bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural relationships in the two-zinc insulin hexamer
Authors: Dodson E. J. , Dodson G. G. , Hodgkin D. C. , Reynolds C. D.
Published: December 2009

The refinement of the crystal structure of two-Zn pig insulin using 1.5-Å (1 Å = 0.1 nm) resolution data by Fourier and fast Fourier least-squares methods allows us to make detailed comparisons between the two independent molecules present in the two-Zn insulin dimer and to describe their interactions in the monomer, dimer, and hexamer. The main chain structures for the two molecules agree well except at the N terminus of the A chain and the C terminus of the B chain. The residues along the line of the local two-fold axes, apart from theB25 side chain, conform extremely closely to the two-fold symmetry, although the discrepancies are much more apparent away from this axis. The ability of the insulin molecule to adopt different conformations may be an important factor in the expression of its biological activity.
Unusual chemical properties of the amino groups of insulin: implications for structure–function relationship
Authors: Sheffer Marla G. , Kaplan Harvey
Published: December 2009

The chemical properties of the three amino groups of insulin were obtained at 10 and 37 °C using the competitive labelling technique with acetic anhydride as the labelling reagent. At 10 °C, pK values of 7.9, 7.2, and 7.8 were found for the glycyl A1, phenylalanyl B1, and lysyl B29 amino groups. When compared with standard amino compounds by means of a Brønsted plot, the two amino-termini were found to be 'super-reactive' and the lysyl ε-amino group buried. In the presence of carbon dioxide at physiological pH values, all three amino groups became much less reactive indicating that they had reacted to form carbamino derivatives. Above pH 8 the reactivities of the glycyl amino terminus and ε-amino group increase sharply indicating that insulin is undergoing a conformational change which is most likely a change in its association state. At 37 °C the amino groups do not titrate normally but exhibit sharp increases in reactivity over the physiological pH range with the midpoints in the pH reactivity profiles between pH values of 7.0 and 7.3. This behaviour is interpreted as a rapid disaggregation of insulin to form monomers as a result of the ionization of the amino groups. It is concluded that at physiological pH and temperature all three amino groups are deprotonated.
Evaluation of receptor function in ACTH-responsive and ACTH-insensitive adrenal tumor cells
Authors: Rae Patricia A. , Tsao Jennivine , Schimmer Bernard P.
Published: December 2009

Two variant cell lines (Y6 and OS3), derived from the ACTH-sensitive mouse adrenocortical tumor clone Y1, are defective in the ACTH-sensitive adenylate cyclase system. This study further characterizes the nature of the defects in Y6 and OS3 cells using ACTH1–10, ACTH4–10, and cholera toxin. In Y1 cells, ACTH1–39, ACTH1–10, and ACTH4–10 stimulated steroidogenesis to the same maximum level with Kd′ values of 5 × 10−11 M, 5 × 10−7 M and 10−4 M respectively. ACTH1–10 (0.4 mM) and ACTH4–10 (3.2 mM) increased the accumulation of adenosine 3′,5′-monophosphate (cAMP) in Y1 cells two- to three-fold. Cholera toxin increased steroidogenesis and cAMP accumulation in Y1 cells with Kd′ values of 0.4 ng/mL and 9 ng/mL respectively. Y6 and OS3 cells responded to added cholera toxin with increased cAMP accumulation and increased steroidogenesis but did not respond to ACTH1–39, ACTH1–10, or ACTH4–10 at concentrations effective in Y1 cells. These data are interpreted to suggest that Y6 and OS3 cells are defective in a process or component that links the principal binding regions of the ACTH receptor to the catalytic subunit of the adenylate cyclase system. Attempts also were made to assess the interactions of ACTH with the principal binding regions of the ACTH receptor by analysis of binding of radioactive, iodinated ACTH1–24. ACTH binding, however, showed low affinity, high capacity, and no target-tissue specificity, and was considered not to be useful in evaluating the integrity of the ACTH receptor.
The effect of growth hormone on the metabolism of a tryptophan load in the liver and brain of hypophysectomized rats
Authors: Young Simon N. , Oravec Michael
Published: December 2009

