Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

Studies on Cyanidium caldarium Phycobiliprotein Pigment Mutants

Phycobiliprotein biosynthesis was investigated in four strains of the unicellular rhodophyte, Cyandium caldarium, with different pigment phenotypes. All strains were incapable of synthesizing phycobiliproteins when grown in the dark. Western blotting experiments showed that dark-grown cells of the wild-type and mutant GGB synthesized the α and β subunit polypeptides of allophyocyanin and phycocyanin after exposure to light for 24 hours, whereas cells of mutant IIIC and GGBY did not. Similarly, light promoted the appearance of allophycocyanin and phycocyanin mRNAs in the wild-type and GGB but not in IIIC and GGBY. However, Southern blots of restricted genomic DNA from the wild type, IIIC, GGBY, and GGB, all hybridized with heterologous phycobiliprotein gene probes and revealed that all four strains contained identical Pst, EcoRI, and Dral restriction fragments containing allophycocyanin and phycocyanin genes. Cells of the wild type and GGB incubated in the dark with the heme precursor. δ-aminolevulinate, synthesized allophycocyanin and phycocyanin apoproteins providing strong evidence for the role of a tetrapyrrole in regulation of phycobiliprotein gene expression. However, cells of IIIC and GGBY incubated in the dark with δ-aminolevulinate did not contain detectable quantities of allophycocyanin or phycocyanin apoproteins. The possible role of a tetrapyrrole in phycobiliprotein gene expression and basis for the genetic lesion in mutants IIIC and GGBY is discussed.     

Rate of utilization of glucose and `compartmentation'' of α-oxoglutarate and glutamate in rat brain

1. The rate of incorporation of ¹⁴C into pyruvate, α-oxoglutarate, lactate and glucose of rat tissues was measured after the subcutaneous injection of uniformly labelled glucose. 2. In rat brain the specific radioactivities of lactate and glucose were similar to that of alanine. In liver the specific radioactivity of glucose was considerably higher than that of lactate or alanine. 3. The specific radioactivities of α-oxo acids of rat brain were lower than those of corresponding amino acids, alanine and glutamate. These findings have been explained in relation to metabolic compartments in vivo. 4. The approximate estimated rate of glucose utilization in rat brain in vivo is 0·96μmole/g. of brain/min.     

Catabolism of [1-C]levulinic Acid by etiolated and greening barley leaves

Levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase (EC 4.2.1.24), has been used extensively in the study of ALA formation during greening. When [1-¹⁴C]LA is administered to etiolated barley (Hordeum vulgare L. var. Larker) shoots in darkness, ¹⁴CO₂ is evolved. This process is accelerated when such tissues are incubated with 2 millimolar ALA or placed under continuous illumination. Label from the C-1 of LA becomes incorporated into organic acids, amino acids, sugars, lipids, and proteins during a 4-hour incubation in darkness or in the light. This metabolism is discussed in relation to the use of LA as a tool in the study of chlorophyll synthesis in higher plants.     

Purification, Characterization, and Fractionation of the delta-Aminolevulinic Acid Synthesizing Enzymes from Light-Grown Chlamydomonas reinhardtii Cells

The synthesis of δ-aminolevulinate from glutamate by Chlamydomonas reinhardtii membrane-free cell homogenates requires Mg²⁺, ATP, and NADPH as cofactors. The pH optimum is about 8.3. When analyzed by a Fractogel TSK gel filtration column the δ-aminolevulinate synthesizing enzymes, including glutamate-1-semialdehyde aminotransferase, elute with an apparent molecular weight of about 45,000. The enzymes obtained from the gel filtration column were separated into three fractions by affinity column chromatography. One fraction binds to heme-Sepharose, one to Blue Sepharose, while the enzyme converting the putative glutamate-1-semialdehyde to δ-aminolevulinic acid is retained by neither column. All three fractions are necessary for the conversion of glutamate to δ-aminolevulinate. The δ-aminolevulinate synthesizing enzymes from Chlamydomonas are sensitive to inhibition by heme but not sensitive to inhibition by protoporphyrin.     

