Solubilization and characterization of particulate form of DNA polymerase from adult rat liver nuclei

DNA polymerase was solubilized from adult liver chromatin-membrane complex. The activity of this solubilized enzyme was 20–30 times higher than that of the partially purified cytoplasmic DNA polymerase. The solubilized nuclear particulate enzyme differed from the cytoplasmic enzyme in properties such as template preference, salt effect and pH optimum. ATP stimulated only the cytoplasmic enzyme, but EDTA and spermidine, stimulated the solubilized nuclear particulate enzyme but not the cytoplasmic enzyme. On sucrose density gradient centrifugation the cytoplasmic DNA polymerase sedimented around 9 S and the solubilized nuclear enzyme sedimented around 3–4 S.     

The separation of sheep liver cytoplasmic and mitochondrial aldehyde dehydrogenases by isoelectric focusing, and observations on the purity of preparations of the cytoplasmic enzyme, and their sensitivity towards inhibition by disulfiram.

Preparations of sheep liver cytoplasmic aldehyde dehydrogenase obtained by published methods were found by analytical isoelectric focusing in the pH range 5--8 to contain 5--10% by weight of the mitochondrial aldehyde dehydrogenase. Under the conditions used the pI of the cytoplasmic enzyme is 6.2 and that of the mitochondrial enzyme 6.6. The mitochondrial enzyme can be removed from the preparation by selective precipitation of the cytoplasmic enzyme with (NH4)2SO4. Kinetic experiments and inhibition experiments with disulfiram show that the properties of the two sheep liver enzymes are so different that the presence of 10% mitochondrial enzyme in preparations of the cytoplasmic enzyme can introduce serious errors into results. Our results suggest that the presence of 10 microM-disulfiram in assays may completely inactivate the pure cytoplasmic enzyme. This result is in contrast with a previous report [kitson (1978) Biochem. U. 175, 83--90].     

The Identity of Hexokinase Activities from Mitochondrial and Cytoplasmic Fractions of Rat Brain Homogenates

Cytoplasmic hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from the soluble fraction of a rat brain homogenate by a procedure that included a unique affinity elution of the enzyme from Blue Dextran-Sepharose. The purified enzyme was examined with respect to properties in which the impure cytoplasmic enzyme has been reported to differ from the solubilized mitochondrial enzyme. These included the ability to bind to mitochondria, inhibition by quercetin, effect of pH on activity, and kinetics. In all regards the purified mitochondrial and cytoplasmic enzymes appeared identical. In addition, comparative peptide maps after partial proteolysis showed no detectable differences. These results do not support the view that there exist distinct mitochondrial and cytoplasmic forms of hexokinase, the latter being permanently relegated to a cytoplasmic location and unable to participate in a dynamic equilibrium with the mitochondrially-bound enzyme. Alternatives are proposed to explain previous results that had been interpreted as indirect evidence for the existence of a distinct cytoplasmic hexokinase.     

Mouse liver cytoplasmic malate dehydrogenase: rapid isolation, characterization and immunochemical comparison with hepatoma enzyme.

Cytoplasmic NAD-dependent malate dehydrogenase is decreased in activity in three transplantable mouse hepatomas compared to the activity of this enzyme in liver tissue. This enzyme is composed of several molecular forms of similar size which differ slightly in charge; the total activity and the discernible number of forms of the enzyme are decreased in both hepatoma and fetal liver. Mixing experiments suggest the absence of a significant quantity of unbound inhibitor of enzyme activity in the tumor or an activator in the liver. The liver cytoplasmic enzyme was purified to homogeneity by a relatively rapid method using Blue Sepharose affinity chromatography, which results in a good yield and high specific activity of the enzyme. Cytoplasmic and mitochondrial enzymes bind and elute differentially from this affinity resin. Molecular weight, kinetic constants and amino acid composition of the cytoplasmic enzyme were determined. Monospecific antiserum to the cytoplasmic enzyme has been produced in a goat and used to demonstrate a lack of immunological cross-reactivity between the mitochondrial and cytoplasmic enzyme. The tumor and liver cytoplasmic enzymes possess similar, if not identical, immunological determinants. Immunotitration experiments have been used to demonstrate that liver and hepatoma enzyme are identical in specific activity. Thus, the reduction in level of cytoplasmic enzyme in hepatoma is due to a decrease in the number of molecules per tissue mass.     

