Esther W. Yamada

Colorimetric determination of inorganic pyrophosphate by a manual or automated method.

Abstract A microprocedure for the colorimetric determination of inorganic pyrophosphate (PPi) in the presence or absence of orthophosphate (Pi) has been developed. PPi is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. Pi is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 μ m PPi can be detemined. The extinction coefficients are 2.70 × 104 and 8.76 × 103 for PPi and Pi, respectively, both with thiol reagent present, and 2.77 × 103 for Pi with no thiol reagent. A ten-fold excess of Pi does not interfere with the determination of PPi and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PPi estimations. Trichloroacetic acid and sodium fluoride inhibi color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3′:5′-cyclic monophosphate are stable in the reaction mixture. The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PPi formation or the rate of synthesis of labeled RNA were very similar. Product PPi formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured. An automated version of the method was devised which allows accurate determination of PPi down to 1 μ m (without range expander attachment) at a sampling rate of 20–40 tubes/hr.

Ca2+-binding properties of a unique ATPase inhibitor protein isolated from mitochondria of bovine heart and rat skeletal muscle.

Previous studies showed that Ca2+ induced monomer to active dimer interconversion of a mitochondrial ATPase inhibitor protein from bovine heart or rat skeletal muscle (Yamada, E.W., Huzel, N.J. and Dickison, J.C. (1981) J. Biol. Chem. 256, 10203-10207). Initial equilibrium dialysis measurements of Ca2+ binding showed that this unique protein possesses three binding sites of high affinity with a maximum of one mol of Ca2+ bound/mol of protein monomer. Magnesium (1 mM) did not affect the first association constant but increased the second and third by about 1.2 and 1.5 fold, respectively. That the apparent association constants varied with concentration of protein monomer was in agreement with the self-associating nature of the protein. Scatchard plots at three concentrations of protein intersected at a molar ratio of about 0.5 (Ca2+/monomer). Ka1 and Ka2 values of 4.2 microM and 12.1 microM, respectively, were estimated by extra-polation of apparent constants to infinite dilution of protein. Ka3 (51.3 microM) was estimated by extrapolation of double reciprocal plots of apparent constants versus protein concentration to infinite levels of protein. A model for Ca2+ binding by this self-associating protein is described. Trifluoperazine had no effect on the activity of the inhibitor protein from either tissue.

Isolation of two ATPase inhibitor proteins from mitochondria of rat skeletal muscle.

An ATPase inhibitor protein was isolated from mitochondria of rat skeletal muscle by alkaline extraction and then was purified, It differed in definitive ways from the ATPase inhibitor protein isolated previously by Ca2+-stripping of submitochondrial particles of rat skeletal muscle. The two ATPase inhibitor proteins were shown to be present together in intact mitochondria.

Visualization of dihydrouracil dehydrogenase activity after disc gel electrophoresis

Abstract A rapid and sensitive method for the location of dihydrouracil dehydrogenase after disc gel electrophoresis based on the reduction of nitroblue tetrazolium to form an insoluble dye has been developed. Semiquantitative evaluation of enzyme activity was achieved by means of densitometer tracings of stained gels. The method permits detection of enzyme activity in partially purified extracts after a minimal number of purification steps. Two enzyme bands, differing mainly in charge, were separated giving the first indication that this enzyme may possibly exist in at least 2 isoenzyme forms in liver.

Effect of methotrexate on cyclic AMP levels in cultured L5178Y cells

L5178Y murine lymphoma cells were cultured for 48 hr in Fischers medium containing 10% horse serum. The cells were then treated with 10−6m methotrexate (MTX) for additional periods of time. Cyclic AMP concentrations increased 1.65-fold compared to untreated controls (P < 0.01) after 1 hr of MTX treatment. Cyclic AMP levels were increased further after 3 hr of MTX treatment (P < 0.05), fell by 6 hr (P < 0.05), and remained depressed after 16 hr (P < 0.05). Cell numbers did not increase during MTX treatment. Inhibition by MTX of cyclic AMP phosphodiesterase activity of homogenates prepared from L5178Y cells was not observed. The data are discussed in relation to a possible block in the G1 to S transition of the cell cycle as an early consequence of MTX treatment. Basal cyclic AMP levels of L5178Y cells grown in medium containing 3.3 × 10−5m hypoxanthine for 1 hr were increased significantly over those of control cultures. Thus, variations in basal cyclic AMP levels of cells could be attributed, at least in part, to the purine content of the cells and/or the horse serum of the culture medium. However, the increases in cyclic AMP caused by hypoxanthine and MTX were additive.

