of collagen turnover than urinary hydroxyproline, may prove to be of value as a specific means of studying the metabolism of bone collagen.

Collagen-Mediated Platelet Aggregation EFFECTS OF COLLAGEN MODIFICATION INVOLVING THE PROTEIN AND CARBOHYDRATE MOIETIES

In an effort to elucidate the nature of the collagen-platelet interaction, the effects of collagen modification on platelet aggregation have been studied. We have shown that purified rat skin (salt) soluble collagen is effective at about 20 nM in mediating platelet aggregation in human platelet-rich plasma. This concentration is somewhat greater than that required of several skin insoluble collagens (ca. 10 nM). Both the alpha1(I) and alpha2 chains from rat skin soluble collagen produced platelet aggregation, but only at concentrations of about 13 muM and 55 muM, respectively. In contrast, heat-denatured collagen and chains (e.g., 65 muM alpha1(I) and 160 muM alpha2) failed to induce platelet aggregation and to inhibit platelet aggregation by native collagen. Glycopeptides were prepared from human skin insoluble collagen by extended digestion with bacterial collagenase and trypsin, and were purified by gel filtration into two classes. One class of higher molecular weight contained sialic acid, glucosamine, galactosamine, fucose, mannose, galactose, and glucose, and the other of lower molecular weight consisted primarily of a mixture of galactose and galactosyl-glucose units O-glycosidically linked to hydroxylysine-containing peptides. We found that, after the residual tryptic activity contaminating the higher molecular weight fraction was inhibited, neither of the glycopeptide classes produced nor inhibited native human skin insoluble collagen-mediated platelet aggregation at the highest concentration examined (ca. 1-2 mg glycopeptide per ml of platelet-rich plasma). Highly purified samples of the hydroxylysyl glycosides, hydroxylysylgalactose and hydroxylysylgalactosylglucose (Hyl-Gal and Hyl-Gal-Glc, respectively), were prepared from human urine and labeled at galactose using galactose oxidase followed by reduction with tritiated borohydride. Binding studies with platelet-rich plasma showed that, at concentrations greater than 50 nM, Hyl-Gal gives apparent binding to platelets, but there was no evidence of Hyl-Gal-Glc binding to platelets at concentrations up to 250 nM. At concentrations several hundredfold higher than the equivalents present in the minimum concentration of rat skin soluble collagen required for platelet aggregation, neither Hyl-Gal (at 29 muM) nor Hyl-Gal-Glc (at 18 muM) caused platelet aggregation or inhibited platelet aggregation by native collagen. Also, at a concentration of 85 muM (which represents a concentration about two thousandfold higher than the equivalents in the minimum concentration of soluble collagen required for platelet aggregation) the Gal-Glc-containing 36 residue rat skin soluble collagen alpha1(I)cyanogen bromide #5 peptide had no platelet aggregating or inhibiting activity. Modification of at least 90% of the rat skin soluble collagen carbohydrate by mild periodate oxidation had no effect on the platelet aggregating activity. Human skin insoluble collagen was reacted with periodate under the same conditions, and this had no demonstrable effect on its ability to induce platelet aggregation. This indicates that the normal carbohydrate side chains of these collagens are not required for the platelet interaction that produces the release of ADP and other metabolic constituents and leads to aggregation.Thus, collagen-platelet interactions appear to involve at least two distinct binding sites on the platelet plasma membrane. One is a protein binding site that activates platelet aggregation and has high specificity and affinity for the collagen triple-helical fold or perhaps even for a particular amino acid sequence in the triple helix.

Residual α2-macroglobulin in fetal calf serum and properties of its complex with thrombin

Summary Active α2-macroglobulin (α2M) has been shown to persist in commercial preparations of fetal bovine serum. Reaction with trypsin and the mitogenic protease thrombin leads to cleavage of the polypeptide chain of α2M, with simultaneous conversion to a fast electrophoretic form. The trapped protease is fully active toward low molecular weight synthetic substrates, but reacts slowly if at all with the protein inhibitor hirudin (M.W. 12,200). Addition of methylamine to fetal bovine α2M leads to covalent incorporation of the amine into the protein, but the subsequent and apparently spontaneous conversion from the slow to the fast electrophoretic form seen with human α2M is not observed. Sedimentation velocity studies of intact and modified fetal bovine α2M show that a small shape change results from reaction with proteases but that, in contrast to the human protein, no shape change results from reaction with methylamine.

Chapter III - The Structure and Mechanism of Action of Proteolytic Enzymes

Publisher Summary The study of the enzyme-catalyzed hydrolysis f proteins has played an important role in its application to the problem of protein structure and its relationship to the elucidation of the detailed catalytic mechanism of such reactions. Proteases can be distinguished in the following groups: (1) the serine proteases, which contain a serine residue that is reactive toward diisopropylphosphofluoridate; (2) the thiol proteases, which depend upon the presence in the protein of the reduced form of a specific cysteine residue; (3) the metalloproteinases, which require the presence of specific metal ions for catalytic activity; and (4) the acid proteases, which are characterized by pH optima near pH 1-3. Proteolytic enzymes pose a problem because most of them have no non-amino acid prosthetic group. The interaction with other molecular species, including the specific reactions that result in catalysis, must involve special and unique group conformations of the side-chains of component amino acids. The location of specific amino acid residues within that area of the protein involved in enzyme-substrate interactions, the active center, or their direct implication in the bond-changing sequence of the catalytic mechanism is one of the major goals of the investigations.