A study of the control of NADP(+)-dependent isocitrate dehydrogenase activity during gonadotropin-induced development of the rat ovary

The concentration of cytoplasmic NADP(+)-dependent isocitrate dehydrogenase increased 20.2-fold during gonadotropin-induced development of the immature rat ovary. Measurement was by protein (Western) blotting using polyclonal antibodies raised against purified enzyme from the porcine corpus luteum. The increase in enzyme concentration during development correlated well with the 18.5-fold increase observed for the specific activity of the enzyme in the cytosolic fraction. An immunochemical similarity was demonstrated between the cytoplasmic enzyme from the ovary, testes, placenta, skeletal muscle, brain, liver, kidney, mammary and adrenal gland. However the mitochondrial NADP(+)-dependent isocitrate dehydrogenase from these tissues was found to be immunochemically distinct from the cytoplasmic enzyme. The concentration of the substrate D(+/-)-threo-isocitrate in the ovaries was measured by fluorometry and found to increase 3.1-fold during hormone-induced development. The intracellular concentration of substrate was estimated to be of the same order of magnitude as the enzyme concentration. We conclude that the increase in cytoplasmic NADP(+)-dependent isocitrate dehydrogenase activity observed during the gonadotropin-stimulated development of the rat ovary is due to increased concentration of enzyme rather than to an activation of the enzyme. The activity of the enzyme in vivo appears to be regulated by the availability of the substrate D(+/-)-threo-isocitrate.     

Identification of the membrane form of phenylalanine hydroxylase from the human liver and characteristics of its subunit composition

Phenylalanine hydroxylase was detected among human liver bioptats and autoptats extracted with 0.4% Triton X-100 from the 105,000 g homogenate fraction. In contrast to the membrane form of the rat liver enzyme, human liver phenylalanine hydroxylase is detected both by its enzymatic activity and immunochemically under non-denaturating conditions. The enzymatic activity of phenylalanine hydroxylase makes 5-15% of that of the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction as can be evidenced from rocket immunoelectrophoresis data. Immunoblotting of proteins performed after denaturating electrophoresis of the membrane and cytoplasmic fractions revealed an antigen band with a similar electrophoretic mobility. The subunit composition of the enzyme in both fractions was characterized by two-dimensional electrophoresis with subsequent immunoblotting. It was found that the membrane fraction of the enzyme is represented only by the L-subunit with Mr of 55 kD, whereas the cytoplasmic fraction, besides the predominant L-subunit, also contains 2H-subunits of the enzyme with Mr = 57 kD. These 2H-subunits differ between themselves as well as from the L-subunit by the pI value.     

Studies on the site of synthesis of several soluble enzymes of the cell nucleus

Rats were given radioactive L-leucine intravenously. At various times after injection, the livers were removed and separated into nuclear and cytoplasmic fractions by a nonaqueous technique. Glyceraldehyde-3-phosphate dehydrogenase, aldolase, and lactic dehydrogenase were isolated from each cell fraction by antibody precipitation followed by gel electrophoresis, and the specific radioactivities of the isolated enzymes were determined. In all three cases, the onset of labeling and the rate of incorporation were the same for the nuclear enzyme as for the corresponding enzyme from the cytoplasm. If we assume that equilibration of the enzymes between the cytoplasmic and nuclear pools occurs slowly relative to the labeling times employed, we may conclude that the labeled nuclear enzymes either were synthesized in the nucleus or moved into the nucleus from a cytoplasmic site of synthesis without first passing into the cytoplasmic pool of enzyme. Treatment with puromycin, an antibiotic which depresses incorporation into cytoplasmic proteins to a greater extent than into nuclear proteins, led to a situation in which the specific activities of the nuclear enzymes were several times as high as those of the corresponding cytoplasmic enzymes following a short period of incorporation. These data substantiate the assumption that equilibration between the cytoplasmic and nuclear enzyme pools occurs slowly and provide further evidence that the labeled nuclear enzymes do not arise from the cytoplasmic enzyme pool.     