Growth hormone antagonizes the induction of tryptophan pyrrolase and tyrosine aminotransferase by cortisol. We have shown that, contrary to previous reports, growth hormone is also capable of antagonizing the induction of these enzymes by tryptophan and α-methyl tryptophan. As α-methyl tryptophan is not metabolized appreciably in the rat, our data show that growth hormone does not act indirectly through changes in the liver tryptophan content as was suggested previously. Growth hormone decreases the rate of tryptophan catabolism in vivo after induction of tryptophan pyrrolase by tryptophan and α-methyl tryptophan. Because the rate of catabolism of a tryptophan load is slower in animals treated with growth hormone, tissue tryptophan levels and the rate of synthesis of 5-hydroxytryptamine in the brain are higher in these animals than in those receiving tryptophan alone. Thus, although tryptophan administration raises brain 5-hydroxytryptamine levels, induction of tryptophan pyrrolase in the liver, by the load, limits the extent and duration of the rise in brain 5-hydroxytryptamine synthesis. This has important implications for the clinical use of tryptophan in psychiatric disorders, where tryptophan is given to produce long-lasting elevations of brain 5-hydroxytryptamine.
Effect of thyrotropin on the phosphorylation of thyroid chromosomal proteins
Authors: See Yew Phew , Burrow G. N. , Liew C. C.
Published: December 2009

Thyrotropin (TSH) stimulated the phosphorylation of histone H1 in calf thyroid slices but had no effect on other classes of histones. Phosphorylation of total phenol-soluble nonhistone chromosomal proteins was not affected by incubation with TSH. However, when these phenol-soluble nonhistone chromosomal proteins were analysed by two-dimensional gels involving isoelectrofocusing and dodecyl sulfate – polyacrylamide gel electrophoresis, TSH was shown to stimulate the phosphorylation of two specific groups of phosphoproteins with molecular weights between 35 000 and 45 000 and isoelectric points at pH values of 5.4–6.0. This increase in phosphorylation with TSH stimulation was confirmed by quantitative analysis of one-dimensional isoelectrofocusing gels.
Immunological and biological activities of fragments and analogs of bradykinin
Authors: Redman L. W. , Regoli D. , Tustanoff E. Reno
Published: December 2009

Using a number of analogs and fragments of a short-chain peptide bradykinin, a series of experiments have been carried out to assess the effect of modifications to the basic structure of the parent molecule on its myotropic and immunoreactive properties. Binding kinetics of both an antibody raised against the authentic nonapeptide and its specific biological receptor found in the guinea pig ileum were used to study these alteration effects. Peptide derivatives of bradykinin with an extension at the N-terminal (Lys- and Met-Lys-bradykinin) cross-react with the antibody raised to bradykinin 59 and 70% respectively. On the other hand, internal fragments with intact C-termini (2–9 and 3–9 bradykinin) react with this same antibody to an extent of 250 and 875% respectively, indicating that they are more potent antigens than the vasopressor molecule itself. Other internal fragments, as well as 9-substituted analogs effectively did not interact. These results indicated that the C terminal arginine of bradykinin is indeed essential in the binding mechanism with its antibody. This in turn illustrates the role of the carrier ovalbumin in the development of antiserum to the ovalbumin–toluene–diisocyanate–bradykinin complex.The physiological experiments with the guinea pig bioassay preparations lead to similar conclusions. Most internal fragments of bradykinin are devoid of activity, whereas N-terminal fragments (2–9, 3–9, and 5–9 bradykinin) have retained some activity again indicating a need for an intact arginine residue at the C-terminus of the molecule. Any modification in position 9 results in severe impairment of biological activity. Thus, the C-terminal residue of bradykinin must be conserved in order that the molecule may retain its immunological and physiological activities. Any extensions, deletions, or modifications of this site will severely retard these functions.
Enhancement of microsomal phosphatidate phosphohydrolase and diacylglycerol acyltransferase activity by insulin during growth of rat adipocyte precursors in culture
Authors: Roncari Daniel A. K. , Mack Esther Y. W. , Yip Dominic K.
Published: December 2009

The availability of a propagating adipocyte precursor culture system has provided the opportunity to study biochemical processes under conditions in which any known interacting influences are controlled. We have studied the activity of various triacylglycerol-biosynthetic enzymes during maturation of rat epididymal adipocyte precursors and any possible effect of insulin on enzyme activity. At certain times in culture, the specific activity of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20) is significantly enhanced in cells grown in the presence of added insulin. Under the culture conditions used in this study, the adipocyte precursors acquire several small lipid inclusions and become rounder, but do not assume the signet-ring appearance of mature fat cells during the first 2 weeks of monolayer confluence. Consequently, the effects of the hormone on enzyme activity become evident prior to complete morphological maturation. Phosphatidate phosphohydrolase is believed to be a rate-controlling enzyme in triacylglycerol synthesis in adipose tissue and liver. The fact that the adipocyte precursor microsomal, rather than cytosolic, phosphohydrolase is influenced by insulin suggests that the membrane-bound enzyme is the regulatory phosphohydrolase in intact cells. The enhancement of diacylglycerol acyltransferase activity may be of significance in the reesterification of fatty acids with diacylglycerols, a reaction that by passes the phosphohydrolase step. Thus, in addition to the well-known mechanisms by which insulin promotes triacylglycerol accretion in adipocytes and their precursors, the hormone significantly enhances the specific activity of critical enzymes of triacylglycerol synthesis.
On hepatic mitochondrial monoamine oxidase activity in lipid deficiency
Authors: Kandaswami C. , D'Iorio A.
Published: December 2009

