Sherman R. Dickman

Activation and inhibition of beef pancreas ribonuclease.

A spectrophotometric assay for ribonuclease is described. The method is sensitive to 0.005 γ of enzyme at pH 7·5 and to γ of enzyme at pH 5.0 The salt activation of RNase was systematically studied. The maximum effect was found at an ionic strength of 0.1 and was non-specific for nonovalent cations. Mg++ also activated RNase but to a lesser extent than Na+. Mg++ decreased the solubility of the products of the reaction. The slight inhibition of RNase due to p-chloromercuribenzoate was decreased in the presence of gelatin or EDTA and abolished by a mixture of the two. The presence of N-ethyl-maleimide, iodoacetamide or oxidized glutathione did not result in a decrease of RNase activity. The data indicate that sulfhydryl groups are not required for RNase activity. Treatment of chromatographic fraction A with oxidized or reduced glutathione did not alter the elution pattern of rbonuclease.

Glutathione peroxidase, glutathione reductase and superoxide dismutase in the aging lens

Glutathione reductase (GR) and glutathione peroxidase (GPx) show in bovine lenses a decrease in specific activity; furthermore, the heat lability of both enzymes increases with age monitoring structural changes of the molecules. GR activity was correlated with type of cataract in human lenses. Its decrease is significantly connected with cortical opacities. Superoxide dismutase activity declines in aging and cataractous lenses. These results support the assumption that in old lens tissue the capacity of the antioxidative scavanger system is diminished.

Factors affecting the activity of egg white lysozyme

A turbidimetric assay for lysozyme which employs Sarcina lutea as substrate is described. The method is sensitive to 0.03 μM lysozyme solutions (0.5 μg./ml.). In 0.025 M citrate, lysozyme exhibits a broad pH optimum in the range pH 5.9 – 6.3. One-tenth molar citrate and 0.03 M trichloroacetate markedly inhibited lysozyme. The activity of lysozyme is affected by the ionic strength of the solution. An activation was observed up to an ionic strength of 0.2; above this value an inhibition of lysozyme occurred. Urea and glycerol reversibly inhibit lysozyme. A photoinactivation of lysozyme in the presence of riboflavin was observed.

Effect of magnesium chloride and potassium chloride on the sedimentation characteristics of ribonucleoprotein isolated from dog pancreas

Ribonucleoprotein particles were isolated from dog pancreas under conditions of invariant ionic composition and analyzed by zone sedimentation in sucrose gradients. On the basis of calculated sedimentation coefficients, the particle population was considered as three fractions: monomeric ribosomes and subunits, S 20,w 0 to 100; polysomes, S 20,w 100 to 500; and large ribosomal aggregates, S 20,w greater than 500. The distribution of the particle population was studied under conditions of variable magnesium ion concentration in one series and of variable potassium chloride concentration in another. Monomeric ribosomes dissociated into 57 s and 38 s subunits at concentrations of magnesium ion below 3 m M , The 57 s particle became the predominant species at 0·5m M -Mg 2+ . The polysomal pattern, however, remained relatively stable in this range. The release of subunits from polysomes occurred at magnesium ion concentrations below 1 m M and was responsible for a decreased yield of this fraction. The yield of large aggregates increased markedly above 3 m M -Mg 2+ . The data indicate that ribosomes possess a heterogeneous range of magnesium ion dissociation constants. Magnesium ion concentration was demonstrated to play a dual part in this population of particles; (1) dissociation of monomers at low concentrations; (2) association of the particles into very large aggregates at high concentrations. The yield of the large aggregates was also increased at low concentrations of potassium chloride. Sedimentation analysis of the post-mitochondrial supernatant fraction in the absence of deoxycholate showed 60% as many monomeric ribosomes as in its presence, but the yield of polysomes was greatly reduced. The results suggest that most of the polysomes are bound to the endoplasmic reticulum in the cells of this tissue. Variations in magnesium ion concentration altered the sedimentation pattern of the post-mitochondrial supernatant fraction similarly to that of the isolated particles. The data support the conclusion that polysomes are not random aggregates, since the amount of the latter can be varied independently of that of polysomes by alterations in the ionic composition of the medium.

Elevated Methionine-tRNA Synthetase Activity in Human Colon Cancer

Summary Methionine-tRNA synthetase catalyzes the formation of methionine-tRNA (Met-tRNA) which is necessary for the initiation of protein biosynthesis. This study compares Met-tRNA synthetase activity in extracts of human colon cancer and normal colonic mucosa. Particle-free supernatants were prepared from homogenates of tumors and adjacent normal mucosa. These preparations were incubated with ATP, a mixture of yeast tRNAs, Mg2+, K+, dithioerythritol, and [3H]methionine. Specific activities (nano-moles of [3H]Met-tRNA per milligram of protein) of Met-tRNA synthetase from tumors and normal mucosa were measured and compared. Five patients were studied and in each the tumor sample had a higher specific activity than its normal counterpart. The mean specific activity of Met-tRNA synthetase in the tumor tissue was approximately four times that of the normal mucosa. Methioninyla-denylate, when added to the assay tubes, proved to be a potent inhibitor of Met-tRNA synthetase. Since protein biosynthesis begins with the formation of Met-tRNA, the synthetase may be a rate-limiting enzyme governing protein biosynthesis in tissues. High specific activity of the enzyme would be expected to correlate with rapid cell growth. Inhibition of the enzyme with methioninyladenylate may prove a useful method of regulating protein biosynthesis in tumor cells. The authors express their appreciation to Sylvia Buorge-Berman for technical assistance and to Drs. Leroy Townsend and Arthur Lewis for the synthesis of methioninyladenylate.

