The structures of inhibitors and the substrate suggest the possible involvement of a metal ion at the active center. The enzyme is a sulfhydryl enzyme and is inactive in the oxidized form. It is rapidly activated by reducing agents such as mercaptans or NaBH,.When the racemization of L-proline is carried out in D20, the optical rotation is initially negative. As the reaction proceeds, it becomes positive, and finally zero when equilibrium is reached. This "overshoot" is attributed to a primary isotope effect. sidered for the hydrogen-transfer process: a "onehydrogen-acceptor" and a "two-hydrogen-acceptor" mechanism. In the first mechanism hydrogen is transferred from the substrate to a hydrogen acceptor at the active site with the concomitant formation of an intermediate derived from the substrate. The intermediate could be a carbanion or an oxidized form of the substrate depending upon the chemical mechanism by which the a-carbon-hydrogen bond is broken. In a subsequent step hydrogen is transferred from the acceptor to the intermediate so that either the original substrate isomer or the product isomer is formed. If a single hydrogen-acceptor site participates, it must be flexible enough to interact with the a-hydrogen of both isomers, or the substrate must change its position relative to the hydrogen acceptor during the course of the racemization. The alternative mechanism is the "twohydrogen-acceptor" mechanism. Here two equivalent sites are involved. The sites are so located that one interacts with the a-hydrogen of one substrate-isomer and the other site with the a-hydrogen of the other isomer. Inversion of configuration is achieved by transferring the a-hydrogen from the substrate to one of the acceptor sites and hydrogen from the other acceptor site to the substrate-derived intermediate. The question concerning the number of hydrogen acceptor sites has not been experimentally investigated for any amino acid racemase. The general problem has been considered by Rose (1966) for enzymes which carry out proton shifts.

The turnover of total collagen in several tissues of 12-week-old normotensive and hypertensive rats was estimated by using tritium-labeled proline as a precursor. The effect of reutilization of the label was minimized by treatment with large doses of unlabeled proline subsequent to administering the radioactive imino acid. The collagen from skin, tail tendon, aorta, and mesenteric artery in normotensive animals had a half-life of about 60-70 days. In hypertensive animals the half-lives of skin and tail tendon collagen were unchanged but the half-lives of collagen in the aorta and mesenteric artery were reduced to 17 days.In previous studies from this laboratory we reported that hypertension in rats leads to an increased synthesis and deposition of collagen in arteries (1) and microvessels (2). In one set of experiments, treatment of hypertensive rats with f-aminopropionitrile, an inhibitor of collagen crosslinking, resulted in a lowering of the blood pressure within 3 weeks (3). If collagen deposition itself leads to a maintenance of the elevated pressure as we have proposed (3), then this reversal in such a short time may indicate that in hypertension collagen breakdown as well as synthesis may be accelerated. To investigate this possibility, we set about to measure the decay in specific activity of protein-bound hydroxyproline (collagen) after rats were labeled with L-[2,3-3H]proline. Many other investigators have attempted to measure collagen turnover using radioactive amino acids (4-9); however, no correction was made for reutilization of the labeled amino acid that was used. In this report we have utilized an in vivo pulse-chase experiment to minimize the problem of reutilization and have been able to estimate the turnover of collagen in several tissues of normal and hypertensive rats. Normotensive rats (12-weeks-old) with an average body weight of 373 g were kept in disposable cages in a hood. DOCA/salt hypertension was produced in uninephrectomized 6-week-ole male Wistar rats by twice weekly subcutaneous injection of DOCA (5 mg per rat) (10, 11). Rats were maintained on the standard diet and allowed free access to drinking water containing 1% NaCl. After 6 weeks of treatment their average blood pressure was 200 mm Hg (1 mm Hg = 133 Pa) and their average body weight was 416 g. During the experiments, the normotensive animals gained 120 g in 53 days and 250 g in 111 days, while the hypertensives gained 90 g in 53 days. Of the twenty rats started on the DOCA/salt regimen, five died from respiratory infections and three others could not be used because they were also infected. For this reason only three time points could be measured for the hypertensive group. Blood pressure was monitored weekly by the tail cuff microphone method with an instrument made by Hoffmann-La Roche & Co., Basel, Switzerland. MATERIALS AND METHODSBlood was obtained by either cardiac puncture or tail vein bleeding. Serum proteins were precipitated by the addition of 3 ml of 10% trichloroacetic acid per ml of serum and removed ...

