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Mediante elettroforesi su carta si dimostra la presenza nei lisati di mitocondri di fegato di ratto di almeno 5 componenti proteici. Le colorazioni specifiche dimostrano la presenza di 3 frazioni lipoproteiche e di altrettante glicoproteiche. I lisati dei mitocondri vennero ottenuti qer trattamento delle particelle con Triton X-100.

Diphosphatidylglycerol (cardiolipin) was found in the mitochondria of a number of cell fractions. Its concentration was estimated by neutron activation chromatographic analysis of the deacylated derivative, 1,3-diglycerophosphoryl-glycerol. Microsomes contained little or none of this lipid. The distributions of the other glycerol phosphatides in mitochondria and microsomes were similar. A possible functional role for diphosphatidylglycerol is discussed.

Phospholipids of chromatin isolated from rat liver were studied. The comparable ratios between phospholipid, DNA and protein were investigated. Individual phosphatide in the lipid fraction was identified and estimated by the combined techniques of column, thin-layer, and paper chromatography. Compared with phosphatides of whole nuclei, chromatin fraction contains lower quantity in phosphatidylethanolamine and sphingomyelin but higher in some acidic phospholipids. Phosphopeptides and phosphoproteins in the residual protein of chromatin were analysed and compared with those of other subcellular fractions of rat liver.

The degree of methylation and methylated nucleotide content of hamster-cell mitochondrial tRNA has been examined separately from the cytoplasmic RNA that purifies with mitochondria, via a combination of multipleisotope experiments, preferential inhibition of mitochondrial RNA synthesis by ethidium bromide, and gel electrophoresis under conditions that partially resolve mitochondrial and cytoplasmic tRNA. Mitochondrial tRNA was found to be 32% as heavily methylated as cytoplasmic tRNA and to be deficient in methylated pyrimidine and ribose components present in cytoplasmic tRNA. Both tRNA types contained 1-methyladenine as the sole methylated adenine, but the methylated guanine patterns of the two types differed substantially. Possible evolutionary implications of these findings have been discussed.

—The uptake of l[14C]glutamine by a crude isolated nerve ending fraction of rat brain was found to be linear with time for at least 5 min, profoundly temperature-dependent, apparently half-saturated at a substrate concentration of 0·26 mm, partially inhibited by dinitrophenol and ouabain and elevated [K+], weakly Na+-dependent, poorly inhibited by drugs which block uptake of biogenic amines and more strongly inhibited by glutamic acid (IC50= 0·5mm) than by aspartic acid, GABA, glycine or methionine. The [14C]glutamine taken up appeared to be associated with nerve endings and was released by membrane-disruption; about 20 per cent was associated with free mitochondria. Glutamine, δ-aminolevulinic acid and several other amino acids were poor inhibitors of [3H]GABA-uptake; δ-aminolevulinic acid was a poor inhibitor of [3H]glutamine-uptake, whereas glutamine was a moderately effective competitive inhibitor (Ki= 1 mm). [14C]glutamine and [3H]GABA were released from brain slices by electrical stimulation or 50 mm K+, while labeled δ-aminolevulinic acid, leucine, urea, amphetamine and tyramine were poorly released. [14C]glutamine was not released by unlabeled glutamate or several aromatic amines. We conclude that the neuropsychiatric features of porphyria are not likely due to a ‘false transmitter’ role for δ-aminolevulinic acid although such a role for glutamine in hepatic encephalopathy or other neuropsychiatric diseases should be considered.

1. 1.The cleavage of NAD in crayfish hepatopancreas is catalyzed chiefly by a pyrophosphatase rather than by NAD glycohydrolase (EC 3.2.2.5). This fact was confirmed by the loss in the coenzyme function of NAD for yeast alcohol dehydrogenase without a significant concomitant loss in reactivity towards cyanide and the identification of the reaction products as NMN and AMP by means of paper chromatography and Dowex 1-X2 column chromatography. 2. 2.NAD pyrophosphatase is localized chiefly in mitochondria and microsomes. The enzyme was partially purified by (NH 4 ) 2 SO 4 fractionation. Using this preparation, the K m value for NAD was determined as 1.1·10 −3 M. The activity with reduced NAD was about 3-fold higher than with NAD. ATP and NADP are not cleaved by the crayfish enzyme. 3. 3.NAD pyrophosphatase is inactivated by heating at 60° for 1 min and no stimulation was observed by heating at 40° for 1 min. The pH optimum is 8.4. 4. 4.Enzyme activity is inhibited by mononucleotides such as AMP, GMP and UMP, and AMP inhibition is competitive with substrate. Nicotinamide did not inhibit the enzyme activity at 1·10 −3 M. 5. 5.The possible metabolic significance of AMP inhibition of NAD hydrolyzing enzymes in mammalian tissues is also discussed.

