The isolation and properties of porcine ferritin and apoferritin.

Porcine ferritin and apoferritin were purified to a greater degree of homogeneity than has been reported previously. Porcine ferritin was insoluble in the absence of a reducing agent, possessed a high content of iron, with an average $\text{Fe}\text{N}$ ratio of ~2.5, and contained almost no detectable endogenous apoferritin. The amino acid composition, ultraviolet-absorption spectrum, and ultraviolet-circular dichroism spectrum of porcine apoferritin are very similar to the respective parameters of equine apoferritin. The native and subunit molecular weights of porcine apoferritin are 503,000 and 20,000, respectively.     

The effect of ethanol and acetaldehyde on [14C]pantothenate incorporation into CoA in cultured rat liver parenchymal cells

The effect of ethanol on [¹⁴C]pantothenate incorporation into CoA and on total CoA levels was measured in 3-day-old primary cultures of adult rat liver parenchymal cells. Ethanol decreased the incorporation of radioactivity into CoA a maximum of 67%, 5 mm ethanol was saturating for the inhibitory effect and 0.2 mm ethanol was sufficient for half-saturation. This inhibitory effect did not result from a loss of CoA precursors or from cell death. Ethanol concentrations up to 10 mm did not decrease the ATP content of cells or the total protein content of cells which adhered to the incubation flask. Ethanol (5 mm) had no effect on the cyteine + cystine content of the cells. Intracellular pantothenate concentrations were not affected by 5 mm ethanol, and increasing the pantothenate concentration did not affect ethanol inhibition. Ethanol inhibition of [¹⁴C]pantothenate conversion to CoA could be fully reversed by rinsing the cells free of ethanol. The ethanol inhibition could also be fully reversed by addition of 4-methylpyrazole, indicating that ethanol must be oxidized via alcohol dehydrogenase to exert its inhibitory effect. Acetaldehyde, the immediate product of alcohol dehydrogenase, was also an inhibitor of the incorporation of [¹⁴C]pantothenate into CoA; the maximum inhibition was 63%. Acetaldehyde concentrations maintained between 18 and 103 μm inhibited incorporation by 57%. The inhibition by acetaldehyde did not correlate well with changes in the NADH and NAD⁺ ratio of the cells (as determined by measuring changes in the lactate-to-pyruvate ratio). The ability of glucagon, dibutyryl cAMP + theophylline, or dexamethasone to stimulate [¹⁴C]pantothenate conversion to CoA was not decreased by the addition of ethanol or acetaldehyde, indicating that ethanol inhibition does not occur by reversal of the cAMP-mediated regulatory mechanism for CoA biosynthesis.     

Variable denaturation of ovalbumin by incorporation of amino acid analogs

A novel firefly luciferin- enhanced luminescent procedure for the quantitation of horseradish peroxidase labels has been directly incorporated into established enzyme immunoassays. The procedure is rapid and sensitive and uses readily available reagents. Light emission from the enhanced reaction is high and relatively constant and thus easily measured. The luminescence procedure has been successfully incorporated into immunometric assays for rubella antibody and human IgE and into a competitive immunoassay for digoxin.     

Siderophore utilization and iron uptake by Rhodopseudomonas sphaeroides

The growth of Rhodopseudomonas sphaeroides in iron-deficient medium did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate type. Iron-limited cultures of R. sphaeroides were not able to remove iron from ferric transferrin unless supplemented with 2,3-dihydroxybenzoic acid. R. sphaeroides was shown to take up 59Fe+3 when it was supplied as ferric chloride, ferric citrate, or ferric parabactin, but not when supplied as ferric rhodotorulate or ferric Desferal. When iron was supplied as ferric citrate, citrate was not taken up by the cells. The growth rate of R. sphaeroides under iron-limiting conditions was decreased by the addition of either Desferal or rhodotorulic acid, while the addition of citrate or parabactin did not affect growth.     

The role of iron chelates in hydroxyl radical production by rat liver microsomes,NADPH-cytochrome P-450 reductase and xanthine oxidase

