Karl M. Wilbur

EXPERIMENTAL STUDIES ON CALCIFICATION IN MOLLUSCS AND THE ALGA COCCOLITHUS HUXLEYI

The calcium carbonate crystals of biological systems occur as calcite, as aragonite, and-less commonly-as vaterite. The reasons for the formation of a particular type of crystal in a given organism are not well understood, although studies of crystallization in solutions have indicated factors which may play a role. These include the presence of organic compounds, inorganic salts, and variations in temperature (Saylor, 1928 ; Kitano, 1962; Kitano et al. 1962; Curl, 1962; Wilbur and Yonge, in press). In molluscs calcium carbonate crystals are usually formed in association with an organic matrix, and it has been suggested that the chemical structure of the matrix may determine the type of crystal (Roche et al., 1951; Ranson, 1952; Watabe and Wilbur, 1960). Temperature also appears to have an influence on the type of crystal deposited by animals. Lowenstam (1954a, b ) found that in organisms which deposited both aragonite and calcite the proportion of aragonite was higher in those animals that lived a t higher environmental temperatures. The first, considered in SECTION I, concerns the influence of three conditions on crystal type: (1) the presence of organic matrices taken from aragonitic and calcitic shells; (2) alteration of temperature; and (3) the supply of nitrogen. In investigating the influence of the matrix we have determined the crystal types which form on decalcified matrices taken from various molluscan species and introduced into other species or recalcified in vitro. The effects of temperature on crystal type have been examined in two systems, the regenerating shell of the snail Viviparus intertextus and the alga Coccolithus huxbyi. The effect on crystal type of reducing the nitrogen content of the medium has also been observed in C. huxleyi. SECTION I1 concerns the relation of crystal formation to cell structure in C. huxleyi. This cell provides an especially interesting opportunity for study of this relationship in that one strain normally forms no CaC03 crystals but can be induced to calcify by reducing the nitrogen content of the medium.

Photosynthesis and coccolith formation: Inorganic carbon sources and net inorganic reaction of deposition1

The concept that the formation ofCnC0, coccoliths functions as a photosynthetic adaptation for the use of bicarbonate is evaluated in the coccolithophorids Coccoliths hudeyi and Cricosphneru carterae by two new methods. In the first, carbon fixation is measured at 10-s intervals in the first 2 min after addition of “CO, and II’“CO,- to buffered cultures; this method exploits the relatively long half-time for the hydration or dehydration of dissolved COP. In the second, shifts in pH and alkalinity resulting from carbon fixation by cells growing in liqllid culture are assessed to indicate fluxes of COz and HC03- into cells and these values compared to measurements of l”C incorporation in photosynthesis and carbonate deposition. The data are interpreted in terms of one of several net inorganic reactions of deposition considered. In this reaction, CO, is the substrate of photosynthesis and HCO,- is the form of carbon supplied to the calcification site. CO, resulting from carbonate deposition supplements the COz from the medium

Studies on isolated cell components: I. Nuclear isolation by differential centrifugation

Abstract A method is presented for isolating nuclei of rat and hamster liver in a high state of purity and in a condition optically similar to nuclei within living cells. The isolation procedure consists in the homogenization and differential centrifugation of fresh liver at 0–5 ° in a salt-sucrose solution buffered at pH 7.1. By layering the material to be centrifuged over a relatively large volume of a slightly denser solution the purification can be carried out in 4 or 5 centrifugations. The entire procedure can be completed in about 90 minutes. The yield as determined by measurements of desoxyribonucleic acid is about 5 per cent. The solutions contain KH2PO4, K2HPO4, NaHCO3, and sucrose. The sucrose concentration is varied to give density differences required for layering. Salt-sucrose solutions maintain a large proportion of the isolated nuclei in a nongranular condition for 6 hours at 0 °. Pure sucrose is less satisfactory for maintenance. The protein-desoxyribonucleic acid ratio for the isolated nuclei averages 5.1 with a range of 2.7 to 8.9.