Studies on the isolation and characterization of DNA from yeast mitochondria (Mt-DNA)4 have been extended to mitochondria from respiratory deficient cells (ϱ− petites). Under appropriate conditions assuring the absence of gluocse repression such cells have been found to contain mitochondrial DNA in amounts comparable to those of the wild type. Mt-DNA from a neutral petitite exhibits a buoyant density in CsCl and a thermal transition midpoint (Tm) very close to that of its isogenic wild type. Mt-DNA's from two other isogenic ϱ− strains, known to be 10 and 50% suppressive respectively, show a very sharp thermal transition with a Tm of 66.5 ° in 0.15 m NaCl—0.015 m sodium citrate. Upon quick cooling almost complete renaturation is observed, as evidenced by second-cycle heating, again with a sharp transition and a Tm of 66.5 °. The average value of the buoyant density of this DNA in CsCl (at 20 °) is 1.6685 which is 0.003 g/ml lower than that reported for synthetic or crab d(A — T). There is virtually no change in the density of this Mt-DNA upon heating and quenching, with or without prior exposure to proteases, again confirming the great ease of its renaturation. In alkaline CsCl its density increases by 0.076 g/ml, indicating strand separation, but upon reneutralization the density returns to that of native Mt-DNA. Ready renaturation, as measured by thermal profiles, is found even for partially degraded Mt-DNA molecules. All these observations suggest that ease of renaturation is due to compositional homogeneity and therefore does not require these DNA's to exist as covalently continuous circles. The composition and homogeneity of the Mt-DNA from one of these suppressive strains (D310-2A-184) was confirmed by direct analysis of base composition: It was found to contain an equimolar amount of G and C as well as of A and T with the former two bases accounting for no more than four mole percent. The implications of these findings with regard to the mutagenic events reponsible for the formation of these aberrant DNA's and their possible genetic capabilities are discussed.

1. Maize and barley shoots have been shown to contain phylloquinone, plastoquinone, alpha-tocopherol (and gamma-tocopherol in maize), alpha-tocopherolquinone and ubiquinone-9. 2. No solanesol was detected in any tissue examined. 3. In maize shoots plastoquinone and alpha-tocopherolquinone were localized in the chloroplast; ubiquinone was in the mitochondria. 4. Etiolated (dark-grown) shoots contained smaller amounts of phylloquinone and plastoquinone; alpha-tocopherolquinone was entirely absent; ubiquinone and alpha-tocopherol concentrations were unaffected. 5. On illumination of etiolated shoots the chloroplastidic quinones phylloquinone, plastoquinone and alpha-tocopherolquinone were synthesized in step with chloroplast development. alpha-Tocopherolquinone was not formed at the immediate expense of alpha-tocopherol.

The lipids of freeze-dried liver mitochondria of nonirradiated and x-ray irradiated rats have been examined by chemical analysis, paper chromatography, infrared spectroscopy, and gas-liquid chromatography. A polyglycerolphosphatide fraction of great similarity with bis(phosphatidic) acid has been isolated and tentatively characterized. Fatty acid analysis suggests that this fraction is not composed of a single lipid but of a group of lipids of different fatty acid composition. Ionizing irradiation of the animals causes a significant increase of the mitochondrial polyglycerolphosphatides. As the other fractions were not similarly affected by the irradiation, polyglycerolphosphatide became one of the major lipid fractions of the liver mitochondria of irradiated rats.

A background pattern of intense, polar and basic peptides is generated in a mixture of proteins which limits the applicability of “fingerprinting” by peptide maps as a method of establishing homologies among membrane proteins. In addition, it is observed that in such mixtures of proteins the peptide pattern in the “neutral” portion of the map is characterized by a few, weak, tailing peptides which appear on a smeared background of ninhydrin positive material. It is concluded that several types of control maps must be prepared along with maps of membrane fractions if real homologies are to be identified. Application of such control maps to analysis of a sample of mitochondrial membrane protein and a sample of quasicrystalline protein indicated that both of these preparations are disperse mixtures of proteins.

