Studies on phenotypic variation between Chinese hamster Kupffer cell lines : II. Correlation relationships and coordinate control of enzyme activities

This study examines somatic cell hybrids between parental cells of identical origin which exhibited quantitative differences in enzyme activities. Nine enzymes in cultured Chinese hamster Kupffer cell hybrids were studied. The parental Kupffer cell clones of identical genetic origin differed several-fold in the specific activities of catalase, arginase, microsomal heme oxygenase, peroxidase, β-glucuronidase, alcohol dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. A selective system based on fusion of ouabain and vinblastine resistant clones of the parental cells was used to isolate hybrids. In hybrid clones, the specific activities of catalase, microsomal heme oxygenase, peroxidase and the dehydrogenases were expressed at the level characteristic of the parental clone with high activities of these enzymes. This result implied interaction between the parental genomes and suggested mechanisms regulating the quantitative levels of several enzyme activities. In contrast, the specific activities of arginase and β-glucuronidase in hybrid clones were intermediate between those possessed by the parental cells and indicated that for each parental genome in the hybrid there was autonomous regulation of the levels of these two enzyme activities.     

Mitochondrial and nuclear glutamate dehydrogenases in Chinese hamster ovary cells in culture.

Nuclear glutamate dehydrogenase (EC 1.4.1.3) activity has been demonstrated in Chinese hamster ovary cells. Some characteristics of this enzyme have been examined and compared with those of the mitochondrial glutamate dehydrogenase from the same source. Differences were detected in the extent of the activation by inorganic phosphate, in the pH versus activity curves, in the affinity of the two enzymes for the cofactor NAD+ and in the electrophosretic mobility. A different rate of decay of the two enzymes has been observed in cells grown in the presence of chloramphenicol. Immunological studies show that, as in ox liver, the nuclear enzyme has specific antigenic determinants besides those in common with mitochondrial glutamate dehydrogenase. Finally, experiments of thermal inactivation indicate a higher stability of the mitochondrial enzyme.     

Hybridization of somatic cells derived from mouse and Syrian hamster: Evolution of karyotype and enzyme studies

Somatic hybrids of drug-resistant mutant hamster and mouse cell lines have been isolated and propagated in long-term culture and have been studied in respect to karyotype and three enzymes. During the course of propagation the long-surviving hybrid clones show progressive loss of telocentric chromosomes associated in at least one case with loss of mouse enzyme. Hybrid clones showed hybrid molecules for malate dehydrogenase (MDH), lactate dehydrogenase (LDH), and 6-phosphogluconate dehydrogenase (6PGD) made up by recombination of parental subunits.This work was supported by National Institutes of Health Grant HD 00486.     

Isolation of viable reconstituted cells from human karyoplasts fused to mouse cytoplasts.

Long-term survivors of reconstituted human-mouse cells have been isolated and characterized by utilizing nuclear and cytoplasmic genetic markers. Karyoplasts were derived from the human SV40-transformed fetal lung fibroblast strain WI38 VA13, while cytoplasts were obtained from the mouse fibroblast A9 cell line which was both hypoxanthine-aminopterin-thymidine-sensitive (HAT^s; nuclear marker) and chloramphenicol-resistant (CAP^r; cytoplasmic marker). The fusion products were isolated in medium containing HAT and CAP. Clones initially showed a growth pattern different from either human or mouse parental cell, but after repeated subculturing, morphologically resembled the nuclear donor cell. The human and mouse components in these cells were identified from other possible fusion combinations by karyotypic, enzymatic and mitochondrial DNA (mDNA) analyses. The karyotype, using both Q-banding and C-banding revealed only human chromosomes. Electrophoretic mobility of the enzyme malate dehydrogenase, a nuclear controlled enzyme, confirmed the human nucleus. Buoyant density centrifugation of radioactive labelled isolated mitochondrial DNA from the reconstituted cells provided evidence that the cytoplasm was of mouse origin.     

Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.     

A new immunochemical method for the quantitative measurement of specific gene products in man-rodent somatic cell hybrids

Summary An immunochemical method has been developed for the quantitative determination of species-specific gene products, for instance a-galactosidase and N-acetyl-a-galactosaminidase, in man-rodent hybrid cells and in the parental cell lines. Antisera raised against the purified enzymes are covalently coupled to Sepharose 4B. The gene products are specifically removed from a cell lysate by incubating with the appropriate Sepharose-coupled antiserum. After centrifugation followed by washing of the precipitated Sepharose, the enzymic activity can be quantitatively measured on the Sepharose beads. With this technique it has been demonstrated that the ability of human N-acetyl-a-galactosaminidase (also known as a-galactosidase B) to hydrolyze a-galactosidic linkages is lost when the enzyme is expressed in man-Chinese hamster hybrid cells.     