STUDIES ON SHELL FORMATION. V. THE INHIBITION OF SHELL FORMATION BY CARBONIC ANHYDRASE INHIBITORS

1. The carbonic anhydrase inhibitors 2-benzothiazolesulfonamide (Cpd. I) and 2-acetylamino-1,3,4-thiadiazole-5-sulfonamide (Diamox) (Cpd. II) markedly reduced the rate of deposition of calcium in the shell of the oyster Crassostrea virginica. Treatment of oysters with Cpd. I, 1:80,000, for 7-8 hours reduced the rate to one-tenth the normal value and gave essentially complete inhibition at higher concentrations. The latter result indicates that inhibition by this compound is not limited to carbonic anhydrase. With Cpd. II the maximum inhibition was 50% and was produced at a concentration of 1:100,000 or higher.2. Measurements of shell movements and oxygen consumption of mantle tissue showed that concentrations of carbonic anhydrase inhibitors which had a marked inhibitory action on calcium deposition exerted no general toxic action on the oyster.3. The rate of calcium deposition of the oyster was more than 9 times that of the isolated mantle-shell preparation, confirming an earlier study. The addition of 0...

The inhibition of certain mitochondrial enzymes by fatty acids oxidized by ultraviolet light or ascorbic acid.

Abstract 1. 1. The relation between lipide oxidation and the activity of succinoxidase, choline oxidase, and aminoxidase of rat liver mitochondria has been examined following ultraviolet irradiation, incubation of mitochondria with ultraviolet-irradiated methyl linolenate and methyl linoleate, and incubation with ascorbic acid. 2. 2. Ultraviolet irradiation of mitochondria produced an oxidation of lipide which was directly proportional to the period of irradiation. Succinoxidase and choline oxidase were inhibited in direct proportion to the extent of lipide oxidation. Incubation of irradiated mitochondria with reduced glutathione gave a partial ( 3. 3. Incubation of mitochondria with ascorbic acid resulted in an inhibition of succinoxidase, choline oxidase, and aminoxidase which was directly proportional to lipide oxidation, as in the case of ultraviolet irradiation. Liver slices incubated with ascorbic acid also showed an increased lipide oxidation and decreased succinoxidase activity. 4. 4. Succinoxidase was inhibited by ultraviolet-irradiated methyl linolenate and its water-soluble oxidation products. The unoxidized ester had little or no effect. Methyl linolenate and methyl linoleate oxidized to the same degree by ultraviolet irradiation had equal inhibitory action on succinoxidase. 5. 5. The results indicate that the effects of ultraviolet light on cells may be mediated in part by the oxidation products of lipide constituents.

6 – Shell Formation

I. Summary and Perspective The formation of shell can be described in terms of two major phases: (1) cellular processes of ion transport, protein synthesis, and secretion and (2) a series of physicochemical processes in which crystals of CaCO3 are nucleated, oriented, and grow in intimate association with a secreted organic matrix. In providing the mineral of shell, Ca2+ and HCO3− are first transported across epithelia at the body surface and the mantle epithelium facing the inner shell surface. Movement of these ions is incompletely understood but almost certainly involves active transport of Ca2+. The physicochemical phase takes place in fluid between the outer mantle epithelium and the inner shell surface and on this shell surface. For crystals to be deposited, the following conditions are required (1) concentrations of Ca2+ and CO32- exceeding the solubility product, (2) conditions favoring crystal nucleation, and (3) the elimination of H+ resulting from CaCO3 formation. Following crystal nucleation oriented crystal growth leads to a shell constructed of a variety of crystal patterns. In the process protein secreted by the mantle surrounds the individual crystals and becomes the cement which binds them together as a shell. Our understanding of the processes of the crystal depositional phase of shell formation is fragmentary. However, we can expect that through analyses of the fluid at the site of crystallization and study of effects of organic fractions of the fluid and shell on crystallization, the conditions governing the initiation and control of crystal growth will be further defined. Substances that initiate crystallization have been proposed but have not been tested. Preliminary studies indicate that the compounds at the crystallization site include inhibitors of CaCO3 deposition. A major aspect of shell construction of great interest yet to receive experimental study is the relation of particular segments of the mantle epithelium to the structure and orientation of strikingly different types of crystal patterns. A promising approach to these crystallographic problems appears to be the application of in vitro methods of physical biochemistry to crystal development and orientation in association with fractions of the organic phase of shell. Studies indicating involvement of the nervous system and hormonal and neurosecretory changes associated with shell formation will almost certainly be expanded and contribute to the understanding of mechanisms of mineralization and their control.