A marked decrease in liver mitochondrial monoamine oxidase activity was noticed in rats fed a fat-free diet as compared with that of their controls. In lipid-deprived rats, the specific activity of this enzyme was very low towards different substrates studied. The activity of kynurenine 3-monooxygenase, which like monoamine oxidase is localized on the mitochondrial outer membrane, was similarly depressed under conditions of lipid deprivation. On the other hand no major changes were observed in the activity of the inner membrane enzyme, kynurenine aminotransferase. Mitochondria from fat-free diet-fed rats were deficient in essential fatty acids whereas no appreciable variations were found in the relative proportions of phospholipids in comparison with those of control mitochondria. Mitochondrial monoamine oxidase activity of the deficient rats retained its sensitivities to inhibitor drugs like clorgyline and deprenyl. No changes were noticeable in the substrate specificity of monoamine oxidase in these rats. When we switched the fat-free diet-fed rats to a diet supplemented with a source of essential fatty acids, there was an elevation in the activities of both monoamine oxidase and kynurenine 3-monooxygenase, their levels approaching those of the control rats.
Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol
Authors: Choy Patrick C. , Farren S. Blake , Vance Dennis E.
Published: December 2009

Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 °C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.
An approach to measuring the in vivo transformation of glycerol to a very low density lipoprotein triglyceride
Authors: Kallai-Sanfaçon M. A. , Norwich K. H. , Steiner G.
Published: December 2009

This paper describes a new method which permits measurement of the steady-state rate of transformation of serum glycerol to a very low density lipoprotein (VLDL) triglyceride in vivo in dogs. Although the turnover of glycerol and the turnover of VLDL triglyceride glycerol have both been previously measured, the rate of transformation of the former into the latter has not. While there is considerable dog-to-dog variation in the absolute turnover and transformation rates, the relationship between the various rates is quite constant. Thus, 13% of the serum glycerol which normal fasting dogs utilize is converted to VLDL triglyceride. The remaining 87% is converted to other products. Also, 28% of VLDL triglyceride glycerol in these dogs is derived from serum glycerol. The balance, 72%, is derived from other sources. The procedure described here can be used to quantitate the contribution of glycerol to VLDL in a number of conditions in which glycerol and (or) VLDL triglyceride metabolism is altered, thereby providing another way to gain insight into the metabolism of VLDL. Even more generally, the principles developed here can be applied to estimate the transformation of other precursors to other products in vivo.
Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo
Authors: Behar-Bannelier Monique , Sharma Rajendra N. , Murray Robert K.
Published: December 2009

The use of L-[35S]methionine (500–700 Ci/mmol (1 Ci = 37 GBq)) for labelling the polypeptides of liver rough (R) and smooth (S) endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 μCi of the isotope and killed at various times (2 min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 μg protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.
Resolution of saturated and unsaturated 5β-cholanoic acids by gas–liquid and thin-layer chromatography
Authors: Child P. , Kuksis A. , Myher J. J.
Published: December 2009

The thin-layer and gas–liquid chromatographic properties of the methyl ester acetates were determined for a series of 20 monounsaturated 5β-cholanoic acids representing the simple chemical and enzymic dehydration products of the common bile acids. The unsaturated acids were generally indistinguishable from their saturated analogues by thin-layer chromatography on plain silica gel, but resolution was achieved on silica gel impregnated with silver nitrate for compounds having sterically exposed double bonds. The gas–liquid chromatographic behaviour of the unsaturated bile acids on the OV-225, SE-30, and Poly-S-179 liquid phases was closely similar to that observed for the saturated bile acids. The 5β-cholenoic acids obeyed the general rules of chromatographic mobility based on the overall shape of the molecule and the number and configuration of functional groups, with a constant retention factor attributable to the oleflnic bond. The structural information provided by the chromatographic behaviour of the standard unsaturated bile acids allows a distinction to be made among most of the isomeric 5β-cholenoates. A complete identification of all isomeric olefins is possible when chromatographic and mass spectrometric data are combined.
Multiple effects of serum low-density lipoprotein on cultured human fibroblasts
Authors: Spain M. J. , Wosu L. O. , Kalant N.
Published: December 2009

