George J. Cardinale

Simultaneous incorporation of 18O into succinate and hydroxyproline catalyzed by collagen proline hydroxylase

Abstract The sequential copolymer (Pro-Gly-Pro)∼12 was hydroxylated in an 18O2 atmosphere with a partially purified preparation of collagen proline hydroxylase. Mass spectral analysis demonstrated the incorporation of one atom of 18O into hydroxyproline and one atom into succinic acid. Since the enzyme produces equal amounts of peptidyl hydroxyproline and succinate these findings strongly suggest that one molecule of O2 takes part in the simultaneous oxygenation of peptidyl proline and α-keto-glutarate. A possible mechanism for this reaction is presented.

Prolyl hydroxylase and an immunologically related protein in mammalian tissues

A modified procedure for measuring protein antigenically related to prolyl hydroxylase has been devised which permits determination of antigen in units of enzyme activity. With this enzyme immunoassay it has been possible to show that tissues of the rat and mouse contain, in addition to prolyl hydroxylase, large amounts of an inactive cross-reacting protein, which can be separated from the enzyme by gel filtration and ion-exchange chromatography. The latter protein was first found in cultured fibroblasts. In all instances cross-reacting protein has a molecular weight of about 80,000–100,000, whereas active enzyme has a molecular weight of about 350,000–400,000. The cross-reacting protein may represent an intracellular pool of enzyme precursor which can be activated rapidly in circumstances requiring rapid collagen biosynthesis.

An activatable form of prolyl hydroxylase in fibroblast extracts

Abstract An in vitro increase in prolyl hydroxylase activity has been effected in sonicates of early log phase L 929 mouse skin fibroblasts from either monolayer or suspension cultures. The requirements for activation are identical to those needed for the hydroxylation reaction itself, i.e., ferrous ion, ascorbate and α-ketoglutarate. Catalase, which is not an absolute requirement for the hydroxylation, is also necessary for activation. The activation is time dependent and, under the conditions used, is complete in 3 hr at 30°. Since ferrous ion also appears necessary for the activation in intact cells and since the same level of activation is achieved in intact cells as in sonicates, it appears that the in vitro activation proceeds in the same manner as that seen in cultured cells.

Increased Vascular Collagen Biosynthesis by Hypertension and Reversal by Antihypertensive Drugs

Collagen synthesis was increased in aortas, mesenteric arteries, cerebral microvessels, pial artery, basilar artery and decreased in the heart of rats made hypertensive with deoxycorticosterone acetate-salt or the spontaneously hypertensive animal. The markers of collagen biosynthesis that were elevated were prolyl hydroxylase, prolyl hydroxylase-related antigen, total collagen content and the incorporation of labelled proline into total protein and into collagen. The antihypertensive drugs reserpine and chlorthiazide could both prevent the increase or reduce the increase in collagen synthesis.

The formation of hydroxyproline in collagen by cells grown in the absence of serum.

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggests that reductants other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.

ACTIVATION OF PROLYL HYDROXYLASE IN FIBROBLASTS BY ASCORBIC ACID

Green and Goldberg first reported that in cultured fibroblasts significant amounts of peptidyl hydroxyproline first appeared in late log-phase. They also demonstrated that the addition of lactate to the culture medium in early logphase resulted in a large increase in peptidyl hydroxyproline formation.’ Subsequently, Gribble et al.3 showed that even though L-929 mouse skin fibroblasts synthesized primary collagen chains in early log-phase, prolyl hydroxylase activity did not increase until late log-phase and that it was at this stage that peptidyl hydroxyproline was formed. Other studies by these authors 4 , revealed that the activity of prolyl hydroxylase in early log-phase could be stimulated severalfold if the cells were concentrated to a higher density or if lactate were added to the medium. Since the activation was independent of protein and RNA synthesis, the results indicated the existence of an inactive precursor of prolyl hydroxylase in early log-phase cells that is activated during normal cell growth or by cell crowding or lactate treatment. Further evidence for this hypothesis was obtained by McGee et al.,” who demonstrated that during cell crowding or lactate treatment, the amount of protein that cross-reacted with a monospecific antibody to prolyl hydroxylase remained virtually constant while the enzyme activity increased severalfold. In succeeding work they were able to isolate an enzymatically inactive protein from early log-phase L-929 fibroblasts that cross-reacted with antibody to prolyl hydroxylase. Furthermore, they also demonstrated that this material had a molecular weight about one-third that of the active enzyme and therefore suggested that the cross-reacting protein may be a subunit precursor of the active enzyme. There have been many reports”-l-* that the amount of hydroxyproline formed by cultured fibroblasts and osteoblasts is increased by treatment with ascorbate. Peterkofsky lz reported that ascorbate rapidly deteriorates under tissue culture conditions and that maintenance of adequate ascorbate levels results in a marked increase in peptidyl hydroxyproline in early log-phase cells. To determine whether the increased hydroxyproline formation is due to enzyme activation we investigated the effect of ascorbate on prolyl hydroxylase activity in early log-phase L-929 fibroblasts.

Hypertension-Induced Vascular Fibrosis and its Reversal by Antihypertensive Drugs

It is generally accepted that neuronal and/or humoral (neurohumoral) factors play an important role in the initiation of hypertension and that the increase in blood pressure, if maintained for a length of time, results in a hypertrophy of the vasculature. The increased wall-to-lumen ratio in the vasculature leads to an increase in peripheral resistance. This initiates a cyclic process, for the increased peripheral resistance further aggravates the situation and blood pressure is elevated even more (1). Although there have been great advances in our understanding of both the etiology and therapy of hypertension, almost all of them concern the neuronal or humoral factors involved (i.e., renin, angiotensin, aldosterone, catecholamines, Na+, etc.). However, the biochemical consequences in the vasculature due to elevated blood pressure have only recently begun to be studied.

Inhibition of prolyl hydroxylase activity by bradykinin analogs containing a prolyl-like residue

Abstract A number of substituted bradykinin analogs were prepared in which the proline in position 3 was replaced by analogs of proline. All of the bradykinin analogs, with the exception of l -azetidine-2-carboxyl 3 -bradykinin showed significant ability to inhibit prolyl hydroxylase activity. Addition of an l -glutamyl residue to the amino terminus of 3,4-dehydro- l -prolyl 3 -bradykinin and trans -4-hydroxy- l -prolyl 3 -bradykinin resulted in competitive inhibitors of increased effectiveness with K i , values approximately 10 −4 m . One of the peptides, l -3,4-dehydro- l -prolyl 3 -bradykinin, appeared to serve as a substrate for prolyl hydroxylase.

Morphine and codeine are endogenous components of human cerebrospinal fluid

We have examined cerebrospinal fluid (CSF) from twelve patients who were not on any medication and found them to contain both morphine and codeine in concentrations of 2 to 339 fmol/ml. These are comparable to the concentration of opioid peptides in spinal fluid. Both morphine and codeine are present mainly in conjugated form from which the free alkaloids can be released by acid hydrolysis.