Nerve ending particles isolated from prenatal and postnatal rats by means of density-gradient centrifugation in a ficoll medium actively incorporated [32P]-orthophosphate into nucleotides, phosphoproteins and phospholipids. This process requires Mg2+; is dependent on malate plus pyruvate but not glucose, and is inhibited by dinitrophenol, gramicidin, oligomycin, azide, p-chloromercuribenzoate, CaCl2 but not iodoacetate. It was concluded that the metabolic energy at the synapse is derived largely from the mitochondria of the synaptic complex rather than from its cytoplasm. Thin-layer chromatography and paper electrophoresis revealed that the metabolically active phospholipids, namely phosphatidic acid, phosphatidyl inositol and the polyphosphoinositides which were found to be tightly bound to the phosphoprotein fraction, contained more than 90% of the total radioactivity but constituted less than 9% of the total phospholipids. The highest phosphoprotein and phospholipid content of the nerve endings (expressed in μmoles P/mg nerve ending protein) as well as maximal 32P-incorporation occurred just prior to and continued into the stage when functional changes are formed. It is suggested that an increase in the amount of enzymatic activity coupled with an increase in the permeability of the synaptosomal membrane to inorganic phosphate and other metabolites could trigger the rapid morpho-biochemical and functional changes observed in rat brain during this period of development. A simple density-gradient withdrawing device for the isolation of the submitochondrial particles after density-gradient centrifugation of the conventional mitochondrial fraction was described.

Mitochondrial DNA of Saccharomyces cerevisiae contains a satellite DNA (density, 1.682) that appears to exist as open-ended filaments at least 5 microns long. DNA from intact cells contains circular filaments whose lengths vary from 0.5 to 7 microns, with a great majority at 1.95 microns. The circular DNA has a density similar to that of the major nuclear peak (1.697). When heat-denatured mitochondrial-satellite DNA is renatured, it cross-links to form a molecule that is larger than the native molecule. The formation of cross-links results in hypersharpening of the density profiles in cesium chloride and also leads to failure to pass Millipore filter paper.

1. 1. Myokinase (ATP-AMP phosphotransferase) was isolated from thoracic muscle mitochondria of the American cockroach, Periplaneta americana (L.). 2. 2. The enzyme was purified greater than 100-fold by ammonium sulfate fractionation and column chromatography on Sephadex. 3. 3. The purified enzyme requires magnesium, but exhibits some activity in the presence of manganese or calcium. It is specific for the adenosine nucleotides, and has a pH optimum of 5.8–6.0 depending upon buffer. 4. 4. The reaction is markedly inhibited by fluoride at 2 × 10 −2 M. K m values 3.1 × 10 −4 , 1.5 × 10 −3 and 3.2 × 10 −4 M were obtained for ATP, ADP and AMP, respectively. 5. 5. The equilibrium constant was calculated to be 0.44 in the presence of magnesium. 6. 6. The significance of this enzyme in the metabolism of adenosine nucleotides in insects is discussed.

1. 1. A new method for the detection of guanidines on paper chromatograms was employed in a study of their biological distribution. This survey wasw designed to find a suitable source of invertibrate creatine kinase. 2. 2. The creatine kinase of the marine polychaete Marphysa sanguinea was purified and found to consist of five electrophoretically distinct forms, none of which appear to be associated with particular matter of the cell. 3. 3. Carbohydrate was associated with the isolated enzymes but does not seem to be involved in its activity. It was removed by gel filtration in the presence of 0·1 M KCl. 4. 4. Antibodies to the enzyme produced in rabbits cross-reacted with extracts of a number of invertebrates and, surprisingly, with some fish.

The effects of digitonin and digitoxin on the phospholipid metabolism of mammalian tissue culture cells have been studied. Digitonin stimulates the incorporation of inorganic orthophosphate- 32 P into the total phospholipid. The phospholipid was then separated on silicic acid-impregnated glass paper chromatography, and the specific activity of each phosphatide was calculated. It was found that digitonin caused a linear increase in the specific activity of phosphatidyl inositol and diphosphatidyl glycerol at the time periods studied. The increase in the specific activities of phosphatidyl choline and phosphatidyl ethanolamine was delayed but to the same extent (more than twofold) after 120-min incubation. The specific activities of phosphatidyl serine and sphingomyelin were low and not significantly affected. The increase was similar in specific activity values of phospholipids of digitonintreated HeLa cells as that of the controls in the nuclear, mitochondrial, and microsomal fractions. Digitonin failed to stimulate the incorporation of palmitic-l- 14 C acid into the phospholipids of HeLa cells. A nonsteroid surface-active agent (Triton X-100) did not stimulate the incorporation of 32 P, into the phospholipids of HeLa cells, but other steroids, desoxycholate, and estradiol did stimulate such incorporation of 32 P i . Digitoxin had little effect on the phospholipid labeling of HeLa cells. However, digitoxin stimulated the incorporation of 32 P, into phosphatidyl inositol and sphingomyelin fraction of human heart culture cells. The specific interaction of various steroids with the target-cell membranes and its relation to phospholipid metabolism is discussed.

