RNA biosynthesis in adipose tissue: effect of fasting

RNA metabolism has been examined in intact adipose tissue and isolated fat cells from rats. The lipocyte contains three species of RNA with sedimentation rates corresponding to those of ribosomal and transfer RNA. The de novo biosynthesis of RNA by adipose tissue cells in vitro was demonstrated. The base ratios of the RNA formed indicate that it was synthesized from a DNA template. Actinomycin D administered in vivo and in vitro decreased total RNA synthesis with the most marked effect on the synthesis of the heavy RNA components. Actinomycin D or puromycin added in vitro was not toxic: they did not inhibit total fatty acid biosynthesis or glucose utilization by the fat pad nor did they inhibit the immediate stimulation of fatty acid biosynthesis and glucose uptake by the addition of insulin in vitro. Starvation for 48-72 hr significantly depressed the synthesis of the heavy RNA components as measured by in vitro uridine incorporation into the individual RNA classes. Refeeding the fasted rat with glucose repaired the defect in RNA biosynthesis before the biosynthesis of monoenoic fatty acid was completely restored. Actinomycin D administered at the time of refeeding prevented the repair of monoenoic fatty acid synthesis. It is concluded that RNA metabolism is intimately involved in the control of biosynthetic reactions in adipose tissue.     

Evolution of DNA and RNA as catalysts for chemical reactions

In vitro selection from combinatorial nucleic acid libraries has provided new RNA and DNA molecules that have catalytic properties. Catalyzed reactions now go far beyond self-modifying reactions of nucleic acid molecules. The future application of in vitro selected RNA and DNA catalysts in bioorganic synthesis appears promising.     

林生山黧豆中2,4—二氨基丁酸合成代谢的体外酶学研究

用~3H-天门冬氨酸为底物,林生山黧豆(Lathyrus sylvestris L.)叶片匀浆上清液为粗酶液,进行体外反应。结果表明,天门冬氨酸的放射性掺入到2,4-二氨基丁酸,加入谷氨酸则能抑制这种掺入。将上述粗酶液透析,加入可能的辅助因子,天门冬氨酸的放射性也掺入到2,4-二氨基丁酸。研究证实在体外天门冬氨酸可以作为2,4-二基丁酸合成的底物,在林生山黧豆体内存在催化天门冬氨酸转变为2,4-二氨基丁酸的合成酶(系)。以2,4-二氨基丁酸和γ-氨基丁酸为底物,用氨基酸自动分析仪测定产物含量,结果表明,2,4-二氨基丁酸和γ-氨基丁酸不互相转变。     

In Vitro Enzymological Studies on Anabolism of 2,4-diaminobutyric Acid in Lathyrus sylvestris

An in vitro synthetic reaction system was established with 2,3-3H-aspartic acid (Asp) as a substrate and the homogenate of fiatpea ( Lathyrus sylvestris L. ) leaves as the crude enzyme extract. The results showed that 3H-Asp was incorporated into 2,4-diaminobutyric acid (DABA). The incorporation was inhibited by the addition of glutamic acid (Glu). 3H-Asp was also incorporated into DABA after the cmde enzyme was dialyzed, indicating that Asp as a substrate for DABA synthesis was catalyzed by a group of enzymes which converted Asp to DABA in flatpea. From the in vitro reactions it was proved that DABA and γ-aminobutyric acid (GABA) could not be mutually substituted as substrates.     

Nitrogen Repression of the Allantoin Degradative Enzymes in Saccharomyces cerevisiae

The preparation and fractionation of a highly active and stable in vitro protein-synthesizing system from Bacillus subtilis is described. Potassium satisfied the requirement for a monovalent ion when the initiation factor-dependent binding of formyl-methionyl-transfer ribonucleic acid and synthesis of formyl-methionyl-puromycin were assayed, whereas it inhibited the reactions for polyphenylalanine synthesis. On the other hand, the ammonium ion satisfied the requirement for all assayed reactions. The in vitro experimental evidence suggested that potassium is an inhibitor of one or a few specific reactions involved in peptide chain elongation in B. subtilis.     

Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.     

