Tris-Cl Buffer Research Articles

Properties of an Aggregate Ribonucleic Acid Polymerase from Rat Thymus and Its Response to Cortisol Injection

Abstract Lysis of rat thymic nuclei in hypotonic Tris-Cl buffer, pH 7.8, yielded an aggregate preparation containing the DNA-dependent RNA polymerase activity of the intact nuclei. The enzymic activity was maximal in the presence of all four nucleoside triphosphates and was minimal with added RNase or actinomycin D. Activity was also dependent upon the presence of either Mg2+ or Mn2+. Addition of the optimal concentration of (NH4)2SO4 to the assay medium markedly stimulated the rate as well as the duration of incorporation of nucleoside triphosphates into RNA in the presence of either Mg2+ or Mn2+. The concentration of enzyme was limiting and primarily responsible for the decline in activity as incubation time was extended. At this time, addition of either more enzyme, calf thymus DNA, or inorganic salts resulted in marked stimulation of enzymic rate. Injection of cortisol (5 mg of steroid per 100 g, body weight) 3 hours prior to death caused a significant decrease in the activity of the aggregate polymerase prepared from thymic nuclei of these rats. The decreased activity was also seen with other thymolytic steroids, but not with nonthymolytic steroids. The extent of incorporation of UTP into the trichloracetic acid-insoluble fraction was stimulated by the presence of an optimal concentration of either (NH4)2SO4, KCl, or polyethylene sulfonate. Addition of calf thymus DNA to the assay system with or without (NH4)2SO4 also markedly stimulated the rate of RNA synthesis. Under all of these conditions, however, the enzymic activity of the aggregate preparation from thymic nuclei of rats injected with cortisol was significantly lower than that of a similar preparation from control animals. Base ratio analyses indicated that the composition of the RNA species synthesized by the aggregate RNA polymerase differed considerably from that formed by this preparation in the presence of Escherichia coli RNA polymerase and was also influenced by the nature of the cation used. The inhibitory influence of cortisol on UTP-3H incorporation was still evident even after addition of E. coli RNA polymerase alone to the assay mixture containing the aggregate enzyme. Addition of either E. coli enzyme or (NH4)2SO4 to the Mn2+-catalyzed reaction, immediately after endogenous enzymic activity had plateaued, stimulated RNA synthesis further but did not obliterate the cortisol effect. In contrast, simultaneous addition of E. coli polymerase and the optimal concentration of (NH4)2SO4 to the assay system eliminated the differences in the rates of UTP-3H incorporation between the aggregate enzyme preparations from control and cortisol-treated animals.

Liver 3':5'-nucleotide phosphodiesterase and its activity in rat livers perfused with insulin.

Most (80–90%) of the cyclic 3′:5′-nucleotide phosphodiesterase activity in 0.25 M sucrose homogenates of rat liver was found in the 100,000 ×g× 30 min supernatant. The non-particulate nature of the liver phosphodiesterase activity was also confirmed with beef and human liver. The phosphodiesterase activity of rat liver supernatant showed a pH maximum in the region of 7.0–7.5 in Tris-Cl or cacodylate buffer, with a Km of 62 μM in Tris-Cl buffer. A dependency on added Mg++ was indicated for maximum phosphodiesterase activity in rat liver supernatant. High-speed centrifugation and Sephadex G-200 chromatography studies indicated that phosphodiesterase activity is associated with a protein of high molecular weight (>200,000). Cyclic 3′:5′-nucleotides with a purine base were preferentially hydrolyzed in all fractions of rat liver. Dibutyryl adenosine 3′:5′-monophosphate exhibited complete resistance to rat liver phosphodiesterase hydrolysis in the two assay systems employed. The role of liver phosphodiesterase activity as a regulatory enzyme of adenosine 3′:5′-monophosphate concentration in the action of insulin on liver was also investigated. Neither prior treatment of perfused livers with insulin nor presence of insulin in two different assay systems affected the phosphodiesterase activity in high-speed supernatants.