Phaseolus vulgaris cytoplasmic leucyl-tRNA synthetase. Purification and comparison of its catalytic, structural, and immunological properties with those of the chloroplastic enzyme

The cytoplasmic leucyl-tRNA synthetase was purified from bean (Phaseolus vulgaris) leaves. After ammonium sulfate fractionation and chromatography on Sephadex G-50, DEAE-cellulose, hydroxylapatite, and phosphocellulose, complete purification was achieved by blue Sepharose CL-6B chromatography using specific elution with pure yeast tRNALeu1. The enzyme was purified 1050-fold and had a specific activity of 940 nmol of leucyl-tRNA formed/min/mg of protein. Polyacrylamide gel electrophoresis of the native enzyme showed one band, but the denatured enzyme showed two bands. These two protein bands are structurally related. The smallest protein appears to be a cleavage product from the largest one, suggesting the presence of a sensitive cleavage site in the cytoplasmic leucyl-tRNA synthetase. The cytoplasmic enzyme is a monomer (Mr = 130,000), larger than its chloroplastic counterpart (Mr = 120,000). The two enzymes differ in their substrate (tRNA) specificity, tryptic peptide map, and amino acid composition. Antibodies were raised against the cytoplasmic enzyme and against the chloroplastic enzyme and no cross-immunological reaction was detected, showing that the two enzymes do not share any antigenic determinant. Taken together, these results suggest that P. vulgaris cytoplasmic and chloroplastic leucyl-tRNA synthetases are coded for by different genes.     

The activation of aldehyde dehydrogenase by diethylstilboestrol and 2,2'-dithiodipyridine.

1. The activation of sheep liver cytoplasmic aldehyde dehydrogenase by diethylstilboestrol and by 2,2'-dithiodipyridine is described. The effects of the two modifiers are very similar with respect to variation with acetaldehyde concentration, pH and temperature. Thus the degree of activation is maximal when the enzyme is assayed at approx. 1 mM-acetaldehyde, is greater at 25 degrees C than at 37 degrees C, and is greater at pH 7.4 than at pH 9.75. With low concentrations of acetaldehyde both modifiers decrease the enzyme activity. 2. Diethylstilboestrol affects the sheep liver cytoplasmic enzyme in a very similar way to that previously described for a rabbit liver cytoplasmic enzyme. Preliminary experiments show that the same is true for a preparation of human liver aldehyde dehydrogenase. It is proposed that sensitivity to diethylstilboestrol (and steroids) is a common property of all mammalian cytoplasmic aldehyde dehydrogenases.     

The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membranebound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membranebound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.Abbreviations PMS phenazine methosulfate - MV methyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - CHAPSO [3-(3-cholamidopropyldimethylammonia)-1-(2-hydroxy-1-propanesulfonate)] National Research Council Research Fellow     

Enzymatic activities of cytoplasmic and of microsomal acetyl-CoA carboxylase of rat epididymal adipose tissue; different regulatory effects of a short-term fasting and palmitoyl-CoA on these two enzymes

The existence of a microsomal acetyl-CoA carboxylase in the rat epididymal adipose tissue was demonstrated in vitro in the present study. Its specific activity was of the same order of magnitude as that of the cytoplasmic acetyl-CoA carboxylase. The effect of several experimental conditions on the enzymatic activities of both enzymes were tested; fasting for 24 hr strongly increased (2.5-4 times) the activity of the microsomal enzyme while the cytoplasmic enzyme remained unchanged. Palmitoyl-CoA (1 and 5 microM), an inhibitor of acetyl-CoA carboxylase, had a greater effect on the cytoplasmic (33 and 88% inhibition) than on the microsomal enzyme (0 and 37% inhibition).     