Chemical synthesis and semisynthesis of membrane proteins

Total chemical synthesis and semisynthesis of proteins have become widely used tools to alter and control the chemical structure of soluble proteins, Thus, offering unique possibilities to understand protein function in vitro and in vivo. However, these approaches rely on our ability to produce and chemoselectively link peptide segments with each other or with recombinantly produced protein segments. Access to integral membrane and membrane-associated proteins via these approaches has been hampered by the fact that integral membrane peptides or lipid-modified peptides are difficult to obtain mostly due to incomplete amino acid coupling reactions and their poor handling properties. This article will highlight the advances in the total chemical synthesis and semisynthesis of small viral as well as bacterial ion channels. Recent synthesis approaches for membrane-associated proteins will be discussed as well.     

Genetic Relatedness Studies with Adenovirus-associated Viruses

Adenovirus-associated viruses (AAV) contain double-stranded deoxyribonucleic acid (DNA). DNA from each of the four AAV serotypes was used as template for the in vitro synthesis of complementary (3)H-ribonucleic acids(RNA). An estimation of genetic interrelatedness was made on the basis of hybridization reactions between synthetic AAV RNA and AAV DNA. Heterologous reactions were 27 to 37% of homologous reactions, suggesting that the AAV serotypes are related to about the same extent. AAV-1 synthetic RNA was also reacted with DNA from helper adenovirus types 2, 7, and SV15. Very low levels of RNA binding were observed, but it is not likely that these reactions represent AAV-adenovirus genetic relatedness.     

The germination characteristics of phytochrome-deficient aurea mutant tomato seeds

The role of light reactions in anthocyanin synthesis was studied in both attached and detached corollas of Petunia hybrida (cv. Hit Parade Rosa), the latter grown in vitro in media containing 150 m M sucrose and 50 μ M gibberellic acid (GA). Light was essential for the synthesis of anthocyanin in detached corollas, whereas in intact corollas its effect was only to enhance anthocyanin synthesis. Continuous white light at a fluence rate of at least 20 μmol m⁻² s⁻¹ was needed for anthocyanin synthesis in detached corollas. Blue light was more effective than red or green, and far-red was ineffective. Pigmentation of detached corollas exposed to light was inhibited by the photosynthetic inhibitor 3-(4-dichlorophenyl)-1,1-dimethylurea (DCMU). The chloroplast uncoupler NH₄Cl did not affect anthocyanin synthesis, which was, however, inhibited by the blocking of ATP synthesis in both the chloroplast and the mitochondria by dicyclohexylcarbodiimide (DCCD). Sucrose uptake in vitro was inhibited by DCMU and by darkness, and was promoted equally by blue and red light. The activity of phenylalanine ammonialyase (EC 4.3.1.5) was inhibited in detached corollas grown in the dark or in the light in the presence of DCMU. The activity of chalcone isomerase (EC 5.5.1.6) was not affected by light. These findings suggest that at least two different light reactions are involved in the regulation of anthocyanin synthesis in petunia corollas, namely the high irradiance reaction (HIR) and photosynthesis.     

Biogenesis of retinoic acid from beta-carotene. Differences between the metabolism of beta-carotene and retinal

The ability of beta-carotene to serve as precursor to retinoic acid was examined in vitro with cytosol prepared from rat tissues. The rate of retinoic acid synthesis from 10 microM beta-carotene ranged from 120 to 224 pmol/h/mg of protein with intestinal cytosol, and from 344 to 488 pmol/h/mg of protein with cytosols prepared from kidney, lung, testes, and liver. Retinol generated during beta-carotene metabolism was not the major substrate for retinoic acid synthesis. At low substrate concentrations (2.5 microM), the rates of retinoic acid synthesis in intestinal cytosol from beta-carotene or retinol were equivalent, and at higher concentrations (10 microM) the rates of retinoic acid synthesis from beta-carotene or retinol in intestine, testes, lung, and kidney were comparable. Thus, beta-carotene metabolism may be an important source of retinoic acid in retinoid target tissues, particularly in species such as humans that are capable of accumulating high concentrations of tissue carotenoids. Retinal, considered an initial retinoid product of beta-carotene metabolism, was not detected as a product of beta-carotene metabolism in vitro. A ratio of retinol and retinoic acid different from that observed during beta-carotene metabolism in vitro was observed with incubations of retinal under identical conditions. These data indicated that beta-carotene metabolism is not merely a simple process of producing retinal and releasing it into solution to be metabolized independently.     