1. Kidney-cortex slices from starved rats were incubated with l-[U-(14)C]lactate or l-[U-(14)C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-(14)C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-(14)C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. (3)H from compounds such as l-[2-(3)H]lactate, which form NAD(3)H in the cytosol, appears in part in water; and (3)H from dl-beta-hydroxy[3-(3)H]butyrate, which forms NAD(3)H in the mitochondria, appears in part in glucose, largely on C-4.

When [7α-3H] dehydroepiandrosterone was incubated with the adrenal homogenates of human fetus at 22 to 26 weeks gestational age, 16α-hydroxydehydroepiandrosterone and/or its sulfate was formed as the only detectable metabolite. The 16α-hydroxylase activity was concentrated in the microsomal fraction of the adrenal homogenate. [1,2-3H]Androstenedione, [4-14C] pregnenolone and [7α-3H] progesterone were also 16α-hydroxylated by incubation with the microsomal fraction. Amoung these substrates, progesterone gave the highest yield of 16α-hydroxylated products. By incubation with the microsomal fraction, formation of following steroids were also established: 6β-hydroxyandrostenedione from androstenedione; 17-hydroxypregnenolone, 17,21-dihydroxypregnenolone and dehydroepiandrosterone from pregnenolone; 17-hydroxy-progesterone, deoxycorticosterone, 11-deoxycortisol and androstenedione from progesterone.

The quantitative inter- and subcellular distribution of 3β-hydroxysteroid dehydrogenase (3β-hydroxysteroid:NAD (P)+ oxidoreductase, EC 1.1.1.51) has been studied in testis tissue of the rat. The specific activity in homogenates of isolated interstitial tissue was 150–500 times higher than the specific activity in homogenates of seminiferous tubules. It was concluded that 95–98% of 3β-hydroxysteroid dehydrogenase is located in the isolated interstitial tissue. Subcellular fractionation of testis homogenates and the distribution of marker enzymes indicated that mitochondria contribute 7–15% to total 3β-hydroxysteroid dehydrogenase in rat testis tissue. Nagarse (subtilipeptidase, EC 3.4.21.14) treatment or cyanoketone (2α-cyano-4,4',17α-trimethyl-17β-hydroxy-5-androsten-3-one) inhibited 3β-hydroxysteroid dehydrogenase activity in rat testis tissue. Measurements of steroid production from endogenous substrates in isolated mitochondrial fractions showed that addition of cyanoketone to incubation mixtures or pretreatment of homogenates of interstitial tissue with nagarse caused a acncumulation of pregnenolone at the expense of testosterone.

Pure yeast tRNAPhe was used as a substrate to compare the tRNA methylating activities in Phaseolus vulgaris cytoplasm, chloroplasts and mitochondria, in the presence of S- adenosyl [Me- 3 H]methionine . The resulting [Me-3H]-tRNAPhe was then analyzed, using the techniques of nucleotide sequence determination. Cytoplasmic and mitochondrial enzymes catalyze the methylation (into m5C) of C48 present in the extra-loop, while chloroplast enzyme preparations catalyze the modification (into m1A) of A14 present in the dihydrouridine loop of tRNAPhe.

The toxicity of three fractions (A, B, and C) obtained by ultrafiltration of a peptic: tryptic digest of gluten has been assessed by serial feeding experiments in patients with treated coeliac disease. The first fraction (A), which contains amino acids and oligopeptides, produced no damage to the jejunal mucosa. The other two fractions (B and C) both caused mucosal damage. Fraction B, which contains the products of digestion of smaller molecular weight, consists of polypeptides which are concentrated in the region of 8000 molecular weight. It contains no gliadin (molecular weight 50 000) or gluten. Ultrastructural evidence of damage was visible six hours after challenge with fraction B and by 10 hours histological abnormalities were also present. Ultrastructural abnormalities occurred early in the epithelial cells and preceded changes in the basement membrane and capillaries. The disaccharidases showed a pronounced depression in all three subjects by 24 hours. The rapid onset of damage after challenge, coupled with the evidence of recovery as soon as 72 hours later, is more in keeping with a direct action on the surface epithelial cells rather than an immune mechanism.