Uninduced rat liver microsomes and NADPH-Cytochrome P-450 reductase, purified from phenobarbital-treated rats, catalyzed an NADPH-dependent oxidation of hydroxyl radical scavenging agents. This oxidation was not stimulated by the addition of ferric ammonium sulfate, ferric citrate, or ferric-adenine nucleotide (AMP, ADP, ATP) chelates. Striking stimulation was observed when ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (DTPA) was added. The iron-EDTA and iron-DTPA chelates, but not unchelated iron, iron-citrate or iron-nucleotide chelates, stimulated the oxidation of NADPH by the reductase in the absence as well as in the presence of phenobarbital-inducible cytochrome P-450. Thus, the iron chelates which promoted NADPH oxidation by the reductase were the only chelates which stimulated oxidation of hydroxyl radical scavengers by reductase and microsomes. The oxidation of aminopyrine, a typical drug substrate, was slightly stimulated by the addition of iron-EDTA or iron-DTPA to the microsomes. Catalase inhibited potently the oxidation of scavengers under all conditions, suggesting that H₂O₂ was the precursor of the hydroxyl radical in these systems. Very high amounts of superoxide dismutase had little effect on the iron-EDTA-stimulated rate of scavenger oxidation, whereas the iron-DTPA-stimulated rate was inhibited by 30 or 50% in microsomes or reductase, respectively. This suggests that the iron-EDTA and iron-DTPA chelates can be reduced directly by the reductase to the ferrous chelates, which subsequently interact with H₂O₂ in a Fenton-type reaction. Results with the reductase and microsomal systems should be contrasted with results found when the oxidation of hypoxanthine by xanthine oxidase was utilized to catalyze the production of hydroxyl radicals. In the xanthine oxidase system, ferric-ATP and -DTPA stimulated oxidation of scavengers by six- to eightfold, while ferric-EDTA stimulated 25-fold. Ferric-desferrioxamine consistently was inhibitory. Superoxide dismutase produced 79 to 86% inhibition in the absence or presence of iron, indicating an iron-catalyzed Haber-Weiss-type of reaction was responsible for oxidation of scavengers by the xanthine oxidase system. These results indicate that the ability of iron to promote hydroxyl radical production and the role that superoxide plays as a reductant of iron depends on the nature of the system as well as the chelating agent employed.     

Bone remodelling induced by physical stress is prostaglandin E2 mediated

In vitro cultured bone cells were found to be responsive to hormones and physical forces. A simple device has been developed which enables the direct application of physical forces to tissue culture dishes to which cells are firmly attached. The physical forces created a deformation of the dish. It was found that prostaglandin E₂ synthesis underwent a rapid increase, reaching a maximum after 20 min and then declined. Concurrent with the increase in prostaglandin E₂ was an increase in cyclic AMP production, having a maximum around 15 min. The increase in cyclic AMP was blocked by indomethacin, the prostaglandin E₂ synthesis inhibitor, indicating the dependence of cyclic AMP production on the de novo synthesis of prostaglandin E₂. Prostaglandin E₂ added to cells mimicked the effect of physical forces on the production of cyclic AMP. The increase in cyclic AMP resulted from an early rise in adenyl cyclase activity (within 5 min) and a later (10 min) increase in phosphodiesterase activity. The same physical forces also stimulatedthe incorporation of thymidine into DNA after 24 h. On addition of prostaglandin E₂ the increase in DNA synthesis was also mimicked. Pretreatment of the cells with indomethacin abolished the effect of physical forces on DNA synthesis.The results suggest a stimulus receptor mechanism for physical forces which, like hormonal effectors, are mediated by prostaglandins and stimulate cyclic AMP and DNA synthesis.We believe that physical forces stimulate bone remodelling through such a stimulus receptor system, mediated by prostaglandins.     

A fast block to polyspermy in frogs mediated by changes in the membrane potential

The role of the egg membrane potential in the prevention of polyspermy in Rana pipiens was studied with intracellular microelectrodes and ion-substituted media. At fertilization, the egg membrane potential shifts from a resting value of ?28 to +8 mV in a single step of less than 1 sec. A second, slower shift reaches a maximum amplitude of +17 mV; the membrane potential is positive for a total of 21 min. When the membrane potential of unfertilized eggs exposed to sperm was held at +1 to +22 mV for 30 min by injecting current through a second intracellular electrode, the initiation of the first cleavage furrow was delayed about 20 min, suggesting that the eggs were not fertilized while the membrane potential was positive. Injection of a similar amount of current after fertilization did not delay cleavage. Furthermore, fertilization in ion-substituted media suggests a correlation between the maximum amplitude of the positive-going shift and the incidence of polyspermy. Up to 25% of eggs were polyspermic when inseminated in the presence of NaI, and the maximum amplitude was reduced to ?20 mV when eggs were fertilized in 40 mM NaI. In contrast, fertilization in 40 mM NaCl reduced the maximum amplitude only to +6 mV, and produced no polyspermy. In solutions of NaBr, intermediate effects on the membrane potential and polyspermy were seen. Comparable results were obtained with the toad, Bufo americanus. We conclude that the membrane potential shift prevents polyspermy.     