Multiple phenylalanyl-transfer ribonucleic acid synthetase activities in the cytoplasm of Neurospora crassa.

Several tRNA's specific for a particular amino acid have been shown to exist in multiple, or isoaccepting, forms. There is considerable interest in establishing whether multiple aminoacyl-tRNA synthetases also exist. We present evidence that the cytoplasm of Neurospora crassa contains three chromatographically separable phenylalanyl-tRNA synthetases distinct from mitochondrial phenylalanyl-tRNA synthetase. In addition to differences in chromatographic properties the three enzymes exhibit different affinities, in Tris-Cl buffer, toward purified species of valine and alanine tRNA's isolated from Escherichia coli. The two major chromatographic fractions have very similar sedimentation characteristics, which makes a monomer-dimer relationship unlikely.

Studies on polynucleotides, 88. Enzymatic joining of chemically synthesized segments corresponding to the gene for alanine-tRNA.

A principal aim of current work in this laboratory is the development of methods for the synthesis of long-chain bihelical DNA's with specific nucleotide sequences. An immediate objective is the total synthesis of the gene for yeast alanine transfer RNA (tRNA).1 2 In approaching this problem, we wanted to exploit the ability of polynucleotide chains to form ordered bihelical complexes by virtue of base-pairing. Thus, it was hoped to join end-to-end relatively short chemically synthesized deoxyribopolynucleotides while these were held together in properly aligned bihelical complexes. In chemical work, syntheses of two icosanucleotides and several shorter deoxypolynucleotide chains corresponding to segments of the ala-tRNA gene (Fig. 1) have recently been completed.3 For the joining reactions, the recently discovered DNA-joining enzymes4 appeared to be highly promising, and a recent study5 on them using deoxyribopolynucleotides with repeating nucleotide sequences showed that very short oligonucleotide chains were adequate for the joining reaction to occur. In extending this work, we have now demonstrated the enzymatic joining of the chemically synthesized deoxyribopolynucleotides with specific sequences (Fig. 1) when these are provided in appropriate combinations. The present paper documents these findings. Materials and Methods.-Deoxyribopolynucleotides: All of the deoxyribo-, oligo-, and polynucleotides shown in Figure 1 are chemically synthesized products. Except for the tetranucleotide, d-T-C-T-C,6 syintheses of these polynucleotides remain to be published.3 In the incubation mixtures for the DNA-joining enzymes, only the short oligonucleotidic components, e.g., d-T-C-T-C, d-T-C-T-C-C, contained 5'-phosphate end groups and these were labeled with P32 as shown in Figure 1. The 5'-P32-labeled products were prepared by phosphorylation of the 5'-OH groups using y-P32-labeled adenosine 5'-triphosphate (ATP) and polynucleotide kinase as described previously.5 We are grateful to Dr. S. Chang for preparation of the y-labeled ATP and of the polynucleotide kinase. Assay of the joining reactions and isolation of the products: The standard incubation mixture (0.015-0.06 ml) contained per ml: 8 mM tris(hydroxymethyl)aminomethane (Tris)-Cl buffer, pH 7.6, 5 mM MgCl2, 64 AiM ATP, 8 mM dithiothreitol, 0.4 OD2E each of Icosa-I and Icosa-1I, and equivalent amounts of 5'-P32-labeled short oligonucleotides. In the experiments with T4-joining enzyme, the concentration of the enzyme was 1100 units/ml and in some experiments subsequent additions of comparable level were made. The E. coli joining enzyme was used at 200 units/ml. (Both enzymes were tested for specific activity side by side by usinlg the assay described previously.5) Inicubations were at 5-20g. Aliquots (I -5 Al) rernioved at differernt intervals were t,akent to 0.08 ml with (.1 M Tris-Cl buffer, pH 8.0, aad after the solutions had beent boiled at 100' for 2 inlIl tlhev were inieubate(d with 25 Ag of bacteriat alkalinie phosphlatase at 70' for 30 min. The mixtures were then applied to O-(diethylaminoethyl)cellulose paper strips for chromatography and the strips were counted for radioactivity as described before.5 The joined product which remained at the origin was eluted with 1 M triethylammonium bicarbo