The effects of serum low-density lipoproteins (LDL) were studied in cultures of human skin fibroblasts grown in medium supplemented with human serum deficient in lipoproteins and in platelet factor. The LDL led to a temporary increase in the rate of cell replication, to increases in the cell content of protein and cholesterol, to an increase in average cell size, and to an increased secretion of glycosaminoglycans. The increases in cholesterol and protein were proportional to the increase in cell size, suggesting that the additional protein and cholesterol were of a structural, rather than a storage, nature. The increase in cell protein during the first few days of exposure to LDL was due to a decrease in the rate of protein degradation. Ultrafiltration of the serum to remove substances of molecular weight < 30 000 did not reduce the basal rate of cell proliferation but did prevent the stimulation of proliferation by LDL; it did not alter the effect of LDL on cell protein and cholesterol, indicating that the latter responses are independent of the mitogenic action. The response of cells from diabetic donors did not differ from that of normal cells.
Purification of factor EF-P, a protein that stimulates peptide-bond synthesis with certain aminoacyl-tRNA analogues
Authors: Glick Bernard R. , Green Robert M. , Ganoza M. Clelia
Published: December 2009

Factor EF-P is a nonribosomal (soluble) protein of Escherichia coli that stimulates peptide bond synthesis when certain aminoacyl-tRNA analogues are used. The purification of this protein to apparent homogeneity is described here. EF-P has a molecular weight of about 21 000, a Stokes radius of 27 Å (1 Å = 0.1 nm), and a frictional coefficient of 1.48, suggesting an asymmetric structure. By this and a number of other criteria, EF-P is a new factor that controls peptide bond formation during protein biosynthesis.
Lysyl dipeptide and tripeptide model systems for racemization studies in amino acid and peptide chemistry
Authors: Benoiton N. Leo , Kuroda Keiji , Cheung S. T. , Chen Francis M. F.
Published: December 2009

Four series of diastereomeric peptide pairs (X-Lys, Lys-Y, Gly-X-Lys, and Gly-Lys-Y; where X and Y = Ala, Leu, Val, Ile, Phe, and Pro) have been synthesized by conventional procedures using benzyl-based protecting groups. Chromatographic separation of each pair, except Gly-Lys-Pro, has been achieved by elution from a 15-cm Aminex A-5 resin column using pH 5.5 buffer for tripeptides, pH 6.5 buffer for dipeptides, and pH 7.5 buffer for phenylalanyl peptides. The isomers have been identified by chromatography of the L–L isomers. Examples of the use of the model peptides for optical purity determinations and as tests for racemization are presented. The series Gly-X-Lys allows comparison of the tendencies to racemize of residues X during couplings. The separations of other peptides where Y = MeVal and X = N- and O-methylhydroxyamino acids are also described.
Aspartate transcarbamoylase: loss of homotropic but not heterotropic interactions upon modification of the catalytic subunit with a bifunctional reagent
Authors: Chan William W.-C. , Enns Caroline A.
Published: December 2009

The role of conformational changes in the allosteric mechanism of aspartate transcarbamoylase from Escherichia coli was studied by reacting the isolated catalytic subunit with the bifunctional reagent tartryl diazide. Two derivatives differing moderately in substrate affinity were obtained depending on whether the reaction was conducted in the presence or absence of the substrate analogue succinate and carbamoyl phosphate. The modification was not accompanied by aggregation or dissociation. The modified catalytic subunits retained the ability to reassociate with unmodified regulatory subunits and produced hybrids similar in size to the native enzyme. These hybrids were appreciably sensitive to the allosteric effectors ATP and CTP but unlike native enzyme showed no cooperativity in substrate binding. The Michaelis constants of these hybrids for aspartate were intermediate between that of the isolated catalytic subunit and that of the relaxed state. Activation by ATP was caused by a reduction in Km to the value characteristic of the relaxed state whereas CTP inhibited by lowering the Vmax. The properties of the hybrids are strikingly similar to the modified enzyme obtained by Kerbiriou and Hervé from cells grown in the presence of 2-thiouracil. However, the crucial modifications are found in the regulatory subunits of the enzyme studied by these authors whereas they are located in the catalytic subunits of the hybrids reported here. Our results suggest that interactions between the catalytic and regulatory subunits have considerable effects on the state of the substrate binding sites in the native enzyme.
Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli
Authors: Dickie Peter , Weiner Joel H.
Published: December 2009

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic–lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme, whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, present in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Sephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.
The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5′-dithiobis-(2-nitrobenzoic acid) and 4,4′-dithiodipyridine
Authors: Talgoy Michael M. , Bell Alexander W. , Duckworth Harry W.
Published: December 2009

Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4′-dithiodipyridine (4,4′-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4′-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4′-PDS, subsequent reactivity toward the other reagent is greatly decreased, (iv) Following modification, the DTNB and 4,4′-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4′-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4′-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4′-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4′-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4′-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.
Presence of herpes simplex virus type 2 DNA in hamster cells oncogenically transformed by ultraviolet-inactivated virus
Authors: Bibor-Hardy V. , Kessous A. , Simard R.
Published: December 2009