Modulation of adenine nucleoside excretion and incorporation in adenosine deaminase deficient human lymphoma cells

The availability of a human lymphoma cell line deficient in adenosine deaminase, adenosine kinase and methylthioadenosine phosphorylase enabled us to compare the effects of nucleoside transport inhibitors on the excretion of endogenously generated adenosine, deoxyadenosine and 5'-methylthioadenosine. The nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole blocked the efflux of adenosine, but not deoxyadenosine or 5'-methylthioadenosine. The inhibitors also prevented the uptake of exogenous adenosine, but not deoxyadenosine or 5'-methylthioadenosine, by human lymphoblasts. The results show (i) that the transport inhibitors modify adenine nucleoside efflux and influx similarly, and (ii) that the effects of the compounds on the excretion and uptake of these three physiologically important adenine nucleosides are distinctly different.     

The effect of prostaglandins E1 and E2 on the human erythrocyte as monitored by spin labels

The effects of the prostaglandins PGE₁ and PGE₂ on the deformability of the human erythrocyte were studied using spin-labeled erythrocytes. Two magnetic resonance parameters were measured: (1) The orientation relaxation time, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, for the erythrocyte, and (2) the order parameter, S, for a fatty acid spin label bound to the membrane. Prostaglandins PGE₁ and PGE₂ exhibited opposite effects on both $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S. PGE₂ made the cell less deformable (increases of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S) and PGE₁ made the erythrocyte more deformable (decrease of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S).     

Influence of membrane fluidity on transport mediated by ubiquinones through phospholipid vesicles

Coenzymes Q₁₀ and Q₃ are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ₁₀ and CoQ₃ reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.     

The incorporation of [14C]acetate into the constituent fatty acids of monogalactosyldiglyceride by isolated spinach chloroplasts

The incorporation into diglycerides of the acyl products synthesized from acetate by spinach chloroplasts was greatly stimulated by the addition of glycerol 3-phosphate. When UDP-galactose was added also, monogalactosyldiglycerides became the major products. Palmitate biosynthesis was stimulated about twofold by these additions, while oleate biosynthesis decreased slightly, so that oleate:palmitate ratios were in the range 0.6 to 0.8 rather than about 1.6 when glycerol 3-phosphate and UDP-galactose were not added. On the other hand, Triton X-100 greatly stimulated both oleate and palmitate biosynthesis to give oleate:palmitate ratios of about 2.0. The proportions of oleate and palmitate in the newly synthesized diglycerides, or in monogalactosyldiglycerides when exogenous UDP-galactose was added, did not always reflect the proportions of these two fatty acids synthesized from acetate. When oleate:palmitate ratios were ?1, equal amounts were incorporated into diglycerides or into monogalactosyldiglycerides. When oleate:palmitate ratios were <1, incorporation of palmitate into diglycerides and monogalactosyldiglycerides exceeded that of oleate. 1-Oleoyl, 2-palmitoyl glycerol compounds were the principal products under all conditions but 1,2-dipalmitoyl compounds were also quantitatively important when glycerol 3-phosphate alone, or glycerol 3-phosphate together with UDP-galactose, was added. The distribution of label in the constituent glycerol and fatty acid moieties when monogalactosyldiglycerides were synthesized from diglycerides is consistent with galactosylation occurring without modification or exchange of fatty acids. The distribution of 16- and 18-carbon acyl residues between the 1 and 2 stereospecific positions of newly synthesized monogalactosyldiglyceride was typical of the endogenous polyene monogalactosyldiglycerides. However when palmitate synthesis was in excess of oleate synthesis some palmitate was esterified in position 1, whereas in the endogenous monogalactosyldiglycerides hexadecatrienoate is confined to position 2.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.     

Electronic spectra for nitrosyl(protoporphyrin IX dimethyl ester)iron(II) and its complexes with nitrogenous bases as model systems for nitrosylhemoproteins

Soret and visible absorption spectra for nitrosyl(protoporphyrin IX dimethyl ester)iron(II) (Fe(PPIXDME)(NO] and its complexes with nitrogenous bases (imidazoles, pyridines, aliphatic amines, and cyclic secondary amines) as model systems for nitrosylhemoproteins have been measured in various solvents. As the solvent polarity increases, the Soret and visible absorption bands for the five-coordinate Fe(PPIXDME) (NO) were shifted to shorter wavelengths. Accompanying the coordination of a nitrogenous base to the vacant axial position of Fe(PPIXDME)(NO), the Soret band becomes sharp and the band maximum is shifted to longer wavelengths. The band positions for the six-coordinate Fe(PPIXDME)(NO)(Base) complex are not sensitive to the pi-bonding ability of the axial ligand trans to NO group. The electronic spectra of five-coordinate Fe(PPIXDME)(NO) and six-coordinate Fe(PPIXDME)(NO)(Base) complexes are interpreted in relation to the structural information. The comparison of the spectra for model systems with those for nitrosylhemoproteins is discussed.     