The malate dehydrogenase ( l -malate:NAD+ oxidoreductase, EC 1.1.1.37) isoenzymes of Euglena gracilis Z occur in two groups, an anodally migrating cluster of three cytoplasmic soluble malate dehydrogenase isoenzymes, which probably occur in the cytosol, and a cathodally migrating group of two particulate isoenzymes which exist in particulate cell components, probably mitochondrial malate dehydrogenase. Comparisons of some physical and kinetic properties are described. Although the molecular and kinetic properties are similar, the soluble malate dehydrogenase is much less stable than mitochondrial malate dehydrogenase. The latter supports the conclusion that the proteins are different. Cells grown heterotrophically in the dark have approximately three times more soluble malate dehydrogenase than cells grown photoautotrophically, whereas there is no significant difference between mitochondrial malate dehydrogenase quantities. This differential regulation between isoenzymes is found when the activities are measured in terms of dry weight of cells, on a unit soluble protein basis, or per cell. Cells transferred abruptly from autotrophic growth conditions to heterotrophic conditions, or vice versa, showed a lag in initiation of soluble malate dehydrogenase modification which correlates with lag in growth. Modulations of the isoenzyme levels in cells changed from one nutritional mode to another were studied under a variety of conditions. In other experiments, no significant alteration of soluble malate dehydrogenase was observed to occur in the absence of growth; removal of any essential parameter for growth (e.g., CO2, light, glucose) inhibits soluble malate dehydrogenase changes, i.e., greening of etiolated cells in the absence of CO2 does not cause a significant reduction of soluble malate dehydrogenase. Analogues of substrates did not induce the soluble malate dehydrogenase, and the effects of inhibitors upon the enzyme changes was studied with paradoxical results.

The metabolism of d - and l -lysine has been studied in the chicken using both 14C and 15N labels. In contrast to the rat, the chicken actively metabolized d -lysine and l -pipecolate. The data indicate that d -lysine is degraded via conversion to pipecolate, α-aminoadipate, α-ketoadipate, and eventually to CO2. The α-nitrogen atom of d -lysine is the first one removed from the carbon skeleton by this route. l -Lysine is metabolized by two alternate pathways which converge at α-aminoadipate. The major pathway, which results in the retention of the α-nitrogen atom of l -lysine in α-aminoadipate, includes saccharopine as a precursor of α-aminoadipate and is thus identical to l -lysine catabolism in rats. The pathway which is indicated to be the minor one includes l -pipecolate as a precursor of α-aminoadipate and results in the retention of the ϵ-nitrogen atom of l -lysine in α-aminoadipate. Even though saccharopine is indicated to be an intermediate in l -lysine breakdown, a large percentage of an intramuscularly injected dose is excreted unchanged in the urine.

The formation of labeled phosphatidylglycerol from L-α-glycerophosphate-2-3H and CDP-D-diglyceride has been demonstrated in homogenate and mitochondria isolated from rat ventral prostate. In addition, labeled phosphatidylglycerophosphate was detected as a minor biosynthesized lipid. Homogenates and mitochondria isolated from prostates after castration of rats have a decreased ability to incorporate L-α-glycerophosphate-2-3H and CDP-D-diglyceride into phospholipids, but this ability is restored after the administration of testosterone to the castrated animals. The composition of synthesized phospholipids by homogenates and mitochondria isolated from normal and sham-operated rats, and from normal and castrated rats treated with testosterone, is very similar; the major labeled lipid (ca. 90%) being phosphatidylglycerol. This composition is very different when mitochondria isolated from prostates of castrated rats were incubated with the same substrates: labeled phosphatidylglycerol (ca. 55-58%) and phosphati...