Immunological Difference between Glucose-6-P Dehydrogenase and Hexose-6-P Dehydrogenase from Human Liver

SHAW and Barto¹ have demonstrated the presence of an autosomally inherited glucose-6-P dehydrogenase (G6PD) in the deer mouse. Subsequently, Ohno et al.² found a similar enzyme in trout and showed that this enzyme and the autosomally inherited mouse enzyme differed from the sex-linked G6PD in possessing marked catalytic activity with galactose-6-P. This autosomally inherited G6PD was therefore named hexose-6-P dehydrogenase (H6PD)^2,3. It was shown to oxidize glucose-6-P, galactose-6-P, mannose-6-P and 2-deoxy glucose-6-P with a K_m of the order of 10^?5 M. It also oxidizes glucose with a K_m of 0.7 M³. It appears to be identical to the so-called “glucose dehydrogenase”. The enzyme utilizes both NAD and NADP and is microsome-bound. G6PD is localized in the soluble fraction of the cells of various tissues. Although it has been shown that two dehydrogenases from liver have different substrate specificity, molecular weight and elec-trophoretic mobility^3,4, it has been suggested that the two enzymes are merely isozymes and they might be interconvertible^5–7. We have now partially purified the two enzymes from human liver and show that they have different immunological properties.     

Sensitivity of isoenzyme analysis for the detection of interspecies cell line cross-contamination

Summary The analysis of the gel electrophoresis banding patterns and relative migration distances for the individual isoforms of intracellular enzymes, such as lactate dehydrogenase, purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase, is used routinely in the biopharmaceutical industry for confirmation of cell line species of origin. In the present study, the sensitivity of the technique (AuthentiKit™, Innovative Chemistry, Marshfield, MA) for determining interspecies cell line cross-contamination was examined. Extracts were prepared from a CHO-K1 line (AA8, Chinese hamster), MRC-5 (human) cells, and L929 (mouse) cells and from several proportional mixtures of the various binary combinations of cells. The isoenzymes were analyzed according to standard procedures for the technique. Contamination of MRC-5 cells with CHO-K1 or with L929 cells was clearly detectable with each enzyme analyzed. Similarly, the contamination of L929 or CHO-K1 cells with MRC-5 cells was readily apparent with each enzyme. On the other hand, contamination of CHO-K1 cells with L929 cells was only detected with lactate dehydrogenase analysis, and contamination of L929 cells with CHO-K1 cells was not detected with any of the four enzymes examined. For the latter case, the analysis of an additional enzyme (peptidase B) was required. The results indicate that interspecies cross-contamination should be detectable with isoenzyme analysis if the contaminating cells represent at least 10% of the total cell population.     

Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids.

Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liver-specific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.     

Intracellular localization and properties of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa.

Glycerokinase and glycerol-3-phosphate dehydrogenase activities have been examined in cell extracts obtained from Neurospora crassa after growth in media containing glycerol. The glycerokinase is located in the cytosol and has been partially purified by ion exchange and gel-filtration chromatography. The molecular weight of the enzyme has been estimated by sucrose density centrifugation to be approximately 120,000. No effect of either fructose-1,6-bisphosphate or other sugar phosphates on enzyme activity was observed. The G3P dehydrogenase activity in cell extracts is apparently catalyzed by a flavin-linked enzyme as no dependence for either NAD⁺ or NADP⁺ could be demonstrated. The enzyme is located primarily in the mitochondria and is not removed from mitochondrial membranes by treatment with digitonin. Separation of digitonin-treated mitochondria on discontinuous sucrose gradients indicated that the enzyme is located on the mitochondrial inner membrane. The synthesis of both enzymes is under some form of catabolite repression since increased specific activities could only be observed in cells grown on acetate, but not glucose, sucrose, or xylose.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.     

Oscillations of enzyme activities during the cell-cycle of a glucose-repressed fission-yeast Schizosaccharomyces pombe 972h

1. Increased specific activities of cytochrome c oxidase, catalase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase and malate dehydrogenase were observed during glucose de-repression of Schizosaccharomyces pombe. 2. The cell-cycle of this organism was analysed by three different methods: (a) harvesting of cells at intervals from a synchronous culture, (b) separation of cells by rate-zonal centrifugation into different size classes and (c) separation of cells by isopycnic-zonal centrifugation into different density classes. 3. Measurement of enzyme activities during the cell-cycle showed that all the enzymes assayed [cytochrome c oxidase, catalase, acid p-nitrophenylphosphatase, NADH-dehydrogenase, NADH-cytochrome c oxidoreductase, NADPH-cytochrome c oxidoreductase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase (NADP) and fumarate hydratase] show periodic expression as ;peaks'. 4. Cytochrome c oxidase shows a single maximum at 0.67 of a cycle, whereas succinate dehydrogenase exhibits two maxima separated by 0.5 of a cell-cycle. 5. All other enzymes assayed showed two distinct maxima per cell-cycle; for catalase, malate dehydrogenase and NADPH-cytochrome c oxidoreductase there is the possibility of multiple fluctuations. 6. The single maximum of cytochrome c oxidase appears at a similar time in the cycle to one maximum of each of the other enzymes studied, except for NADH dehydrogenase. 7. These results are discussed with reference to previous observations on the expression of enzyme activities during the cell-cycle of yeasts.     

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.     