Intracellular localization and chromatography of mouse pancreas ribonucleases.

A number of tissues have been homogenized in 0.25 M sucrose, and various intracellular particulates have been separated by differential centrifugation according to the general procedure of Schneider and Hogeboom,’ or modifications thereof.2 In many instances, the distribution of enzymes in these fractions has yielded interesting data on the localization of a given enzyme or substance and on its presumed metabolic function. As a result of this work, it has been demonstrated that certain cellular components are present exclusively in certain particulates. Thus, desoxyribonucleic acid (DNA) has been found only in nuclei,3 certain enzymes of the respiratory chain (succinoxidase and cytochrome oxidase) have been found in mitochondria,’ and glucosed-phosphatase has been detected in liver micro~ornes.~ I t becomes possible, therefore, if one assumes a similar intracellular enzyme localization in other tissues, to utilize enzyme activities as “markers” for organelles. Van Lancker and Holtzer5 have recently applied such reasoning in a study of the intracellular distribution of DNA, ribonucleic acid (RNA), amylase, desoxyribonuclease (DNase), cytochrome oxidase, and acid phosphatase in mouse pancreas. In other work we have reported that mouse pancreas ribonuclease (RNase) can be chromatographically separated into two major fractions and one minor fraction.6 I t became possible, therefore, to determine whether each chromatographic peak was derived from one intracellular site, or whether a specific organelle contained more than one type of chromatographically distinct RNase. In the course of these studies it was observed that certain treatments exerted marked effects on the chromatographic behavior, as well as on the total RNase activity of some of the fractions. These results will be presented and their significance discussed.

Ribonuclease assay based on uridine phosphate determination.

Abstract A ribonuclease assay is described in which undegraded RNA and nucleotides containing amino groups are precipitated by uranyl acetate at pH 4.0 at the end of the incubation. Uridine derivatives are measured as sole products of the reaction. The influence of pH, time, ionic strength, RNA concentration, and RNase concentration on the reaction has been studied.

Primary Amide Groups of Human Hemoglobin.

Summary The hemoglobin from sickle cell anemic individuals does not differ significantly in primary amide content from normal hemoglobin. Normal human hemoglobin was found to contain 26 primary amide groups per mole.

Detachment of ribosomes from dog pancreas polysomes without concurrent polypeptide synthesis.

Abstract 1. Ribonucleoprotein particles were isolated from dog pancreas cytoplasm and shown to contain polysomes by zone sedimentation velocity analysis, sensitivity to ribonuclease, amino acid incorporation and electron microscopy. The parameters of amino acid incorporation into protein were determined, using as precursor either aminoacyl-tRNA or an amino acid mixture. 2. It was found that release of monomeric ribosomes from polysomes during incorporation from free amino acids was independent of polypeptide synthesis. Ribonuclease activity does not appear to be responsible for this effect. This breakdown of polysomes during incubation in the incorporation mixture was dependent on the presence of GTP and ATP. Polysomes were stabilized under these conditions by NH 4 + , K + , Mg 2+ , or by aminoacyl-tRNA. Since the components responsible for the breakdown phenomena were those participating in the known reactions of protein synthesis, the release of ribosomes may result from the malfunction of the mechanism that links movement of mRNA through the ribosomal decoding site to amino acid incorporation into polypeptides.

Effects of urecholine and atropine on canine pancreas slices. On incorporation of 14C-adenine and 14C-orotic acid into acid-soluble metabolites and RNA.

Abstract The incorporation of 8- 14 C-adenine into cytoplasmic and nuclear RNA of canine pancreas slices proceeds at a much faster rate than that of 6- 14 C-orotic acid. The drug, urecholine, inhibits the conversion of adenine into nuclear RNA. This inhibition is blocked by the cholinergic antagonist, atropine. The incorporation of labeled adenine or orotic acid into the acid-soluble pool of the tissue is likewise inhibited by urecholine, and this effect is also prevented by atropine. By means of thin-layer chromatographic separation of the components of the acid-soluble pool, it was shown that urecholine decreased the pool size of ATP and ADP whereas that of AMP was but very slightly decreased. Intracellular concentration of IMP was unchanged, but that of hypoxanthine was increased. The specific activities of ATP and ADP were moderately decreased, that of AMP was unaffected, but those of IMP and hypoxanthine were markedly increased. The amount and specific activity of hypoxanthine in the medium were increased. Atropine blocked the effect of urecholine on ATP and ADP pool size and concentration, but it resembled urecholine in increasing the specific activity of IMP. Atropine also blocked the effect of urecholine on hypoxanthine pool size and specific activity in both the tissue and the medium. Atropine, however, increased the pool size of IMP.