Radio-labeled dihydromorphine, morphine, methadone and levorphanol were administered to rats in pharmacologically active doses by the intracisternal route of administration, and the rats were killed 0.5 or 1 hr later. The drugs were localized in the fraction of brain containing the pinched-off presynaptic nerve-endings, the synaptosomes, as well as in the soluble portion of the tissue. The 14 C-labeled narcotic antagonists, naloxone and nalorphine, were also localized in the same particulate fraction of brain. The amount of drug in the synaptosomal fraction was dependent on the dose of drug, on the time between drug administration and sacrifice, and on the region of brain, and was specific for each narcotic agonist and antagonist. The relevance of this localization of narcotic analgesic drugs in the synaptosomal fractions of rat brain to the mechanism of action of the drugs remains to be explored.

1. 1. Phosphatidylcholine biosynthesis in Tetrahymena pyriformis was investigated. Experiments in vivo using [Me-14C]methionine showed incorporation of radioactivity into the choline portion of phosphatidylcholine. No methylation of 2-aminoethylphosphonic acid was detected. Incorporation of the intact choline molecule into lecithin was demonstrated when the cells were grown on [Me-14C]choline. 2. 2. The transfer of the methyl group of [ Me- 14 C ] S - adenosyl - l - methionine to form lecithin was demonstrated in cell-free systems. The activity was localized in the microsomal cell fraction. The enzyme system accepted exogenous phosphatidylmonomethylethanolamine as substrate but not phosphatidylethanolamine or phosphatidyldimethylethanolamine. 3. 3. CDP-choline: diglyceride phosphocholinetransferase activity was localized in the mitochondria. The enzyme was twice as active with the optimal concentration of Mn2+ as Mg2+; the enzymatic activity was inhibited by low concentrations of Ca2+ or Hg2+.

The distribution and amino acid composition of the chloroform-methanolsoluble proteolipids and residual proteins in the subcellular fractions of human cerebral white and grey matter has been investigated. Proteolipid protein was over 5 times higher in white matter than in grey matter. It represented nearly 30% of total protein in white matter but less than 5% in grey. Nuclear fractions contained more than 55% total protein whilst nuclear and mitochondrial fractions together accounted for over 80%. The amino acid spectra of proteolipid protein and non-lipid protein showed variations between subcellular components ; differences were also seen between proteolipid protein and residual protein amino acid patterns within the same subcellular fractions.

Rat brain homogenates incubated with noradrenaline for 5 min showed reduced incorporation of 32P into phospholipids. Subcellular fractionation studies indicated that this effect of noradrenaline occurred only in the synaptosomal fraction. Incubation with noradrenaline for 20–120 min increased the incorporation of 32P into phospholipids. This effect occurred in both the synaptosomal and mitochondrial fractions and was inhibited by the α-blocking drug thymoxamine. Noradrenaline did not significantly increase 32P incorporation into phosphoproteins. The results are discussed in terms of a possible transmitter role for noradrenaline in the brain.

The properties and behavior of a "microsomal" (100,000 x g particles) chitin synthetase of Mucor rouxii were investigated. The enzyme utilizes uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc) as glycosyl donor and is strongly and specifically activated by free N-acetyl-d-glucosamine (GlcNAc). A variety of GlcNAc analogues were tested as activators but were found ineffective. A small proportion of free GlcNAc was incorporated into chitin; however, the activation of chitin synthetase by GlcNAc is probably an allosteric effect. Seemingly, UDP-GlcNAc is also a positive allosteric effector of the enzyme. Diacetylchitobiose was also synthesized in the reaction mixtures. However, the kinetics of its formation do not support the proposal that this dimer is an intermediate in chitin synthesis. Added primer was not essential for the operation of the microsomal chitin synthetase. Nevertheless, addition of higher N-acetylchitodextrins produced large stimulations indicative of primer shortage in the particles. Certain lipid extracts of M. rouxii proved to be highly stimulatory for chitin synthesis. However, efforts to obtain evidence for a lipid intermediate proved negative. Chitin synthetase was also stimulated by ATP, CTP, and GTP but UDP was inhibitory. Optimum pH was about 6.5. The enzyme was exceedingly susceptible to Mg++ concentration. Microsomal chitin synthetase was relatively stable in the frozen state if suspended in phosphate buffer but not in Tris buffer. Mitochondrial (10,000 x g) enzyme, by contrast, rapidly decayed under similar conditions.