RNA polymerase activity of erythroblasts isolated from regenerating or erythroblastosis-infected avian bone marrow

1. 1. DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) activity of nuclei isolated from six erythroblast-rich fractions of regenerating (“regenerating”) avian bone marrow was compared to that of six comparable preparations obtained from erythroblastosis-infected (“viral”) bone marrow. 2. 2. Both initial rates of RNA synthesis and net RNA synthesis were measured since it was found that the effect of (NH4)2SO4 (0.35 M) on each was different. Both were found to be correlated with the per cent erythroblasts present in “viral” or “regenerating” fractions, in the presence or absence of (NH4)2SO4. 3. 3. Under the standard conditions of assay the initial rates of RNA synthesis catalysed by nuclei of “viral” erythroblasts was twice that of nuclei of “regenerating” erythroblasts (P < 0.001). This difference was even more pronounced in the presence of (NH4)2SO4 and became 3-fold; although (NH4)2SO4 had a negligible effect on the initial rates of “viral” nuclei it decreased those of “regenerating” nuclei. 4. 4. The net amount of RNA synthesized by nuclei of “viral” erythroblasts was 1.3 times that synthesized by nuclei of “regenerating” erythroblasts (P < 0.01). (NH4)2SO4 had no effect on the net synthesis of RNA of “regenerating” nuclei but resulted in a 2.4-fold increase in the net synthesis of “viral” nuclei. Thus, in the presence of (NH4)2SO4, net synthesis of RNA of “viral” nuclei was 2.4 times that of “regenerating” nuclei (P < 0.001). 5. 5. These data indicate that increases in DNA-dependent RNA polymerase activity occur after infection of erythroblasts of avian bone marrow with erythroblastosis virus.

Antigenic reactivity of ribosomal protein S6 and the calcium-binding ATPase inhibitor protein of mammalian mitochondria

SummaryPhosphorylation of ribosomal protein S6 of mammals precedes activation of cell growth in numerous biological systems. We have cloned a cDNA for ribosomal protein S6 from T-47D human breast cancer cells by immunoscreening a λgt11 expression library with antibody raised against the mitochondrial Ca2+-binding ATPase inhibitor protein (CaBI) of bovine heart mitochondria (Yamada & Huzel: J Biol Chem 263: 11498–11503, 1988). Similar clones were obtained by the immunoscreening of a rat heart expression library. In agreement with others, the open reading frames of the cDNAs from the two species coded for the same amino acid sequence. No difference in S6 of the human neoplastic cells compared to that of non-neoplastic cells was found. However, common antigenic determinants in S6 and CaBI were indicated. Accordingly, S6 was purified from rat liver ribosomes and antiserum prepared. Immuno-dot blot and Western blot analyses showed high specific reactivity between S6, the cloned chimeric β-galactosidase fusion protein from a cDNA clone, and CaBI with anti-S6 and anti-CaBI antibodies. The antibodies also showed a high degree of discrimination for S6 and CaBI. Neither interacted with the other ribosomal proteins nor with another ATPase inhibitor protein from bovine heart mitochondria. Neither interacted with the Ca2+-binding proteins, calmodulin, oncomodulin, Protein C, or Factor X. Prothrombin was weakly reactive with anti-CaBI but not with anti-S6. Thus, the results fulfill the specific criteria for the concept and operational definition of common protein epitopes in S6 and CaBI. However, neither prothrombin nor S6 fusion protein inhibited mitochondrial ATPase activity even at 20 times the concentrations at which CaBI gave 97% inhibition.