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METABOLIC ACTIVITIES AND CLEAVAGE OF EGGS OF THE SEA URCHIN, ARBACIA PUNCTULATA A REVIEW, 1932-1949

1950; Marine Biological Laboratory (MBL); Volume: 98; Issue: 3 Linguagem: Inglês

10.2307/1538668

1939-8697

M. E. Krahl,

Microbial Metabolic Engineering and Bioproduction

1. This review deals with the general question of relation of metabolic activities to cleavage in the Arbacia egg. According to current biochemical concepts the answer to this question would be found in complete information about the following subjects:a. The identity and interrelationships of the catalysts by which the eggs derive energy from foodstuffs.b. The reactions by which energy is transferred to make possible the synthesis of structural and functional elements of the egg, such as carbohydrates, proteins, lipids, coenzymes, nucleic acids and other substances, many of which may yet be unknown.c. The chemical and Physical chemical rearrangements of these structural functional elements which lead to the biological event of cleavage.The third subject is completely beyond investigation by present techniques, and the second is just being opened to investigation in the living egg by application of isotopic labelling techniques. This review is concerned, therefore, chiefly with the nature and interrelationships of the catalysts by which Arbacia eggs derive energy from foodstuffs.2. The metabolic catalysts which have been shown to he available in the Arbacia egg for deriving energy from foodstuffs are:a. An enzyme with some, but not all, of the properties of the cytochrome oxidase of other animal tissues. After extraction from the egg it oxidizes cytochrome c, but whether this activity is related to its biological function in the egg is an open question, as no biochemically significant quantity of cytochrome c has yet been conclusively demonstrated in the eggs, although it is present in the sperm.The enzyme is poisoned by carbon monoxide, cyanide, azide, and sulfide, but not by copper-binding reagents. Its relative affinities for oxygen and carbon monoxide are, in the cell-free state, the same as those for cytochrome oxidase of mammalian muscle but in the intact Arbacia egg, these relative affinities are intermediate between those of the usual cytochrome oxidase and those of other iron-porphyrin compounds such as hemoglobin.b. The coenzymes, flavin-adenine dinucleotide, diphosphopyridine nucleotide, and diphosphothiamine. The properties of the enzymes of which these coenzymes are parts have not been investigated.c. A mechanism for oxidative disposal of pyruvate.d. An enzyme complex which can oxidize oxalacetate, α-ketoglutarate, or succinate with concomitant disappearance of inorganic phosphate, presumably via generation of "high energy" Phosphate bonds and esterification of suitable phosphate acceptors.e. The following enzymes which have undefined roles with respect to energy production and to chemical syntheses in the egg: dehydrogenases for various substrates other than those already mentioned; phosphatases; ribonuclease: desoxyribonuclease: catalase; peptidases.The distribution of various enzymes in the egg has been studied with very suggestive findings. In the unfertilized or recently fertilized egg the total reducing activity, cytochrome oxidase, and desoxyribonuclease are quantitatively most abundant in the cytoplasmic matrix, i.e. in solution or upon the small granules less than 1 µ in diameter. As development proceeds, a progressively greater fraction of the enzymes are associated with larger granules of the type which stain as mitochondria. In view of these observations and the fact that the capacity for oxidative phosphorylation is found, in the adult mammalian tissues so far studied, to be associated with mitochondrial particles, the suggestion arises that localization of enzymes on specific particles may parallel development of specific function.3. What is the actual pathway connecting enzyme activities with cleavage? Such answers to this question as are available come largely from experiments designed to block a specific metabolic enzyme system and to observe the degree to which the egg can or cannot cleave when deprived of this enzyme or group of enzymes.A note of caution should be emphasized here. The problem of explaining physiological effects in chemical terms is an exceedingly difficult one. It involves the demonstration not only that a given effect of a reagent upon an enzyme is sufficient to explain the physiological effect, but also the demonstration that the chemical effect offered as explanation is the only one possible, i.e. is a necessary explanation. Only the sufficiency criterion is usually met. In view of these considerations it is clear that conclusions about the relation of enzymes to function, which are based on action of inhibitors, must be only provisional. With this reservation in mind, the main findings about enzymatic activities and cleavage may be summarized.a. For energy to support cleavage, eggs of Arbacia are completely dependent on aerobic processes. Like other animal cells, the eggs can form lactic acid but this process cannot support cleavage.The nature of the foodstuffs oxidized is not settled; evidence for oxidation of carbohydrate or its phosphorylated breakdown products, of fats, or of amino acids has been offered, but is not conclusive.b. Evidence for participation of two enzyme systems in oxidative energy-yielding reactions has been presented.(1) The cytochrome oxidase system. The principal indication for participation of this oxidative step is that both the inhibition of oxygen consumption and the inhibition of cleavage by carbon monoxide are light reversible. Cytochrome oxidase is the only metabolic catalyst known to be subject to light-reversible inhibition by carbon monoxide.(2) An oxidative phosphorylation system. The principal evidence is the highly specific parallel between inhibition of cleavage and inhibition of a cell-free phosphorylating system from the eggs by a series of substituted phenols. Supporting evidence is found in the fact that these agents likewise block phosphorylation and phosphate transfer in the intact fertilized egg.c. In an effort to reveal the metabolic steps essential for cleavage, comparisons of the metabolic activities of unfertilized and fertilized eggs have been made.The oxygen consumption of intact Arbacia eggs increases upon fertilization to a degree which depends on the temperature, by a factor of about 10 times at 11° C., by a factor of about 2 times at 30° C., and negligibly at 32° C. The higher respiration of the fertilized egg is relatively more sensitive than that of the unfertilized both to inhibitors of cytochrome oxidase, such as cyanide and carbon monoxide, and to inhibitors which act at other loci, such as narcotics.The concentration of cytochrome oxidase is the same in unfertilized and fertilized eggs, and in great excess relative to oxygen consumption of intact eggs. The concentration of total dehydrogenase activity, as measured by ability to reduce ferricyanide, is likewise unchanged upon fertilization.The ability to dispose of pyruvate, on the other hand, appears to be increased following fertilization.From these and related facts, all observers appear to agree that the increase in oxygen consumption upon fertilization is the consequence of the introduction of a new or accelerated step between some substrate, perhaps pyruvate, and cytochrome oxidase. The nature of the new or accelerated step, a frequent subject of speculation, is unknown. It is not known to what extent, if any, the energy liberated by the excess oxygen consumption is used for synthetic processes and for cleavage.The precise mechanism by which oxidative energy is made available for cleavage remains to be defined.4. In the interest of completeness, and to provide a foundation for the further study of relation of metabolic activities and cleavage, the effects of all agents so far tested upon oxygen consumption and cleavage of Arbacia eggs have been recorded, even though their effects upon specific enzymatic activities frequently still await investigation. Examples of the viewpoints from which these substances have been investigated, and of the agents employed, are:a. To demonstrate participation of various enzyme systems in yielding energy for cleavage: poisons for cytochrome oxidase such as cyanide, carbon monoxide, azide, sulfide; poisons for phosphorylation processes such as substituted phenols, azide, usnic acid; and poisons for other metabolic enzymes such as naphthoquinones and iodoacetate.b. To learn more about the mechanisms by which physiologically active substances affect growth and cell division: acids and bases, carbamates, colchicine, nitrogen mustards, sulfonamides, penicillin.c. To determine the form in which toxic ionizable substances penetrate living cells such as Arbacia eggs: substituted phenols, barbiturates, local anesthetics, other weak acids and bases. These agents appear to gain access to the interior of the Arbacia egg chiefly, if not entirely, as undissociated molecules.