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2)

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO₂ fixation for more than 60 hours. The incorporation of ¹⁴CO₂ under saturating CO₂ conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to ¹⁴C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.     

The incorporation of carbon dioxide into milk citrate in the isolated perfused goat udder

1. Isolated perfused goat udders supplied with glucose, acetate and amino acids were infused for several hours with NaH¹⁴CO₃. 2. Lactose, milk-fat fatty acids and glycerol had very little radioactivity. The specific radioactivity (counts./min./mg. of C) of milk citrate was 9–16% that of the carbon dioxide in the perfusion fluid and 19% that estimated for tissue carbon dioxide. The specific radioactivity of tissue citrate resembled that of milk citrate. 3. The radioactivity in citrate was predominantly in C-6, suggesting some carboxylation of α-oxoglutarate in addition to carboxylation of C₃ compounds. 4. [1-¹⁴C]Glutamate was infused in a similar experiment, and milk citrate radioactivity was predominantly in C-1+C-5. 5. The results are discussed in relation to the contribution of glucose and acetate carbon to citrate. The implications of the carboxylation of α-oxoglutarate are considered.     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.     

Induction of alpha-amylase in barley endosperm by substrate levels of glutamate and aspartate

Incubation of embryoless barley (Hordeum vulgare) half-seeds for 24 hours with 0.1 m glutamate or aspartate resulted in the release of 17 to 48% as much α-amylase as did incubation with 260 mμm gibberellin. With incubation periods of 48 to 51 hours these amino acids were on the average about half as active as response-saturating concentrations of gibberellin, and in some experiments they were essentially as active. Citric acid cycle intermediates, glycolytic pathway intermediates, and cofactors of these pathways failed to induce α-amylase synthesis, while the following compounds were active: asparagine, homoserine, diaminopimelate, isoleucine, methionine, glutamine, ornithine, citrulline, argininosuccinate, and δ-aminolevulinate. However, threonine, lysine, β-alanine, alanine, γ-aminobutyrate, α-ketobutyrate, proline, arginine, glycine, leucine, and putrescine were inactive. Two patterns were noted in the list of active and inactive compounds: (a) all of the active compounds contain an amino group and are biosynthetically derived from citric acid cycle intermediates; and (b) biosynthetic precursors of the amino acids arginine, proline, threonine, and lysine were active whereas these amino acids were not.     

Biosynthesis of oleic, arachidonic and docosahexaenoic acids from their C18 precursors in the yolk sac membrane of the avian embryo

The yolk sac membrane (YSM) of the chicken embryo is known to express δ-9 and δ-6 desaturase activities, suggesting that biosynthesis of the unsaturated fatty acids 18:1n-9, 20:4n-6 and 22:6n-3 might occur during the transfer of yolk lipids across the YSM. If so, this biosynthesis could help to satisfy the demands of the embryonic tissues for these unsaturates. To assess the ability of the YSM to perform these conversions, pieces of the tissue were incubated in vitro with the precursor fatty acids, ¹⁴C-18:0, ¹⁴C-18:2n-6 or ¹⁴C-18:3n-3, and the recovery of radioactivity in the respective products, 18:1n-9, 20:4n-6 and 22:6n-3, was determined. After 4 h of continuous incubation, radioactivity from these precursors was incorporated primarily into triacylglycerol and phospholipid of the tissue pieces. Only small proportions (0.3–4.7%) of this incorporated radioactivity were, however, recovered as 18:1n-9, 20:4n-6 or 22:6n-3. The majority of the incorporated label was retained in the form of the precursor fatty acids. After a 1-h pulse incubation with the ¹⁴C precursors, followed by a 3-h chase incubation in the absence of exogenous label, the conversion of incorporated radioactivity to the end product unsaturates was again relatively low (0.5–8.1%). Thus, although conversions of the precursors to the end product fatty acids were detectable in this system, the biosynthesis of these unsaturates is apparently a quantitatively minor pathway in the YSM. Nevertheless, since the amount of 18:2n-6 in the yolk lipids far exceeds that of 20:4n-6, the conversion of even a small proportion of the former to the latter fatty acid could significantly increase the supply of 20:4n-6 to the embryonic tissues.     

Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum)

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-¹⁴C]oleic acid and [1-¹⁴C]acetate as precursors. In 4-day aged tissue, [1-¹⁴C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H₄), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that ¹⁴C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO₄ treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-¹⁴C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that ¹⁴C was contained in alkanols and alkane-α,ω-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C₂₀, which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C₁₆, C₁₈, C₂₀, C₂₂, and C₂₄-α,ω-diols. Soluble lipids derived from [1-¹⁴C]acetate in the aged tissue also contained labeled very long acids from C₂₀ to C₂₈, as well as C₂₂ and C₂₄ alcohols, but no labeled ω-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C₂₁ and C₂₃ were the major components with lesser amounts of C₁₉ and C₂₅. The amount of ¹⁴C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-¹⁴C]oleic acid, respectively. Incorporation of [1-¹⁴C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-¹⁴C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the α-ω-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.     

Carbon and Nitrogen Isotopes from Top Predator Amino Acids Reveal Rapidly Shifting Ocean Biochemistry in the Outer California Current

Climatic variation alters biochemical and ecological processes, but it is difficult both to quantify the magnitude of such changes, and to differentiate long-term shifts from inter-annual variability. Here, we simultaneously quantify decade-scale isotopic variability at the lowest and highest trophic positions in the offshore California Current System (CCS) by measuring δ¹⁵N and δ¹³C values of amino acids in a top predator, the sperm whale (Physeter macrocephalus). Using a time series of skin tissue samples as a biological archive, isotopic records from individual amino acids (AAs) can reveal the proximate factors driving a temporal decline we observed in bulk isotope values (a decline of ≥1 ‰) by decoupling changes in primary producer isotope values from those linked to the trophic position of this toothed whale. A continuous decline in baseline (i.e., primary producer) δ¹⁵N and δ¹³C values was observed from 1993 to 2005 (a decrease of ∼4‰ for δ¹⁵N source-AAs and 3‰ for δ¹³C essential-AAs), while the trophic position of whales was variable over time and it did not exhibit directional trends. The baseline δ¹⁵N and δ¹³C shifts suggest rapid ongoing changes in the carbon and nitrogen biogeochemical cycling in the offshore CCS, potentially occurring at faster rates than long-term shifts observed elsewhere in the Pacific. While the mechanisms forcing these biogeochemical shifts remain to be determined, our data suggest possible links to natural climate variability, and also corresponding shifts in surface nutrient availability. Our study demonstrates that isotopic analysis of individual amino acids from a top marine mammal predator can be a powerful new approach to reconstructing temporal variation in both biochemical cycling and trophic structure.     

The metabolism of glyoxylate by cell-free extracts of Pseudomonas sp

1. Extracts of Pseudomonas sp. grown on butane-2,3-diol oxidized glyoxylate to carbon dioxide, some of the glyoxylate being reduced to glycollate in the process. The oxidation of malate and isocitrate, but not the oxidation of pyruvate, can be coupled to the reduction of glyoxylate to glycollate by the extracts. 2. Extracts of cells grown on butane-2,3-diol decarboxylated oxaloacetate to pyruvate, which was then converted aerobically or anaerobically into lactate, acetyl-coenzyme A and carbon dioxide. The extracts could also convert pyruvate into alanine. However, pyruvate is not an intermediate in the metabolism of glyoxylate since no lactate or alanine could be detected in the reaction products and no labelled pyruvate could be obtained when extracts were incubated with [1-¹⁴C]glyoxylate. 3. The ¹⁴C was incorporated from [1-¹⁴C]glyoxylate by cell-free extracts into carbon dioxide, glycollate, glycine, glutamate and, in trace amounts, into malate, isocitrate and α-oxoglutarate. The ¹⁴C was initially incorporated into isocitrate at the same rate as into glycine. 4. The rate of glyoxylate utilization was increased by the addition of succinate, α-oxoglutarate or citrate, and in each case α-oxoglutarate became labelled. 5. The results are consistent with the suggestion that the carbon dioxide arises by the oxidation of glyoxylate via reactions catalysed respectively by isocitratase, isocitrate dehydrogenase and α-oxoglutarate dehydrogenase.     