Purification of RNA using an anti-RNA monoclonal antibody.

Studies of the synthesis and modification of RNA employ many types of in vitro reactions. Often, the RNA product must be concentrated or purified away from other reaction components such as salts, unincorporated nucleotides, protein, or DNA. Here I describe an immunological approach suitable for the isolation of RNA from in vitro reactions. A variety of RNAs of differing size and nucleotide sequence were immunoprecipitated with a monoclonal antibody specific for RNA. RNA binding took place in seconds with nearly quantitative recoveries. Immunoprecipitation was more efficient than ethanol precipitation in removing unincorporated nucleotides. Proteins which do not bind to RNA remained soluble. The immunoprecipitated RNA sample was solubilized directly with a buffered solution suitable for gel electrophoresis under denaturing conditions. Thus, RNAs can be rapidly concentrated for electrophoresis in a single step. Antibody-RNA binding was reversible under nondenaturing conditions in the presence of excess rRNA. This procedure serves as a novel means of purifying RNA and RNA-binding proteins from in vitro reactions.     

Adenovirus DNA synthesis in vitro in an isolated complex.

DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.     

Estradiol benzoate induced changes in protein synthesis and lysosomal hydrolases in the pituitary of male rats

In vitro protein synthesis, lysosomal hydrolases activity and peroxidase activity in the anterior pituitary were estimated in adult male rats treated with 50 micrograms of estradiol benzoate (EB) for 1 day or 7 days. Pituitary protein synthesis, protein and RNA content increased after 7 days. A significant increase in total and membrane-bound acid phosphatase was noted after 1 day or 7 days of EB treatment whereas total beta-glucuronidase activity decreased in both 1 and 7 day group. Cathepsin activity increased after 7 days and pituitary peroxidase system did not change by EB treatment. These findings suggest that immediate change in the enzyme milieu may be one of the first reactions by which EB expresses its feedback control.     

Identification of an intermediate of delta-aminolevulinate biosynthesis in Chlamydomonas by high-performance liquid chromatography

The first committed intermediate of the chlorophyll biosynthetic pathway is delta-aminolevulinic acid (ALA). In plant cells, ALA is formed from glutamate by a pathway not yet clearly defined. One of the proposed pathways involves the reduction of glutamate to glutamate-1-semialdehyde (GSA) via a glutamyl-tRNA intermediate. GSA is then converted to ALA by an aminotransferase. We are studying this pathway using partially purified components from Chlamydomonas reinhardtii in in vitro reactions with [3H]L-glutamate as the substrate and analysis of the radioactive reaction products via HPLC. In reactions either lacking GSA-aminotransferase or containing gabaculine (an inhibitor of aminotransferase), a radioactive intermediate is formed which cochromatographs with synthetic GSA. As observed previously for ALA synthesis, the synthesis of this intermediate has an absolute requirement for RNA, ATP, and active enzymes, while the requirement for NADPH is less stringent. Both the accumulated intermediate and the synthetic GSA can be converted to ALA by the aminotransferase without any additional substrates or cofactors. These results support previous observations that GSA or a very similar compound is an intermediate of ALA synthesis.     

Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ.     

Novel formation of alpha-amino acids from alpha-oxo acids and ammonia in an aqueous medium

In the course of a study of possible mechanisms for chemical evolution in the primeval sea, we found the novel formation of alpha-amino acids and N-acylamino acids from alpha-oxo acids and ammonia in an aqueous medium. Glyoxylic acid reacted with ammonia to form N-oxalylglycine, which gave glycine in a 5-39% yield after hydrolysis with 6N HC1. Pyruvic acid and ammonia reacted to give N-acetylalanine, which formed alanine in a 3-7% overall yield upon hydrolysis. The pH optima in these reactions were between pH 3 and 4. These reactions were further extended to the formation of other amino acids. Glutamic acid, phenylalanine and alanine were formed from alpha-ketoglutaric acid, phenylpyruvic acid and oxaloacetic acid, respectively, under similar conditions. N-Succinylglutamic acid was obtained as an intermediate in glutamic acid synthesis. Phenylacetylphenylalanineamide was also isolated as an intermediate in phenylalanine synthesis. Alanine, rather than aspartic acid, was produced from oxaloacetic acid. These reactions provide a novel route for the prebiotic synthesis of amino acids. A mechanism for the reactions will be proposed.     