131 I and 3 H from monoiodotyrosine are incorporated into trichloroacetic acid-insoluble protease-digestible material by supernatant fractions from rabbit liver cytoplasm and from disrupted rabbit liver and sheep thyroid mitochondria. 1. 1. Ribosomes, Mg 2+ , ATP, and GTP are not required for the reaction and neither puromycin nor ribonuclease are inhibitory. Boiling of the extracts or the addition of tyrosine markedly diminish incorporation. There is a high degree of dependence upon either pyruvic or α-ketoglutaric acid and a lesser dependence upon pyridoxal phosphate. 2. 2. Monoiodotyrosine as such is not demonstrable in acid hydrolysates or proteolytic digests of the acid-insoluble product. 3. 3. In the presence of pyruvic or α-ketoglutaric acid and pyridoxal phosphate monoiodotyrosine is extensively converted to a series of closely related compounds which can be separated from the monoiodotyrosine by gel filtration with Sephadex G-25. 4. 4. Label from this group of compounds is incorporated into acid-insoluble pronase-digestible material in the presence of various extracts or of albumin. Incorporation does not require pyruvic acid and boiling of the proteins increases their activity. The data suggest that monoiodotyrosine is first converted to an intermediate by transamination and that this intermediate is subsequently incorporated into protein in a chemical reaction.

Cultured human synovial cells secrete hyaluronic acid (HA) into the culture medium. Glucosamine-6-3H was shown to be a direct and relatively specific precursor of HA-3H by the following observations: the susceptibility of nondialyzable radioactivity in the medium to hyaluronidase, its migration with hexuronic acid on zone electrophoresis in polyvinyl chloride, its exclusion from Sephadex G-200, and the localization of radioactivity to glucosamine after hydrolysis of the labeled polysaccharide. The presence of intracellular HA-3H was established by sequential extraction of labeled cells and by radioautography of synovial cell cultures digested with hyaluronidase in situ. When cells were exposed to medium lacking glucose, glucosamine-3H-uptake was enhanced; and this made possible electron microscopic radioautographic studies. These studies demonstrate the early and continued presence of HA-3H within the Golgi apparatus.

Amino acid incorporating mitochondria, supplied with formyl-group donor, show a puromycin dependent formation of initial peptides. When the puromycin concentration is increased from 1 to 10 mM the length of the puromycin peptides formed is reduced up to the predominant formation of formyl-methionyl-puromycin. A method for measuring the total production of these peptides has been developed, allowing an assay of the initiation reaction. The number of initiation acts is higher than that of mitochondrial ribosomes present. Under suitable conditions the rate of formation of puromycin peptides is constant and largely accounts for the rate of synthesis of the polypeptide chains.

— The concentration of methylhistamine in whole brain of guinea pig is 72 ng (about 0-5 mμ mole/g). The greatest portion is found in the crude mitochondrial fraction.

SUMMARY A study was conducted on the in vivo incorporation of l-[14C]-serine into the lipids and proteins of the various subcellular fractions of the developing rat brain before and during the stage of active myelination. The total radioactivity in the various fractions at 12 days of age was higher than that at 3 days, while the radioactive specific activity was reversed. The specific activities of the proteins and lipids were higher at 3 days of age with the exception of the subcellular fraction containing myelin. At both ages the lipids of the various cellular fractions had similar specific activities, a finding that suggests a common source for lipid biosynthesis. Incorporation of radioactivity into the various phospholipids was in the following order: phosphatidyl serine > phosphatidyl ethanolamine > phosphatidal serine > sphingomyelin and phosphatidyl choline. Of all the phospholipids, the plasmalogens increased most in total radioactivity during the period when meylination was most active. Serine-containing phospholipids appear to be most tightly bound to proteins. The brain mitochrondrial fraction contained most of the phosphatidyl serine decarboxylase activity with some activity in the nuclei. Biosynthesis of phosphatdyil ethanolamine through decarboxylation of phosphatidyl serine could take place in rat brain. Four unidentified radioactive metabolites were found in the acid-soluble fraction in addition to l-[14C]serine.

A mitochondrial oxidase system of Neurospora crassa, able to utilize dihydroorotic acid as a primary substrate, was found and characterized. Another substrate for this system was isolated from yeast extract and identified as 5 N-methylformamidodihydroorotic acid. No linkage of this oxidase system to the cytochrome chain or to oxidative phosphorylation could be detected. The possible biological origins and metabolic significance of 5 N-methylformamidodihydroorotic acid are discussed.