The Biosynthesis of Some Isothiocyanates and Oxazolidinethiones in Rape (Brassica campestris L.)

The incorporation of the radioactivity from acetate-1-¹⁴C, acetate-2-¹⁴C, dl-methionine-1-¹⁴C, dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, dl-allyl-glycine-2-¹⁴C, and dl-2-amino-5-hydroxyvalerate-2-¹⁴C into the aglycones of progoitrin, gluconapin, and glucobrassicanapin of maturing rape plants (Brassica campestris L.) was investigated. Radioactivity from dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, and acetate-2-¹⁴C were incorporated into the 3 major thioglucosides. The other organic compounds were poorly incorporated except for dl-allylglycine-2-¹⁴C into glucobrassicanapin. The results obtained suggest that the rape plant can synthesize amino acids by the condensation of acetate (as acetyl CoA) to α-keto acids to yield a homologue of the original amino acid. These newly formed amino acids are then employed to synthesize the 3 major thioglucosides.     

Metabolic Pathway of alpha-Ketoglutarate in Citrus Leaves as Affected by Phosphorus Nutrition

The uptake and metabolism of α-[5-¹⁴C]ketoglutarate by phosphorus-deficient and full nutrient (control) lemon (Citrus limon) leaves were studied over various time intervals. After 45 minutes in P-deficient leaves, the bulk of incorporated ¹⁴C appeared in organic acids and much less in amino acids, while in the control leaves, the ¹⁴C contents of organic and amino acids were equal. In P-deficient leaves, after longer incubation times the ¹⁴C content of organic acids and amino acids increased, while that of CO₂ and residue fractions remained low. In full nutrient leaves the ¹⁴C content of amino acids and organic acids decreased after longer incubation time and increased in the insoluble residue and CO₂. In full nutrient leaves the organic and amino acid metabolism were closely related and accompanied by protein synthesis and CO₂ release, while in P-deficient leaves an accelerating accumulation of arginine and citric acid was linked together with inhibition of protein synthesis and CO₂ liberation.     

Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

Active-site residues in rat kidney γ-glutamyltransferase (EC 2.3.2.2) were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by β-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the γ-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[¹⁴C]acetamide. One of these possessed a carboxy group, which formed a [¹⁴C]glycollamide ester, and the other was cysteine, shown by isolation of S-[¹⁴C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[¹⁴C]acetamide. 4. Isolation of the γ-[¹⁴C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The γ-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the γ-[¹⁴C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the γ-[¹⁴C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with γ-glutamyl-ε-lysine. 5. A scheme for the catalytic mechanism is proposed.     

Abscisic Acid localization and metabolism in barley aleurone layers

Aleurone layers of Hordeum vulgare, cv. `Himalaya' took up [¹⁴C]-abscisic acid (ABA) when incubated for various times. Radioactivity accumulated with time in a low speed, DNA-containing pellet accounting for 1.6 to 2.3% of the radioactivity recovered in subcellular fractions at 18 hours. Thin layer chromatography of ethanolic or methanolic extracts of the cytosol, which contained greater than 95% of the radioactivity taken up by layers, revealed that labeled ABA was metabolized to phaseic acid (PA) and 4′-dihydrophaseic acid (DPA) and three polar metabolites Mx₁, Mx₂, and Mx₃. ABA was not metabolized by endosperm, incubated under conditions used for layers, indicating that metabolism was tissue-specific. Layers metabolized [³H]DPA to Mx₁ and Mx₂. ABA, PA, and DPA-methyl ester and epi-DPA-methyl ester inhibited synthesis of α-amylase by layers incubated for either 37 or 48 hours. These layers converted the methyl DPA and epi-methyl-DPA esters to their respective acids. DPA did not inhibit Lactuca sativa germination or root and coleoptile elongation of germinating Hordeum vulgare seeds, or coleoptile elongation of germinating Zea mays seeds.     