The regulation of hepatic gluconeogenesis using alpha-ketoisovalerate and propionate as precursors

The regulation of gluconeogenesis from alpha-ketoisovalerate and propionate was investigated in perfused livers from fasted rats. With alpha-ketoisovalerate as the gluconeogenic precursor, infusion of beta-hydroxybutyrate and acetate stimulated the rate of alpha-ketoisovalerate decarboxylation, but inhibited the rate of glucose production. Oleate, on the other hand, inhibited both alpha-ketoisovalerate decarboxylation and glucose production. When propionate was the primary gluconeogenic substrate, oleate, beta-hydroxybutyrate, and acetate infusion did not significantly alter hepatic glucose production. The present studies suggest that gluconeogenesis from alpha-ketoisovalerate is regulated at the level of various dehydrogenases prior to formation of propionyl-CoA, but subsequent to the branched-chain alpha-keto acid dehydrogenase reaction.     

Oxidized saccharides as inhibitors of alpha-glucan synthesis by Streptococcus mutans glucosyltransferase

Specific inhibition by periodate-oxidized dextrans of the synthesis of alpha-glucan by S. mutans glucosyltransferase prompted a search for structurally related inhibitors that might be effective as anticaries agents. Clinical dextran derivatives in which from 5 to 50% of the D-glucose units were oxidized acted as potent and specific enzyme-inhibitors, as did 10%-oxidized derivatives of dextran fractions ranging in mol. wt. from 10(4) to 2 X 10(6). Within these limits, differences in oxidation or molecular weight did not significantly affect the high inhibitory potency of the derivatives. In contrast, periodate oxidation of (1 leads to 6)-alpha, (1 leads to 3)-alpha-, and (1 leads to 4)-alpha-linked oligosaccharides containing less than approximately 15 D-glucose units, and of sucrose and structurally related trisaccharides, yielded derivatives that were poor inhibitors. Enzymic hydrolysis of oxidized dextrans caused a loss of their inhibitory power and indicated that, to act as specific inhibitors, oxidized molecules must contain at least 16 to 20 D-glucosyl residues. The similar, minimum size required in order that unoxidized oligosaccharides may act as efficient acceptors in the glucosyltransferase reaction suggests that the inhibitory potencies of oxidized derivatives may reflect their relative abilities to bind at the acceptor site of the enzyme.     

The environment of the sulfhydryl group in human plasma albumin as determined by spin labeling

A series of spin labels, varying in chain length between the maleimide attaching group and the nitroxide free radical, has been used to investigate the environment of the sulfhydryl group in human plasma albumin. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined and the location of the sulfhydryl was assessed. The effect of bound fatty acids on the sulfhydryl environment was also determined. The environment was found to be analogous to that in the bovine protein, that is, a crevice approximately 9.5 Å deep and not affected in the native state by fatty acids.     

The role of hydroperoxides as calcium release agents in rat brain mitochondria

Hydroperoxides have previously been shown to induce Ca2+ release from intact rat liver mitochondria via a specific release pathway. Here it is reported that, in rat brain mitochondria, a hydroperoxide-induced Ca2+ release is also operative but is of minor importance. Hydroperoxide stimulates Ca2+ release in the presence of ruthenium red about twofold at a Ca2+ load of 40 nmol/mg mitochondrial protein. After addition of hydroperoxide, Ca2+ release from brain mitochondria can still be evoked by Na+. In the presence of succinate and rotenone, hydroperoxide induces only a very limited oxidation of pyridine nucleotides, most probably due to the low level of glutathione peroxidase (EC 1.11.1.9) and glutathione reductase (EC 1.6.4.2) found in brain mitochondria. Similar to liver mitochondria, a NADase (EC 3.2.2.5) activity is found in brain mitochondria. Its localization and sensitivity toward ADP and ATP, however, is different from that of the liver mitochondrial enzyme.     

Transfer of reducing equivalents into mitochondria by the interconversions of proline and Δ1-pyrroline-5-carboxylate

Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers ³H from [1-³H]glucose to NADP⁺ to Δ¹-pyrroline-5-carboxylate and yields [³H]proline. The formation of [³H]proline depends on the presence of NADP, Δ¹-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and Δ¹-pyrroline-5-carboxylate reductase. The production of [³H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to Δ¹-pyrroline-5-carboxylate by mitochondrial proline oxidase. Δ¹-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by Δ¹-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of Δ¹-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.