Examination of phospholipids of heavy mitochondrial, and other particulate fraction of livers from whole-body X-ray irradiated fasted rats and controls showed that the increase of phosphatidylglycerol previously reported actually took place in fractions with “lysosomal” and “microsomal” properties. Study of the in vivo incorporation of (P 32 ) orthophosphate into mitochondrial phospholipids demonstrated that phosphatidylglycerol is more strongly labeled than cardiolipin and that it retains a greater part of that label much longer in mitochondria from the irradiated rats than from controls. It is concluded that phosphatidylglycerol plays a more active metabolic role than cardiolipin and that it may be involved in protein metabolism, which can be affected by ionizing irradiation.

— We have studied the subcellular distribution of exogenous and endogenous serotinin in slices from the hypothalamus and midbrain of several species. In a procedure which appears to label the endogenous pools, tissue slices were incubated with low concentrations of [3H]5-HT (5 × 10-8 M), for 45 min, when there was apparent equilibrium between [3H]5-HT of tissue and medium. After the tissue slices were homogenized in 0-32 M-sucrose and subjected to differential centrifugation, the distribution of exogenous and endogenous 5-HT in pellets and supernatant fluid was similar. In some experiments, the crude mitochondrial pellets were resuspended in 0-32 M-sucrose, layered on linear, continuous density gradients of sucrose (1 -5-0-32 M), and centrifuged for short times (incomplete equilibrium centrifugation). The subcellular distribution of particulate tritium, total tritium, and particulate endogenous 5-HT was the same in portions of the gradients containing synaptosomes. The peak distribution of [3H]5-HT in sucrose gradients was separable from the peak for [14C]GABA by four to five fractions; potassium (a marker for cytoplasm occluded within synaptosomes) occurred in the regions of the gradients containing most of the labelled compounds. The distribution of monoamine oxidase activity (a mitochondrial marker) overlapped the distribution of [3H]5-HT after a 15 min centrifugation but appeared in denser regions of the gradient after centrifuging for 2 h. Particles containing [3H]5-HT and [I4C]NE were slightly but consistently separable in synaptosomal fractions isolated from the hypothalamus or midbrain of rat, guinea pig and hamster.

Adrenal “mitochondrial” preparations of domestic duck ( Anas platyrhynchos ) were incubated with 14 C labelled androstenedione (Δ 4 A) *** , testosterone (T) and 11-deoxycorticosterone (DOC) as model substrates in the presence of NADPH generating system. Δ 4 A was converted to 19-OH-Δ 4 A (1%), 11β-OH-Δ 4 A (38%) and testosterone (32%). 11β-OH-Δ 4 A (3%), 11β-OH-T (60%) and 18-OH-T (2%) were the characterizable metabolites of T. Among the many unidentified metabolites, using T as a precursor, a compound presumed to be 19-OH-T was also isolated. The C 21 labelled substrate (DOC) gave rise to 19-OH-DOC (

In our previous paper (1) some hexosamine intermediary factors taking part in the biosynthesis of mucin have been described. However, it was not established whether N -acetylglucosamine was the mucin biosynthesis metabolite or the degradation product of macromolecular compounds. The present work deals with the activity of N -acetylglucosamine kinase of the human gastric mucosa.

1. Incubation conditions were established in experiments with human-prostate homogenates for almost complete conversion of [4-(14)C]testosterone into at least ten transformation products. 2. Whole homogenates of tissue with benign hypertrophy were shown to contain 3alpha-, 3beta- and 17beta-hydroxy steroid dehydrogenases, Delta(4)-3-oxo steroid 5alpha- and 5beta- reductases and unidentified hydroxylases. 3. Most of the 17beta-hydroxy steroid-dehydrogenase activity was located in the mitochondria, which showed little other activity. 4. The 3alpha- and 3beta-hydroxy steroid dehydrogenases and the 5beta-reductase were located in the high-speed supernatant and required supplementation with NADPH for activity. 5. The 5alpha-reductase was located in both microsomal and high-speed supernatant fractions and also required supplementation with NADPH.

1. When dilute aqueous emulsions of radioactive cholesterol are exposed to daylight, extensive photolysis may take place. This results chiefly in the formation of substances more polar than cholesterol, some of which are probably acidic. Substances less polar than cholesterol are formed to a smaller extent. Photolysis is greatest when the emulsions are strongly acidic or strongly alkaline. 2. Photolysis takes place rapidly if radioactive cholesterol stored in the dry state, either on glass or on filter paper, is exposed to daylight. 3. If precautions are taken to minimize exposure to daylight during analysis and storage of the samples, the amount of non-enzymic alteration of cholesterol in biological experiments may be negligible.