Chlorophyll Turnover in Skeletonema costatum, a Marine Plankton Diatom

[³H]- and δ-[¹⁴C]Aminolevulinic acids were incorporated into the chlorophylls of Skeletonema costatum, a marine plankton diatom. In the stationary phase of growth, the tetrapyrrole-based pigments reached steady-state labeling after 10 hours. Under conditions of exponential cell division and chlorophyll accumulation, ³H was rapidly lost from the labeled chlorophylls and was replaced with ¹⁴C derived from δ-[4−¹⁴C]aminolevulinic acid. The kinetics of isotope dilution suggests recycling of tetrapyrrole precursors and/or two pigment pools, containing both chlorophyll a and chlorophyllide c, one which turns over rapidly (10 hours) and another which turns over more slowly (100 hours). Calculation of turnover times varied from 3 to 10 hours for chlorophyll a and from 7 to 26 hours for chlorophyllide c. The data suggest the dynamics of chlorophyll metabolism in S. costatum and explain the diatom's ability to undergo light-shade adaptation within a generation time.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. General features

Four intrinsic soluble proteins are synthesized and secreted by sexually mature guinea-pig seminal-vesicle mucosa, which comprises a monolayer of a homogeneous columnar epithelial cell. All four proteins can be extracted readily in 154mm-NaCl from the organ's luminal constituents in which they are present in high concentration. They are referred to as proteins 1, 2, 3 and 4 in order of their elution during DEAE-cellulose column chromatography. Specific primary antibodies were harvested from goats that had been inoculated with the purified vesicular proteins; secondary antibodies were obtained from a donkey inoculated with goat γ-globulins. Double-antibody-immunoprecipitation techniques were developed to precipitate the vesicular proteins. Thus proteins newly synthesized from ¹⁴C-labelled amino acids could be precipitated and the incorporated radioactivity assessed. Isolated seminal-vesicle mucosa, incubated in only a buffered salt solution containing glucose, readily synthesized the soluble secreted proteins from added [¹⁴C]lysine plus [¹⁴C]glycine, [¹⁴C]histidine plus [¹⁴C]glutamate, [¹⁴C]glutamine alone and [¹⁴C]arginine alone. The rates of incorporation (d.p.m./mg of total soluble protein) of labelled lysine and glycine and of labelled arginine were linear with time over 180min. With the other labelled precursors, rates diminished between 60 and 180min. Labelled protein could be detected after only 10–15min of incubation. Only 4–9% of the newly synthesized protein remained associated with the mucosa; the remainder was found in the cell-free incubation medium. The isolated seminal-vesicle mucosal preparation will provide a unique opportunity to study the synthesis and secretion of abundant cell-specific proteins by this androgen-dependent tissue.     

Promotion of seed germination by cyanide

Potassium cyanide at 3 μm to 10 mm promotes germination of Amaranthus albus, Lactuca sativa, and Lepidium virginicum seeds. l-Cysteine hydrogen sulfide lyase, which catalyzes the reaction of HCN with l-cysteine to form β-l cyanoalanine, is active in the seeds. β-l-Cyanoalanine is the most effective of the 23 α-amino acids tested for promoting germination of A. albus seeds. Aspartate, which is produced by enzymatic hydrolysis of asparagine formed by hydrolysis from β-cyanoalanine, is the second most effective of the 23 amino acids. Uptake of aspartate-4-¹⁴C is much lower than of cyanide.