Herpes simplex virus type 2 (HSV-2) DNA has been detected by molecular hybridization in hamster fibroblast cells oncogenically transformed by ultraviolet-irradiated virus. At early passages after cloning in soft agar, about 40% of the HSV-2 genome was present in all the transformed cell lines at one to six copies per cell. In cell lines derived from tumors induced by these cells, the same percentage of the HSV-2 genome was also found with more uniform number of copies (between two and three). Thus the presence of viral DNA seems to be necessary for the maintenance of the transformed state in these cell lines.
The major intracellular alkaline deoxyribonuclease activities expressed in wild-type and Rec-like mutants of Neurospora crassa
Authors: Chow T. Y.-K. , Fraser M. J.
Published: December 2009

Over 95% of the deoxyribonuclease (DNase) activity of log-phase mycelia of Neurospora crassa is expressed as single-strand (ss) specific endonucleolytic activity. This activity is associated with three nucleases (D1, D2, and D3) which, after partial purification from extracts, express activity with double-strand (ds) DNA as well. All three enzymes also degrade RNA at approximately the same rates that they degrade ss-DNA. D3 has been identified as endo–exonuclease, an enzyme previously shown to have endonuclease activity with ss-DNA and RNA and exonuclease activity with ds-DNA, both of which are inhibited by ATP. D3 is inhibited by ATP, is relatively resistant to p-hydroxymercuribenzoate (PHMB), and sediments with an apparent molecular weight of 75 000. D2 has the properties of the previously described mitochondrial nuclease. It is a relatively unstable Mg2+-dependent endonuclease with no appreciable strand specificity for DNA. In addition, it is not inhibited by ATP and is strongly inhibited by PHMB and by ethylenediamine tetraacetic acid (EDTA). It also sediments with an apparent molecular weight of 75 000. The properties of D1 are quite variable from one preparation to another. Freshly isolated D1 sediments with an apparent molecular weight of 180 000. It often shows some inhibition by ATP, but is relatively resistant to both PHMB and EDTA. However, on 'ageing,' the properties of D1 gradually convert to those of D2 with concomitant decrease in molecular weight, loss of inhibition by ATP, and increase in sensitivities to PHMB and EDTA. The results indicate that D1 is very likely a second form of the mitochondrial enzyme. Evidence was obtained for the presence of protein inhibitor(s) in crude extracts which may account for the masking of the ds-DNase activities of these enzymes in extracts.Two Rec-like mutants of Neurospora (uvs-3, and nuh-4) are deficient mainly in expressed levels of D3, the endo–exonuclease. However, the levels of inactive endo–exonuclease precursor in these two mutants are higher than in the wild type. There may, therefore, be some defect in the conversion of precursor to active enzyme in these two mutants. Another mutant, which is not sensitive to mutagens relative to the wild (nuh-3), has depressed levels of both endo–exonuclease and the mitochondrial enzyme. Nuh-3 has some defect in the conversion of D1 to D2. Proteinases probably play some role in vivo in these enzyme conversions.
Studies of two temperature-sensitive mutants of Mengo virus
Authors: Lee Patrick W. K. , Colter John S.
Published: December 2009

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.
The ribosomal RNA of the trypanosomatid protozoan Crithidia fasciculata: physical characteristics and methylated sequences
Authors: Gray M. W.
Published: December 2009

When extracted and analyzed under conditions which maintain noncovalently associated RNA–RNA complexes, the bulk cellular RNA of Crithidia fasciculata contains species of apparent molecular weights 1.3, 0.825, 0.08, 0.065, and 0.045 × 106 in addition to 5 S rRNA and tRNA. Heat denaturation results in the disappearance of the 1.3 × 106 dalton RNA and the appearance of three new species having molecular weights of 0.67, 0.575, and 0.059 × 106. In addition, the apparent molecular weight of the 0.825 × 106 dalton component is reproducibly lowered to 0.81 × 106 after heat treatment. With the exception of tRNA, all of the RNA species are present in close to equimolar amounts in either undenatured or heat-denatured C. fasciculata bulk cellular RNA. On the basis of previous observations on the ribosomal RNA of the closely related organism, Crithidia oncopelti (Spencer, R. & Cross, G. A. M. (1976) J. Gen. Microbiol. 93, 82–88), the 1.3 and 0.825 × 106 dalton RNA's are considered to be components of the large and small subunits, respectively, of C. fasciculata ribosomes, but the subunit localization of the other RNA's described here has not yet been determined.O2′-Methylnucleosides account for about 1.4 mol% of the total nucleoside constituents of unfractionated C. fasciculata rRNA. Quantitative analysis suggests that the rRNA molecules in a C. fasciculata ribosome contain a total of 95–100 O2′-methyl groups, distributed in 80–85 Nm-Np sequences (including four 'hypermodified' Nm-Np, each containing a modification of a base or base–sugar linkage in addition to sugar methylation), six different Nm-Nm-Np sequences, and one Nm-Nm-Nm-Np sequence. While the specific pattern of O2′-methylation in the rRNA of C. fasciculata is distinct, both qualitatively and quantitatively, from the pattern observed in other organisms, Crithidia rRNA does contain certain 'universal' O2′-methylated sequences which appear to have been extensively conserved in evolution.The base-methylated nucleoside, N6, N6-dimethyladenosine(m26A), has been isolated from both C. fasciculata and wheat embryo rRNA in the form of the alkali-resistant dinucleotide, m26A-m26Ap. This dinucleotide and its enzymatic degradation products have been characterized by examination of their ultraviolet absorption spectra and electrophoretic and chromatographic properties.
Wheat embryo ribonucleates. XIII. Methyl-substituted nucleoside constituents and 5′-terminal dinucleotide sequences in bulk poly(A)-rich RNA from imbibing wheat embryos
Authors: Kennedy T. D. , Lane B. G.
Published: December 2009

