Studies on Cell Metabolism and Cell Division

Abstract

1. An enzyme capable of oxidizing reduced cytochrome c (i.e. a cytochrome oxidase) has been obtained from Arbacia eggs. In 0.02 M hydroquinone, the cytochrome oxidase was half activated at a cytochrome c concentration of approximately 4 x 10–6 M. The concentration of the cytochrome oxidase was found to be nearly the same in unfertilized and fertilized eggs, the amount of the enzyme—as measured by means of its activity toward cytochrome c as a representative substrate—being more than sufficient to account for the highest rate of oxygen utilization yet observed in the intact, living, fertilized eggs, and of the same order as that in certain rat tissues. 2. The Arbacia cytochrome oxidase was strongly inhibited by carbon monoxide in the dark, the inhibition being almost completely reversed by light. The inhibition constant was not greatly altered by variation in the concentration of cytochrome c or the concentration of hydroquinone used as reductant for the cytochrome c, having a value of 3 to 5 under the conditions used. The inhibition constant was about 2 with p-phenylenediamine as reductant for the cytochrome c, but apparently had the surprisingly low value of about 0.5 with 0.02 M cysteine as reductant. 3. The cytochrome oxidase was completely inhibited by sufficiently high concentrations of sodium cyanide, sodium azide, and sodium sulfide. It was also completely inhibited in 0.6 M sodium chloride. It was not inhibited by two inhibitors of copper containing enzymes, 8-hydroxyquinoline and sodium diethyldithiocarbamate. It was also not significantly inhibited by 2,4-dinitrothymol, 2,4-dinitro-o-cyclohexylphenol, phenylurethane, 5-isoamyl-5-ethylbarbituric acid, or iodoacetic acid. 4. Quantitative examination of the fertilized eggs showed that cytochrome c, if present at all, occurred in a concentration of less than 2 micrograms per gram of wet fertilized Arbacia eggs. On the basis of these data and those of Fig. 2, above, it seems safe to conclude that cytochrome c cannot carry a significant fraction of the oxygen consumption of fertilized Arbacia eggs. It was also found that, in contrast to similar preparations from certain other animal tissues, the Arbacia cytochrome oxidase preparation displayed no succinic dehydrogenase activity when tested manometrically in the presence of excess cytochrome c. 5. Extending previously reported (3) experiments with other inhibitors, the effects of sodium azide and sodium sulfide on the respiration and cell division of fertilized Arbacia eggs were determined, the eggs being initially exposed to the reagents 30 minutes after fertilization at 20°C. With either reagent cleavage was completely blocked by a concentration of reagent which reduced the respiration to approximately 50 per cent of the normal level. 6. On the basis of certain theoretical considerations regarding the possible mechanism of action of cyanide and other respiratory inhibitors it is suggested that a fraction of the respiration apparently concerned with supplying energy for division processes in the fertilized Arbacia egg may be keyed into the respiratory cycle through a carrier having a somewhat higher potential than those which carry the larger portion of the egg respiration. The theory is also employed in an effort to resolve a number of hitherto apparently paradoxical observations regarding the effects of cyanide, azide, and carbon monoxide on cell respiration.

The Effects of Caffeine on Oxygen Consumption and Cell Division in the Fertilized Egg of the Sea Urchin, Arbacia Punctulata

“…Relationship of caffeine concentration to the degree of cleavage in- cytochrome oxidase point from the evidence of Krahl, Keltch, Neubeck, and Clowes (1941) and others. I have no direct evidence that caffeine acts specifi- cally on the cytochrome oxidase enzyme but it should be noted that the caffeine maximum inhibition of 02 consumption is above 60 per cent and cyanide maximum inhibition is above 70 per cent.…”

“…Ball and Meyerhof (1940) indicated the presence of an oxidative enzyme upon which respiratory inhibitors act in unfertilized Arbacia eggs. Krahl, Keltch, Neubeck, and Clowes (1941) reported a cytochrome oxidase enzyme, in the fertilized Arbacia egg, which can oxidize reduced cytochrome C. These authors demonstrated that this enzyme, when acting with cytoghrome C as a substrate, is inhibited completely by sodium cyanide or sodium azide, just as these agents depress cell division in the fertilized living Arbacia egg. In contrast, the total respiration can be inhibited only to a maximum of 70 to 80 per cent by cyanide and only about 50 per cent by azide.…”

Nitric Oxide and Hydrogen Sulfide Regulation of Ischemic Vascular Growth and Remodeling

“…Earlier works between 1877 and 1884 identified H 2 S as a “bacterial product” with its roles in intestinal and periodontal diseases, and more recently in regulating bacterial resistance to antibiotics. It was not until the 1940’s that H 2 S was identified as an inhibitor of cytochrome c oxidase (169). Only in the last two decades, have positive effects of H 2 S as a regulator of physiological functions, including the cardiovascular and nervous system, been identified.…”

A symposium on respiratory enzymes

“…See, however, Haber and Weiss ( 18 ) . 90 A SYMPOSIUM ON RESPIRATORY ENZYMES peroxide reaction contains the enzyme in the ferric state (61).…”