Separation and identification of urinary metabolites of [formula omitted] acid in man

The metabolic fate of 14 C-p-n- butoxyphenylacethydroxamic acid (Droxaryl) is studied in Man after oral and rectal administration of the drug. The fractionation of the urinary metabolites was undertaken by ion-exchange chromatography on DEAE-Sephadex A-25 columns (0·01 M Tris-Cl buffer, pH 7·2, elution with a linear gradient of NaCl). The identification of the degradation compounds shows that the main metabolic routes seem to be the conjugation with glucuronic acid (about 70%) and with sulfuric acid (about 5–10%). Hybrid compounds cannot be excluded. Important degradation reactions occur also in the functional group of Droxaryl. The main differenne in the metabolic behaviour of Droxaryl after oral or rectal administration in Man is the higher amount of unchanged drug in the urine of rectally treated patients.

Uridine Diphosphate Sugar Hydrolase: PURIFICATION OF ENZYME AND PROTEIN INHIBITOR

The partial purification of a uridine diphosphate sugar hydrolase from Escherichia coli ATCC 12793 and a specific protein inhibitor of this enzyme are described. The products of the hydrolysis are uridine, inorganic phosphate, and sugar 1-phosphate. The enzyme appears to be identical with the 5'-nucleotidase in this organism. In addition, these cells contain a specific adenosine diphosphate-glucose pyrophosphatase. The 5'-nucleotidase is relatively nonspecific, but the uridine diphosphate sugar hydrolase activity cleaves only uridine nucleotides at a significant rate. Other nucleoside diphosphate sugars containing adenine, guanine, or cytosine as the base are cleaved at less than 5% of the rate of the uridine nucleotides, although the apparent affinity of these nucleotides for the enzyme is the same as that of the uridine nucleotides. Both enzymatic activities show an absolute requirement for the addition of a divalent cation. At pH 8.0 in Tris-Cl buffer, this requirement can be satisfied in order of decreasing affinity by Co++, Mn++, and Mg++, while at pH 5.1 in acetate buffer only Co++ and Mn++ are effective. These same metals protect the enzyme against heat inactivation. Similar, if not identical, enzymes are present in several other strains of E. coli, and a similar enzyme is also present in Salmonella weslaco. All of these enzymes are released into the medium when cells are converted to spheroplasts, but the inhibitor remains in the spheroplast. In Salmonella typhimurium LT-2, a soluble adenosine diphosphate-glucose pyrophosphatase and a particle-bound nucleoside diphosphate sugar pyrophosphatase of broader specificity which is not associated with 5'-nucleotidase activity are present. Some of the properties and possible functions of these enzymes are discussed.

Inactivation of hemagglutinating capacity of type A influenza virus by trypsin treatment.

SummaryThe effect of trypsin on hemagglutinating capacity of 6 type A strains of influenza virus of human origin has been determined under different experimental conditions. With both virus and enzyme in tris-Cl buffer and incubation at 37°C the hemagglutinating capacity of all strains was rapidly inactivated. Minimum concentration of enzyme and minimum time required for inactivation was not the same for all strains. However, with all strains, lowering the incubation temperature or adding sodium or calcium chloride to the trypsin-virus mixtures either reduced the rate of the reaction or completely inhibited it. These changes in test conditions did not in all instances affect all strains to the same extent. With 5 of the 6 strains the effect of added salt appeared to be more closely associated with the concentration than the type of ion present, whereas with the sixth strain the type of ion was a significant factor. The findings illustrate the importance of both the strain examined and the experimental con...