Amino acids of the organic matrix of neogastropod shells

Abstract 1. 1. Analyses of the organic matrix of the shell of the neogastropods Littorina irrorata, Polinices duplicatus, Thais floridana, Pila virens, Campeloma decisum and Nassarius obsoletus have shown differences in amino acid content between the soluble and insoluble fractions of each species as well as interspecific differences in both fractions. 2. 2. Glycine, glutamic and aspartic acids, serine and alanine were present in highest concentration in both soluble and insoluble fractions. 3. 3. The soluble fraction of each species had a higher aspartic and glutamic acid content than the insoluble fraction. 4. 4. Glycine, phenylalanine and tyrosine were consistently higher in the insoluble fraction than in the soluble fraction. 5. 5. The insoluble matrix of the shell of Nassarius differed from that of the other five species in containing unusually high or low concentrations of several of the amino acids. 6. 6. The soluble and insoluble fractions of molluscan shell matrix are thought to have different functions in shell formation.

EFFECTS OF AMINO ACIDS, MAGNESIUM, AND MOLLUSCAN EXTRAPALLIAL FLUID ON CRYSTALLIZATION OF CALCIUM CARBONATE: IN VITRO EXPERIMENTS

The extrapallial fluid (EPF), the fluid component of the shell-forming system of molluscs, has been examined for its effect on the rate of CaCO3 crystal formation in vitro. The medium was an artificial inorganic extrapallial fluid supersaturated with respect to CaCO3. EPF of the bivalves Crassostrea virginica and Mercenaria mercenaria strongly inhibited CaCO3 crystallization in high dilution. The inhibitory material had negatively charged groups as indicated by the removal of inhibition subsequent to passage through a DEAE-Sephadex column and the restoration of inhibition in material eluted from the column with 2.0 M NaCl. Polyaspartic and polyglutamic acids with COO- groups also inhibited crystal formation whereas polyamino acids lacking those groups and free amino acids were without effect. The inhibition by the acidic polyamino acids suggests that polypeptides known to be present in EPF are one possible cause of inhibition of crystal formation by EPF. Mg present in EPF was also strongly inhibitory. The...

The chemical composition of the periostracum of the molluscan shell

Abstract 1. 1. Fifteen amino acids were present in the periostracum of each of twenty-seven species of gastropods and bivalves. 2. 2. Glycine usually accounted for 40 per cent or more of the amino acids of the periostracum of marine and fresh-water bivalves and of fresh-water and terrestrial gastropods. 3. 3. The periostracum of marine gastropods has less glycine and higher ranges of lysine, arganine, aspartic acid and serine than that of fresh-water species. 4. 4. The amino acid composition of Potamopyrgus periostracum was altered by culturing in different salinities. 5. 5. Electron microscopic examination of the periostracum of twelve genera of gastropods showed that fresh-water species had two layers, terrestrial species had a single layer and marine species had a single or incomplete layer.

STUDIES ON SHELL FORMATION. IV. THE RESPIRATORY METABOLISM OF THE OYSTER MANTLE

1. The oyster mantle has been studied with respect to endogenous respiration, activity of various oxidative enzymes and response to intermediates of the tricarboxylic acid cycle, and respiratory inhibitors.2. The endogenous respiration has been found to be similar for different mantle regions, though small differences were present. Endogenous respiration was not significantly altered by changes in the pH of the sea water medium between 6.0 and 8.4 or by isolation of mantles in sea water without added substrate for periods of a few days.3. Isocitric, succinic and malic dehydrogenases, fumarase, and cytochrome oxidase were present in the mantle. Aconitase could not be demonstrated. Oxaloacetic decarboxylase was found in very high concentration. Its presence may be of significance with respect to the formation of shell carbonate.4. Isocitrate, succinate, malate, and oxaloacetate stimulated respiration. Enzyme assays and the effects of added substrates indicated the presence of the major portion of the tricar...