Stimulation of thiouracil binding at the iodination system in beef thyroid microsomes

Abstract 1. 1. It has been shown previously by many investigators that beef thyroid microsomal fractions enzymically iodinate in vitro in the presence of glucose and glucose oxidase. [ 14 C]Thiouracil has now been found to be maximally bound to microsomal fractions under these same conditions. Both iodination and thiouracil binding are inhbited in a very similar pattern by thiols (mercaptoethanol, glutathione) as well as thioureylenes (1-methyl-2-mercaptoimidazole, thiourea). 2. 2. Both iodination and thiouracil binding still occur if glucose oxidase is replaced by 0.1 mM H 2 O 2 . 3. 3. Thiouracil has been shown previously 1 to react rapidly and at low concentrations with the sulfenyl iodide group in a model protein. This reaction of model sulfenyl iodide was inhibited by specific thiols and thioureylenes at concentrations comparable in all respect to that effective in the thyroidal system. 4. 4. After I 3 -pretreatment of the microsimal fraction the subsequent uptake of [ 14 C]thiouracil was increased, consistent with previously reported data on model sulfenyl iodides. The relative inhibition by thiols and thioureylenes of this [ 14 C]-thiouracil binding by iodine pretreated microsomal fractions was also characteristic of the sulfenyl iodide group. 5. 5. These data provide additional support for the hypothesis 1–3 that a sulfenyl iodide group takes part in the iodinating mechanism of the thyroid gland.

The site of acetylation of glyceraldehyde-3-phosphate dehydrogenase by p-nitrophenylacetate

Abstract The major site of acetylation of glyceraldehyde-3-phosphate dehydrogenase [ d -glyceraldehyde 3-phosphate; NAD oxidoreductase (phosphorylating) EC 1.2.1.12] by p -nitrophenylacetate is the sulfhydryl group of the cysteine residue in the peptide, Ala-Ser-CySAc, derived from the acetylated enzyme by pepsin digestion. If all of the peptides analyzed come from the same section of the peptide chain of the protein, then the sequence about this site of acetylation appears to be Ala-Ser-CySAc-(Asp, Thr, Thr). The relationship of these results to the peptide Ser-CM-CyS-(AspNH 2 , Thr) obtained by Harris and Park from pepsin digests of the enzyme after reaction with iodoacetate is discussed. These data do not provide support for the earlier concept that glutathione or a glutathione-like portion of the peptide chain was the site of similar reactions.

Platelet-Collagen Adhesion — Membrane Fluidity and the Development of High Affinity Adhesion through Multiple Interacting Sites

Multiple, linked interactions between the platelet surface and collagen fibers have been implicated in the initiation of platelet secretion and subsequent aggregation. The formation of such multiple simultaneous interactions could give rise to high affinity adhesion of platelets to collagen even though the affinity of the individual interactions may be much weaker. This concept has been tested by measuring the adhesion of platelets to collagen under conditions which could effect the formation of multiple interactions. Adhesion is markedly diminished at 4 degrees C but not at 23 or 37 degrees C. Metabolic inhibitors such as 2-deoxyglucose and Antimycin A do not inhibit adhesion although they virtually abolish subsequent aggregation. Brief formaldehyde fixation of platelets greatly reduces adhesion. These results are consistent with the concept that the formation of multiple linked interactions between the platelet surface and collagen are important in platelet-collagen adhesion and that mobility of platelet membrane components is required for the clustering of these interactions in focussed regions on the platelet surface.

Attaching human fibroblasts secrete a type I procollagen rich in 3-hydroxyproline.

Abstract The predominant collagenous protein secreted during the attachment of freshly trypsinized human foreskin fibroblasts was found to be Type I procollagen. Evidence is presented that both the α1 and α2 chains exhibit a 3-hydroxyproline/4-hydroxyproline ratio 4–5 fold higher than that of normal Type I collagen. These findings suggest that caution should be exercised in assigning an observed increase in the 3-hydroxyproline/4-hydroxyproline ratio to the synthesis of a basement membrane type collagen.

Scanning electron microscopic visualization of a microexudate prepared by the release of cells by urea.

Abstract The scanning electron microscope (SEM) was used to examine the residue remaining after human fibroblasts, permitted to attach to plastic dishes in the absence of serum, were removed with buffered 1 M urea. The ‘urea carpet’ is entirely different from the “substrate-attached material” (SAM) of Culp [4, 5, 6] in that it contains no “footpad” material. Furthermore, the SEM pictures clearly indicate that urea carpet stimulates the adhesion and spreading of newly added fibroblasts.

Comparison of collagen and glycosaminoglycan synthesis in attaching control and diabetic human skin fibroblasts

SummaryCultured fibroblasts derived from normal subjects and juvenile diabetics attach in the absence of serum to plastic culture dishes and secrete macromolecules, including collagenous components, hyaluronic acid, and proteoglycans into the medium and onto the plastic surface where they form a microexudate carpet. Most diabetic fibroblasts examined did not spread as well as normal cells during a 4-hr interval after the initial attachment. There were no significant differences between normal and diabetic cells with respect to proline and lysine incorporation and lysine hydroxylation. The percentage glycosylation of hydroxylysine was marginally higher in the media proteins of diabetic cells, but glycosylation in both normal and diabetic cells was elevated over that typically observed in human skin collagen.Collagenous components were estimated to constitute approximately 15–20% of the microexudate carpet fraction in both normal and diabetic cell strains. Diabetic fibroblasts exhibited a marginally lower ratio of heparan sulfate to chondroitin sulfate in the cell surface to matrix microexudate carpet fraction (trypsinate) than did normal fibroblasts. The hyaluronate and chondroitin sulfate contents of this fraction of diabetic cells were not significantly different from those of normal cells.