The presence of terminal deoxynucleotidyl transferase in the N-methyl-N-nitrosourea induced leukaemia in BDF1 mice and its effect on the accuracy of the DNA polymerases.

DNA polymerases have been prepared from leukaemic and normal spleens and their fidelity in copying a polyd AT).polyd(AT) template assessed. The leukaemic cytoplasmic DNA polymerases were less accurate than the controls whereas no difference in accuracy was observed in the nuclear DNA polymerases. The preparations of leukaemic cytoplasmic DNA polymerase also contained the enzyme terminal deoxynucleotidyl transferase. When this enzyme was removed by further purification the accuracy of the cytoplasmic DNA polymerases increased to that of the controls.     

Localization of angiotensin converting enzyme (dipeptidyl carboxypeptidase) in swine sperm by immufluorescence

Testis and epididymis are known to have high amounts of angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). We investigated the localization of the enzyme in these tissues by an immunofluorescent technique and found that the enzyme was localized in the spermatids and residual bodies in the Sertoli cells of the testis. Furthermore, the enzyme was shown to be present in the cytoplasmic droplet of epididymal sperm and also in detached cytoplasmic droplets in semen. The enzyme was not detected in the interstitium of testis and epididymis except for the endothelial cells of the vessel.     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Propranolol-induced inhibition of rat brain cytoplasmic phosphatidate phosphohydrolase

Propranolol, a cationic amphiphilic drug, caused enhanced incorporation of labeled precursor into phosphatidic acid and its metabolites in rat cerebral cortex mince, suggesting increased biosynthesis or reduced degradation. Inhibition of phosphatidate phosphohydrolase could explain the observed drug-induced accumulation of phosphatidic acid and other acidic lipids. Propranolol exhibited differential effects on the free and membrane-bound forms of phosphatidate phosphohydrolase. The drug inhibited cytoplasmic enzyme in a dose-dependent manner only when membrane-bound substrate was used but had practically no effect on the membrane-bound enzyme irrespective of the nature of the substrate used or on the cytoplasmic enzyme when free substrate was used. Brain cytoplasmic enzyme obtained from rats sacrificed 30 min after intraperitoneal injections of propranolol did not show any inhibition. Propranolol bound to membranes may prevent cytoplasmic enzyme action, probably by decreasing the availability of substrate through the formation of stable lipid-drug-protein complexes.     

Localization of the membrane-bound hydrogenase in Alcaligenes eutrophus by electron microscopic immunocytochemistry

Abstract The membrane-bound hydrogenase was localized in cells of Alcaligenes eutrophus by electron microscopic immunocytochemistry. Post-embedding labeling performed on ultrathin sections revealed that the enzyme was located predominantly (80%) at the cell periphery in autotrophically and heterotrophically grown bacteria harvested from the exponential phase of growth. In the stationary growth phase, however, only 50% of the enzyme was found at the cell periphery; the remaining 50% was distributed over the cytoplasm. The relative amount of electron microscopic label per cell as seen by application of the protein A—gold technique was higher in cells grown autotrophically as compared to cells grown heterotrophically on fructose. Derepression of the enzyme was followed electron microscopically in a substrate-shift experiment (growth on fructose, followed by a shift to glycerol). Major amounts of the enzyme appeared to undergo a reattachment to the cytoplasmic membrane under these conditions, starting with a reduced location of the enzyme in the cytoplasm and an accumulation in cell areas close to the cytoplasmic membrane. These findings indicate that the 'membrane-bound' hydrogenase (i.e., that material enriched as membrane-bound enzyme according to the appropriate activity test) is not, in fact, membrane bound or membrane integrated but membrane associated. It may or may not interact with the cytoplasmic face of the cytoplasmic membrane, depending on the growth phase and conditions.     