(1) N6-Methyladenosine, not previously detected as a component of plant mRNA, is shown to be present in the poly(A)-rich RNA which is isotopically labeled when wheat embryos imbibe water in a medium which contains [methyl-3H]methionine.(2) N6-Methyladenosine and 7-methylguanosine 'caps' can constitute more than 85% of the radioactivity when specimens of poly(A)-rich [methyl-3H]RNA are freed of most of the radioactivity contributed by contaminating rRNA, N6-methyladenosine accounting for 75–80% and 7-methylguanosine accounting for 20–25% of this radioactivity.(3) There are three principal 5′-terminal dinucleotide sequences (type 0 'cap' structures) in the poly(A)-rich RNA from imbibing wheat embryos: m7GpppA, m7GpppG, and m7GpppC.(4) The proportional distribution of radioactivity among the 5′-terminal dinucleotide sequences varies with the time of labeling of the imbibing wheat embryos (30 min and 24 h), m7GpppA being the most 'rapidly labeled' sequence and m7GpppC being the most 'slowly labeled' sequence.(5) Poly(A)-rich mRNA from imbibing wheat embryos is distinct from the corresponding RNA from any other organism yet studied in terms of its component methyl-substituted nucleosides and 5′-terminal dinucleotide sequences. The possible significance of these differences, as well as signal similarities between the poly(A)-rich mRNA of imbibing wheat embryos and other organisms, are subjects of discussion.
A novel assay for endonucleases acting at apurinic sites and its use in measuring AP endonuclease activity in repair-deficient mutants of Saccharomyces cerevisiae
Authors: Futcher A. Bruce , Morgan A. Richard
Published: December 2009

A quick and convenient assay for depurination and AP endonuclease activities has been developed. (The term 'AP endonuclease' refers to a nuclease that acts on apurinic and probably apyrimidinic sites on DNA.) It is based on the observation that different topological forms of DNA, such as open circular DNA and covalently closed circular DNA, bind different amounts of the fluorescent intercalator ethidium bromide, and can therefore be distinguished by their fluorescence, This assay has been used to measure AP endonuclease activity in 22 repair-deficient mutants of Saccharomyces cerevisiae. All 22 had normal or nearly normal AP endonuclease activity. The AP endonuclease activity was partially characterized.
Renal adaptation to phosphate deprivation in the Hyp mouse with X-linked hypophosphatemia
Authors: Tenenhouse H. S. , Scriver C. R.
Published: December 2009

The mechanism of renal adaptation to variation in dietary inorganic phosphate (Pi) was investigated in intact Hyp/+ (heterozygous) mice and +/+ (normal homozygous) female siblings. Hyp/+ mice were selected for expression of the X-linked Hyp allele, when fed the control diet (0.6% P), by evidence of persistent postweaning hypophosphatemia (1.78 ± 0.08 mM, mean ± SE, versus 2.68 ± 0.19 mM in +/+ siblings (p < 0.01)). Hyp/+ mice had an elevated fractional excretion index for Pi (FEIPi) (0.570 ± 0.024, mean ± SE) on this diet versus +/+ siblings (0.352 ± 0.025, p < 0.001). Renal cortex content of Pi (~46 nmol/mg protein) and net radioisotopic uptake of Pi by slices were similar in Hyp/+ and +/+ mice. Purified brush border membrane vesicles (BBMV) from Hyp/+ kidney cortex transported labelled Pi (100 μM) by a Na+-dependent mode at about one-half the rate (p < 0.001) observed in +/+ mice. Hyp/+ and +/+ mice fed a low P diet (0.03%) maintained their phenotypic differences in vivo and in vitro. Both adapted to chronic (> 2 week) Pi deprivation with a striking reduction (40- to 50-fold) of FEIPi (p < 0.001) and a fall in plasma [Pi] (p < 0.001). Neither the renal Pi content nor uptake of Pi by slices changed in deprived Hyp/+ and +/+ mice. On the other hand, BBMV uptake by Na+-dependent cotransport increased 200% (p < 0.001) during Pi deprivation in both the Hyp/+ and +/+ mouse. D-glucose transport did not increase. We conclude that renal adaptation to phosphate deprivation is achieved by modulation of a component of Na+-dependent cotransport in brush border membrane that is not controlled by the X-linked gene.
Photooxidation of NADH by 2,3-butanedione: a potential source of error in studies on active site arginyl residues
Authors: Homyk Mona , Bragg P. D.
Published: December 2009

Incubation of NADH or NADPH with 2,3-butanedione in aqueous solution results in photooxidation of the reduced pyridine nucleotides under conditions of ordinary laboratory lighting. Maximum rates of photooxidation are obtained at pH 7 and with light at a wavelength of 410 nm. This reaction could lead to artifactual results in experiments on the role of arginyl groups in enzymes in which a reduced pyridine nucleotide is used to protect the active site residues from modification by 2,3-butanedione.
Affinity chromatography of Ruta graveolens L. O-methyltransferases. Studies demonstrating the potential of the technique in the mechanistic investigation of O-methyltransferases
Authors: Sharma Satish K. , Brown Stewart A.
Published: December 2009

Two discrete furanocoumarin (5- and 8-) O-methyltransferases and a caffeic acid 3-O-methyl-transferase from cell cultures of Ruta graveolens L. have been copurified by affinity chromatography on 1,6-diaminohexane agarose (AH-Sepharose 4B) linked with 5-adenosyl-L-homocysteine (SAH). The furanocoumarin O-methyltransferases, which transfer a methyl group from S-adenosyl-L-methionine (SAM) to the 5- or 8-hydroxyls of linear furanocoumarins, were not retarded by 5-(3-carboxypropanamido)-xanthotoxin (CPAX) immobilized to AH-Sepharose 4B, but addition of SAM to the irrigant buffer led to complete retardation of both enzymes on this affinity system. An analogous phenomenon was observed for the caffeic acid O-methyltransferase, with a ferulic acid ligand coupled to the same insoluble support. SAH was as effective as SAM in promoting binding of the furanocoumarin O-methyltransferases to CPAX and caffeic acid 3-O-methyltransferase to immobilized ferulic acid, respectively. The strong and specific adsorption of these enzymes was abolished by exclusion of SAM or SAH from the irrigant buffer. It is concluded that the enzymes bind first to SAM or SAH, and that this binding process in turn induces the binding site for their specific phenolic substrates or their analogs. Based on these findings, a compulsory–ordered kinetic mechanism for the action of these O-methyltransferases is postulated.
Phosphoester specificity of purified human liver alkaline phosphatase
Authors: Seargeant L. E. , Stinson R. A.
Published: December 2009

Kinetic parameters for the hydrolysis of a number of physiologically important phosphoesters by purified human liver alkaline phosphatase have been determined. The enzyme was studied at pH values of 7.0 to 10.0. The affinity of the enzyme for the compounds was determined by competition experiments and by their direct employment as substrates. Phosphodiesters and phosphonates were not hydrolysed but the latter were inhibitors. Calcium and magnesium ions inhibited the hydrolysis of ATP and PP1 and evidence is presented to show that the metal complexes of these substrates are not hydrolysed by alkaline phosphatase. A calcium-stimulated ATPase activity could not be demonstrated for the purified enzyme or the enzyme in the presence of a calcium-dependent regulator protein. Nevertheless, the influence of magnesium and calcium ions on the ATPase activity of alkaline phosphatase means that precautions must be taken when assaying for Ca2+-ATPase in the presence of alkaline phosphatase.The low substrate Km values and the hydrolysis which occurs at pH 7.4 mean that the enzyme could have a significant phosphohydrolytic role. However, liver cell phosphate concentrations, if accessible to the enzyme, are sufficient to strongly inhibit this activity.
Biochemical properties and kinetic parameters of dihydroxyphenyialanine – 5-hydroxytryptophan decarboxylase in brain, liver, and adrenals of cat
Authors: Bouchard Sylvie , Roberge Andrée G.
Published: December 2009