Collagen synthesis is increased in the aortas, mesenteric arteries, and to a lesser extent, in the hearts of rats either made hypertensive with desoxycorticosterone acetate-salt or that are spontaneously hypertensive. Several markers of collagen biosynthesis were shown to be increased, including prolyl hydroxylase (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase), prolyl hydroxylaserelated antigen, total collagen content, and the incorporation of [3Hiproline into total protein and into collagen.The antihypertensive agents chlorothiazide and reserpine, when administered before the onset of hypertension in the rats treated with desoxycorticosterone acetate-salt, prevented or diminished the increase in collagen biosynthesis. When reserpine was given after the onset of hypertension, prolyl hydroxylase activity was decreased concomitant with the decrease in blood pressure. Treatment with reserpine is particularly effective in diminishing arterial prolyl hydroxylase activity.

Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) is a mixed-function oxygenase that hydroxylates peptidyl proline with the simultaneous and stoichiometric decarboxylation of a-ketoglutarate to succinate and CO2. It has been found that highly purified preparations of the enzyme can decarboxylate a-ketoglutarate in the absence of a peptidyl proline substrate. The uncoupled decarboxylation proceeds at only a fraction of the rate of the whole reaction and or study requires substrate quantities of the pure enzyme, as well as oxygen, ferrous ion, and ascorbate. No hydroxyproline is formed under these conditions. Immobilized antiserum to prolyl hydroxylase was found to remove both activities from enzyme preparations. However, addition of free antiserum during incubation inhibits only the complete reaction. Poly(Lproline), a specific inhibitor of prolyl hydroxylation, enhances the uncoupled decarboxylation of a-ketoglutarate without itself being hydroxylated. All of these findings prove that a-ketoglutarate can serve as substrate in the absence of peptidyl proline and is most likely the initial site of attack by oxygen. In the coupled reaction an oxidized form of the keto acid, perhaps a peroxy acid, then attacks prolyl residues in the unhydroxylated substrate. Prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) catalyzes the hydroxylation of peptidyl proline with the concomitant decarboxylation of a-ketoglutarate. For each mole of proline hydroxylated, one mole of a-ketoglutarate is stoichiometrically decarboxylated to succinate and carbon dioxide. The enzyme also has an absolute requirement for ferrous ion and a reducing agent (1).The hydroxyl group of the hydroxyproline is derived from molecular. oxygen (2, 3). After the specific and absolute requirement of the enzyme for a-ketoglutarate was discovered (1, 4), several other oxygenase systems that require a-ketoglutarate were described (5-9). Lindblad et al. (10) reported that 180 from molecular oxygen is incorporated not only into carnitine during the y-butyrobetaine hydroxylase reaction, but also into the succinate derived from a-ketoglutarate. Cardinale et al. (11) were able to demonstrate that prolyl hydroxylase catalyzes the incorporation of equal amounts of 180 into peptidyl hydroxyproline and into the succinate derived from aketoglutarate. On the basis of such findings, Holme et al. (12) proposed as a mechanism for the hydroxylation of y-butyrobetaine that a peroxide bridge is formed between the a-ketoglutarate and y-butyrobetaine. Such an intermediate could then dissociate to yield the hydroxylated 'y-butyrobetaine (carnitine) and succinate. It was proposed (12) that a hydroperoxide is first formed on y-butyrobetaine; this hydroperoxide could then attack a-ketoglutarate and the intermediate would then decompose to carnitine, succinate, and CO2. An analogous reaction mechanism, involving the initial formation of a hydroperoxide on the number four carbon of the prolyl residue, can be postulated for prolyl hydro...

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