Processing of DNA base damage by DNA polymerases. Dihydrothymine and beta-ureidoisobutyric acid as models for instructive and noninstructive lesions

The processing of unrepaired DNA lesions is a key to understanding and predicting the biological end points of particular DNA damages. In this study, we prepared single-stranded f1 phage (f1-K12) DNA containing dihydrothymine or beta-ureidoisobutyric acid as models for instructive or noninstructive base lesions and assessed the potential biological consequences of these lesions in vitro and in vivo. To determine the effect of the two lesions on in vitro DNA synthesis, the extent of DNA synthesis was measured by 3H-labeled nucleotide incorporation, and the newly synthesized DNA was analyzed by DNA sequencing gels. The results showed that dihydrothymine in the template was at most a weak block to in vitro DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (Pol I) and T4 DNA polymerase. In contrast, beta-ureidoisobutyric acid constituted a very strong (probably absolute) replicative block in vitro. With Pol I, termination bands were observed either opposite or one base prior to (3' to) the putative beta-ureidoisobutyric acid depending on its position in the template. However, when DNA synthesis was catalyzed by Pol I lacking a 3'----5' exonuclease activity, termination bands were only observed opposite beta-ureidoisobutyric acid, with purine nucleotides being incorporated preferentially opposite the lesion. With T4 DNA polymerase that contains a very active 3'----5' exonuclease activity, DNA synthesis was arrested almost exclusively one base prior to (3' to) the putative beta-ureidoisobutyric acid site in the template. We also measured survival of transfecting DNA containing dihydrothymine or beta-ureidoisobutyric acid in an attempt to correlate the in vitro data with in vivo processing. In keeping with the results obtained in vitro, dihydrothymine present in transfecting f1-K12 DNA did not constitute an inactivating lesion. On the other hand, it took 0.9 beta-ureidoisobutyric acid residues per molecule to inactivate transfecting f1-K12 DNA, indicating that the lesion was an absolute replicative block in vivo. When host cells were ultraviolet-irradiated to induce the SOS response, a slight increase (about 2-fold) in survival of transfecting f1-K12 DNA containing beta-ureidoisobutyric acid was observed. The potential effects of the structures of base lesions on lesion-polymerase interactions are discussed.     

Lipase-Catalyzed Synthesis and Hydrolysis of Cholesterol Oleate in Aot/Isooctane Microemulsions

We have examined a lipase-catalyzed bidirectional ester synthesis/hydrolysis reaction in a water-in-oil microemulsion system. The reactants were cholesterol (alcohol), oleic acid (acid) and cholesterol oleate (ester), and the solvent system consisted of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water. The reactions were assayed by using [³H]oleic acid, [³H]cholesterol, or [³H]cholesterol oleate for the synthesis and hydrolysis reactions, respectively (separate incubations). The lipase that we used derived from Candida cylindracea, and was used at a concentration of 0.1mg/ml microemulsion. The reactions were performed at 22°C as the reactions proceeded more slowly at higher temperatures. With the initial reactant concentrations set to 10 mM cholesterol, 1 min oleic acid, and 1 mM cholesterol oleate, it was observed that the optimal [H₂O]/[AOT] ratio was at about 9 both for the esterification reaction and for the hydrolysis reaction (after 24 h). The hydrolysis reaction was slower than the synthesis reaction at all [H₂O]/[AOT] ratios studied (0-20), but the difference in reaction yield for the synthesis and the hydrolysis reactions became smaller as the reaction time increased (up to 11 days). When the reaction yield was followed as a time function, it was observed that about 80% of the oleic acid was esterified within 3 days of reaction ([H₂O]/[AOT] ratio of 6), whereas the corresponding value of 80% hydrolysis of cholesterol oleate was reached within 11 days. The results of the present study indicate that by choosing optimal reactant concentrations and reaction conditions, it is at least in part possible to determine the direction of the lipase-catalyzed synthesis/hydrolysis reaction.