Biochemical properties and kinetic parameters of nonpurified dihydroxyphenylaianine –5-hydroxytryptophan decarboxylase extracted from brain and two peripheral organs, liver and adrenals, were studied in the cat. This study shows that decarboxylase activity in brain is lower than in peripheral organs and that 5-hydroxytryptophan can be decarboxylated without exogenous addition of pyridoxal-5′-phosphate (PLP). However, the addition of PLP substantially increases the enzyme activity. Excess of coenzyme (>60 μM) induces inhibition in adrenals and liver but not in the central nervous system (CNS). The observed inhibition might be related to the presence of a tetrahydroisoquinoline derivative formed in the medium. Differentiation between mechanisms of action of decarboxylase in the CNS and peripheral organs is suggested.
Biosynthesis, utilization, and translocation of endogenous isomeric spin-labelled radioactive cytidinediphosphodiglycerides from isolated guinea pig liver microsomal to mitochondrial membranes
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

When isolated guinea pig liver microsomal membranes were incubated with isomeric (5-, 12-, and 16-doxyl stearoyl) spin-labelled sn-3-[2-3H]phospfaatidic acid in the presence of CTP and Mg2+, formation of corresponding CDP-[2-3H]diglycerides (in an amount representing 16.5–17.4% of the labelled lipids), which were acceptable substrates in the microsomal biosynthesis of sn-3-[2-3H]phosphatidyl-myo-[U-l4C]inositols, took place. When microsomal membranes containing known amounts of labelled CDP-diglycerides were incubated with unlabeled mitochondrial membranes, reisolated mitochondria contained labelled lipids in an amount which could not be accounted for by the microsomal contamination of reisolated mitochondria, determined by the assay of NADPH – cytochrome c reductase activity, establishing therefore the translocation of labelled CDP-diglycerides (and other labelled lipids) from microsomal to mitochondrial membranes in an amount of ~50% of microsomal content. The rate of loss of paramagnetic lipid species in microsomal and in reisolated mitochondrial membranes was found to be quite different. When reisolated mitochondria containing trans-located isomeric spin-labelled CDP-[2-3H]diglycerides were further incubated with sn-3-[U-14C]glycerophosphate, the formation of labelled phosphatidylglycerophosphate and phosphatidylglycerol was detected. These findings established that the translocation of endogenously formed CDP-[2-3H]diglycerides occurred from isolated microsomal membranes to both outer and inner mitochondrial membranes.
Proteolytic conversion of rat thymus DNA polymerase α to a more active form
Authors: Penner P. E.
Published: December 2009

The relationship between two DNA polymerase α species from mammalian tissues has been resolved with the isolation of a protease from rat thymus which converts the larger α polymerase (7.3S) to a smaller (5.4S) size. The proteolytic activity is present only in the chromatin fraction and the limited proteolysis is accompanied by an increase in activity of the DNA polymerase, possibly consistent with a biological control function for this phenomenon.
The effects of low temperature acclimation of winter rye on catalytic properties of its ribulose bisphosphate carboxylase–oxygenase
Authors: Huner N. P. A. , Macdowall F. D. H.
Published: December 2009

A comparison was made of the kinetics of the carboxylation reaction of bicarbonate–magnesium-activated ribulose biphosphate carboxylase–oxygenase purified from cold-hardened and unhardened winter rye (Secale cereale L. cv. Puma). The activity of the (NH4)2SO4− precipitated enzyme from hardened plants was stable at −20 °C for a month, whereas the form from unhardened plants was reversibly cold inactivated. The [Formula: see text] of the unhardened form increased more rapidly with decreasing pH below 8.2, but the estimated pKa of chemical groups associated with the active site was not affected by the cold hardening. The temperature dependencies of the [Formula: see text] of the two forms of the enzyme crossed at 10 °C with the effect that the catalysis of carboxylation by ribulose biphosphate carboxylase–oxygenase from Puma rye was most efficient in the temperature range to which the plants had been adapted.
Interaction of spironolactone with rat skin androgen receptor
Authors: Boisselle Andrée , Dionne France T. , Tremblay Roland R.
Published: December 2009

Some characteristics of the dorsal skin cytoplasmic androgen receptor (AR) have been studied in male rats. The affinity constant, the binding specificity, and the sedimentation profile of the receptor have been found to be similar to the rat prostate AR. The measurement of the number of binding sites in various hormonal conditions (deprivation) led to the conclusion that this receptor was largely occupied by endogeneous hormones from gonadal and (or) adrenal sources. Administration of spironolactone or canrenone to 7-day-castrated rats was accompanied by a rapid and drastic decrease of available binding sites. This diminution was ascribed to the competitive inhibition of canrenone, the active in vivo metabolite of spironolactone. It is postulated that the antiandrogenic action of spironolactone, at the skin level, is mediated by canrenone which inhibits the formation of specific testosterone and (or) 5α-dihydrotestosterone receptor complex in cytoplasm and consequently in nuclei.