Endocrine control of cytosolic factors stimulating adenylate cyclase in rat lung

Endocrine control of cytoplasmic factors modulating adenylate cyclase activity in rat lung membranes was investigated. Hypophysectomy, adrenalectomy and thyroidectomy showed an adverse effect on the body and organ weights. Lung protein, glycogen and DNA contents were decreased in the endocrine ablated animals which were restored to the normal values on hormone treatment. Phosphodiesterase and phosphorylase activities were increased and decreased in adrenalectomized and thyroidectomized animals, respectively. The activities of these enzymes were restored to normal values on hormone treatment. Adrenalectomy and thyroidectomy affected ATPases differently. Basal adenylate cyclase activity in rat lung membranes was not affected by adrenalectomy and hormone treatment. However, the total enzyme activity was increased by both dexamethasone (DEX) and thyroxine (T4) treatments. The activation of the particulate adenylate cyclase by the cytoplasmic factors was markedly decreased in the lung from hypophysectomized, adrenalectomized and thyroidectomized rats. This decrease in the cytoplasmic activation of adenylate cyclase was restored to or above the control values on hormone treatment. Alteration in the activation of enzyme by cytoplasmic factors did not appear to be due to the change in the responsiveness of the enzyme. Glucocorticoids appeared to have a specific effect on the cytoplasmic factors modulating the enzyme.     

Inhibition of leucyl-tRNA-synthetases by modified ATP analogs

The interaction between modifying ATP analogs containing alkylating or phosphorylating groups in the polyphosphate moiety of the ATP molecule and leucyl-tRNA synthetases from cytoplasm and chloroplasts of Euglena gracilis (strain Z) was studied. It was shown that most of the ATP analogs irreversibly inhibit the cytoplasmic enzyme, having no inhibiting effect on the chloroplast synthetase. The kinetic constants K1 and k2 for the interaction between the most effective irreversible inhibitors and the cytoplasmic enzyme were determined. The data on the protection of the enzyme activity by substrates against irreversible inhibition suggest, that the effect of the adenosine 5'-(beta-chloroethyl phosphate) is directed to the ATP-binding site of the cytoplasmic enzyme, whereas the mixed anhydride of AMP and mesithylene carbonic acid acts predominantly on the binding site of 3'-terminal adenosine of the tRNALeu molecule. ATP analogs may be effectively used for affinity labelling of the cytoplasmic leucyl-tRNA synthetase.     

Isolation of protoplasts of Xanthomonas rubrilineans and their use in the study of localization of aminopeptidases]

The culture of Xanthomonas rubrilineans was able to synthesize a number of intracellular aminopeptidases. To study localization of the enzymes in the cells, a protoplasting procedure was developed providing the yield of 99.7 per cent. The following subcellular fractions were isolated: periplasmic, cytoplasmic and membranous. It was shown that alanine aminopeptidase was a cytoplasmic enzyme and glutamate peptidase was a membrane-bound enzyme.     

Distinct nuclear genes for yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases.

Total methionine-tRNA synthetases from wild type $\text{Saccharomyces}\text{cerevisiae}$ can be fractionated on hydroxylapatite into two peaks: Peak I is the mitochondrial, peak II the cytoplasmic isoenzyme. The specificity towards various tRNAs and the antigenic determinants are not identical. A mutant strain, known for its altered cytoplasmic enzyme, contains a mitochondrial species with the same properties as the wild type mitochondrial enzyme, as well as a cytoplasmic isoenzyme with a K_M for methionine about 300 times higher than the corresponding wild type enzyme. Another strain, obtained by back-crossing the mutant with a wild type strain, retains the enzyme pattern found in the mutant. The results are in favor of two distinct nuclear genes for yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases.