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Glucose reacts nonenzymatically with the NH2-terminal amino acid of the beta chain of human hemoglobin by way of a ketoamine linkage, resulting in the formation of hemoglobin AIc. Other minor components appear to be adducts of glucose 6-phosphate and fructose 1,6-diphosphate. These hemoglobins are formed slowly and continuously throughout the 120-day life-span of the red cell. There is a two- to threefold increase in hemoglobin AIc in the red cells of patients with diabetes mellitus. By providing an integrated measurement of blood glucose, hemoglobin AIc is useful in assessing the degree of diabetic control. Furthermore, this hemoglobin is a useful model of nonenzymatic glycosylation of other proteins that may be involved in the long-term complications of the disease.

The newly discovered coenzyme PQQ can now be measured at picomole levels with redox cycling methods developed in our laboratory. PQQ-peptides have been obtained from digests of the copper quinoenzymes, diamine oxidase, lysyl oxidase and dopamine-beta-hydroxylase. PQQ is present in egg yolk and milk, suggesting its immediate availability for developing embryos and newborn animals. We suggest that PQQ, when exposed in traumatized, ischemic, inflammed or pathological tissues, may catalyze the formation of large amounts of superoxide and should be considered as a source of oxidative stress when planning pharmacotherapeutic intervention. PQQ and quinoproteins play a role in the redox metabolism and structural integrity of cells and tissues.

An unknown amino acid was purified from bovine ligament elastin. The compound was shown by proton NMR to have a pyridinium ring structure similar to isodesmosine, yet containing an olefinic double bond on one additional side chain which was not attached to the pyridinium ring. Mass spectral analysis confirmed the NMR data and indicated a parent compound with a mass of 653. A structure is proposed that is derived from the condensation of five lysine residues. The trivial name of pentasine is proposed for this compound.

PRIMARY STRUCTURE OF THE CoLLAGENS. • . . . . . . . . . . . . . . . • . • . .. ...... ... .... 618 Collagens of chain composition (a1h a2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 619 Collagens of chain composition [a1(II)]a and [al(III)Ja. ... . . ......... . . . . . .. 627 Collagen of chain composition [aI, a2, a3] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 628 BIOSYNTHESIS OF CoLLAGEN MOLECULES...... .. . . • . . .. . .. . . .. .. . . . . . .. . ..... 630 HYDROXYLATION OF PROLINE AND LYSINE RESIDUES IN CoLLAGEN.... . . . . ....... 634 BASEMENT MEMBRANES AND THE GLYCOSYLATION OF CoLLAGENS.... .. . . . .. ..... 637 ELASTIN. . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 640 CROSSLINKS OF CoLLAGEN AND ELASTIN ...... ... ...... 652 Lysine (L) . . .. .. .... .. .. . . ... . ..... . .. . .. . . . ..... 653 o-Hydroxylysine (HL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 653 a-Amino adipic acid a-semialdehyde (AL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 653 a-Hydroxy a-amino adipic acid a-semialdehyde (HAL) . . . . . . . . . . . . . . . . . . . .. .. 654 Dehydrolysinonorleucine (deLNL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 655 Lysinonorleucine (LNL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. 655 "Aldor' (ALAL).... . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. 656 Dehydrohydroxylysinonorleucine (deHLNLa, deHLNLb) . . . . . . . . . . . . . . . . . . . . . 656 o-Hydroxylysinonorleucine (HLNL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 656 Dehydrohydroxylysinohydroxynorleucine (deHLHNL). . .... ...... 656 Hydroxylysinohydroxynorleucine (HLHNL) ... . ... " .. . . .. . . .. .. .. . . . . ..... 657 Syndesine (S). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 657 Dehydromerodesmosine (deM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 657 Merodesmosine (M). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 657 Dehydrohydroxymerodesmosine (deHM) . . . . . . .. . .. . .. . . .... .. .... ......... 657 Dihydrodesmosines (dihydroD) . . . . . . . . . . . . . . . . .. . . . . . . . " ... .. 658 Desmosines (D) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 658 Tetrahydrodesmosines (tetrahydroD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 658 Compound 285 . . . . . . . . . . . . . . . . ...... . . ....... . . . . . .... . . . . . .. . . . . . . . . , 659 Posthistidines . .. . . . . ....... . . . . . . . . .... " .. . . . . . . . . . . . .. .. 659 Compound 16 . . . . . . . . . . . . . . . . . ...... . ..... . . . . " .. .. .. .. .... . . . . ....... 659 COLLAGENASESAND ELASTASES . . • . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . 660 CoNCLUDING REMARKS 660 APPENDIX . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . 664

Kinetic analysis was made of the dialysis of digests of ichthyocol gelatin prepared with collagenase. It was found that most, if not all, of the peptides in such digests are dialyzable, but at greatly different rates. First, there is a class of slowly dialyzable peptides that contain most of the tyrosine, ester groups, and carbohydrate moieties of the parent protein molecule. Second, there is a class of slowly dialyzable peptides, which are rich in the polar amino acids and appear to originate from the polar or “noncrystalline” regions of the protein molecule. Third, there is a class of rapidly dialyzable peptides that are rich in proline and hydroxyproline and appear to originate from the “crystalline” regions of the collagen molecule. After 48 hr., the most slowly dialyzable peptides remain in an aggregate fraction which, operationally, has been designated a “non-dialyzable” fraction. It has been established that the aggregate peptides have, relative to original gelatin, increased contents of glutamic acid, aspartic acid, and lysine, and contain major portions of the carbohydrate, tyrosine, and hydroxylamine-sensitive (“ester-like”) bonds of gelatin. However, they have relatively decreased contents of proline, hydroxyproline, arginine, and leucine. Their Contents of glycine and alanine are relatively unchanged. Dinitrophenylation studies indicated that the average peptide length of the “nondialyzable” peptides was 11–15 residues. On the basis of these and other studies, a representation is made of the polar and nonpolar regions of the collagen molecule.

The action of hydralazine on collagen prolyl hydroxylation was studied in a cell culture system using WI-38 fibroblasts. The prolyl hydroxylation level was determined by a method involving the digestion of collagen by bacterial collagenase and the examination of specific peptides. The presence of low concentrations of hydralazine (0.2 mM) in both “young” and “old” fibroblast cultures strongly inhibited collagen prolyl hydroxylation. The degree of inhibition was greater in serum-deficient cultures. No significant improvement in the degree of hydroxylation was observed by increasing either ascorbate or iron levels in the hydralazine-containing cultures in which hydroxylation was inhibited. Some of the reported side effects of hydralazine seen in patients might be related to its inhibitory effects on mixed function oxidative (MFO) hydroxylation systems. While the ascorbate dependence of the prolyl hydroxylase system of WI-38 decreased with the “age” of the culture, hydralazine inhibition of hydroxylation was dramatic with cultures of all “ages”.

It was found that 0.02 mM was the higher concentration of drug which is not toxic and is compatible with normal cell morphology and growth over a period of a week. Using this concentration of drug there was no consistent stimulation of cell growth. A small increase in cell number was noted, however, in some cultures during the first 24 hours of culture. DpH at a concentration of 0.02 mM in the presence of ascorbic acid decreased collagen prolyl hydroxylation in "old" W1-38 cultures but a concentration of 0.1 mM was required to decrease the values to a similar extent in "young" cultures. A concentration of 0.02 mM DpH had no effect on hydroxylation in an "old" HGF culture. It is speculated that in a responsive individual a reduction of collagen prolyl hydroxylation may play a role in the etiology of DpH-induced gingival enlargement and congenital malformations.