Characteristics of somatic cell hybrids (mouse X Chinese hamster) with different ratios of parental species chromosome sets. IV. Electrophoretic analysis of several enzymes of the dehydrogenase class]

Electrophoretic mobilities in polyacrylamide gel of five dehydrogenases: NADP-dependent malate dehydrogenase (NADP-MDH), 6-phosphogluconate dehydrogenase (6PGD), alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) were investigated in a series of mouse X Chinese hamster somatic cell hybrids. Seven hybrid lines with different ratio of chromosome sets of hamster and mouse: 1:1, 2:1, 3:1 and 1:2 respectively were studied. NADP-MDH and 6PGD of both parental species and intermediate hybrid bands were present in all hybrids except two lines. These lines had only hamster MDH due to the elimination of mouse chromosomes. A correlation was found between the gene dose and the intensity of the expression of the MDH bands. The mouse type ADH was detected in all hybrids. The hamster ADH was found in one of the hybrid lines that lost all mouse chromosomes during cultivation. It is suggested that hamster ADH activity was suppressed in hybrids by the mouse genome. The species origin of GDH and G6PD could not be established due to similarity of electrophoretic mobilities of respective enzymes in parental cells.     

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD⁺-dependent L-lactate oxidation (10^-4 kat kg^-1 protein), as well as NADH-dependent pyruvate reduction (10^-3 kat kg^-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a K_m value of 0.25 mmol l^-1 (pH 7.0, 0.3 mmol l^-1 NADH) for pyruvate and of 13 mmol l^-1 (pH 7.8, 3 mmol l^-1 NAD⁺) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN flavin adenine mononucleotide     

Regulation of glucose 6-phosphate dehydrogenase expression in CHO-human fibroblast somatic cell hybrids

Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.     

Cell growth in arginine- and ornithine-deprived medium and conversion of glutamate to ornithine and arginine in rat hepatoma cells

Summary A subline growing in medium without arginine and ornithine was established from a rat Reuber hepatoma cell line (R-Y121B·cho). The subline designated R-Y117B·cho was able to grow in glutamine, arginine and ornithine-free, glutamate-supplemented medium. Arginine synthesis from glutamate requires four urea cycle enzymes and another two enzymes, glutamate semialdehyde dehydrogenase and ornithine aminotransferase. Since R-Y121B·cho cells have all the urea cycle enzymes, two other enzyme activities were determined. The activities of ornithine aminotransferase and glutamate semialdehyde dehydrogenase were similar in R-Y117B·cho and its parental R-Y121B·cho cells, but R-Y117B·cho cells had higher conversion of glutamate to arginine than parental cells.     

Microscale isoelectric focusing studies of mouse and human hypoxanthine-guanine phosphoribosyl transferases

A microscale isoelectric focusing technique has been developed and used to study hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C. 2.4.2.8, inosinate-guanylate:pyrophosphate phosphoribosyl transferase) activities in mouse and human cell lines. The enzymes of both mouse and human origin are shown to exhibit considerable heterogeneity, but each type has a unique range of isoelectric pH. The enzyme of a mouse × human hybrid cell line, derived by fusion of HGPRT^– parental cells, gives a homogeneous peak of activity, unlike the wild-type enzyme of either parent. The possibility is suggested that this enzyme activity is due to intra-allelic complementation.Centennial Fellow of the Medical Research Council of Canada, 1967–1970.     

Isolation of a lactic dehydrogenase-A-deficient CHO-K1 mutant by nylon cloth replica plating.

A mutant Chinese hamster ovary cell deficient in lactate dehydrogenase A activity has been isolated using a nonselective technique. The method uses histochemical staining to examine colonies directly for enzyme activity and nylon cloth replica plating to recover particular clones. The mutant cell has an apparent Km (pyruvate to lactate) that is nearly tenfold higher than the parental cell, while its Vmax has been reduced more than 80-fold. In mutant cell extracts with added porcine LDH-B enzyme, molecular dissociation and recombination of subunits produces two new active LDH tetramers (A1B3, A2B2). The electrophoretic mobility of at least one of the tetramers (A1B3) was different from those formed in the parental extracts. The evidence suggests the variant cell contains a mutation in the structural gene for LDH-A.     

The intracellular localization of adrenal 3β-hydroxysteroiddehydrogenase/Δ5-isomerase by density gradient perturbation

The intracellular localization of 3β-hydroxysteroiddehydrogenase/Δ⁵-isomerase (Δ⁵-3β-OHD) in bovine adrenal cortex was determined by density perturbation with density gradient centrifugation and the use of marker enzymes. Mitochondrial rich fractions after incubation with succinate and iodonitrotetrazolium (INT) were loaded with insoluble reduced formazan and separated on a linear sucrose gradient. A shift in median density was observed in the INT-treated mitochondria for protein, succinic dehydrogenase activity, and for Δ⁵-3β-OHD activity, but not for 21-hydroxylase, a microsomal enzyme marker. Density perturbation and gradient fractionation reveals a dual location of Δ⁵-3β-OHD in both mitochondrial and microsomal fractions.