Isolation of pig platelet membranes and granules distribution and validity of marker enzymes

A method for the subcellular fractionation of pig platelet homogenates by sucrose density gradient centrifugation is described. The procedure is simple, highly reproducible and yields two major particulate fractions and a soluble phase. One particulate fraction consists almost entirely of membrane fragments and is relatively free from granule contamination. The other particulate zone contains the platelet granules and mitochondria. The distribution on the gradients of the enzymes lactate dehydrogenase, succinate dehydrogenase, 5′-nucleotidase, leucyl β-naphthylamidase and cholinesterase has been studied and organelle localisation further substantiated by electron microscopy. The degree of solubilisation of certain marker enzymes during homogenisation has been investigated and the parallel release of these enzymes with the soluble phase marker enzyme lactate dehydrogenase, suggests they have a true biphasic location between the soluble and particulate components of the cell. No significant difference was found in the molar ratios of cholesterol to phospholipid in the subcellular fractions but the content of each lipid was twice as high in the membrane fraction as in the granule fraction.     

Immunofluorescent localization of glycogenolytic and glycolytic enzyme proteins and of malate dehydrogenase isozymes in cross-striated skeletal muscle and heart of the rabbit.

Specific antisera against glycogen phosphorylase, phosphofructokinase, aldolase, glyceraldehyde-phosphate dehydrogenase, enolase, lactate dehydrogenase, cytosolic and mitochondrial malate dehydrogenase from rabbit muscle were obtained from sheep. The gamma-globulins were used for indirect immunofluorescent localization of the respective enzymes in rabbit skeletal muscle and heart. In stretched skeletal muscle a cross-striation like distribution was observed for all enzymes studied. In the case of mitochondrial malate dehydrogenase this pattern is due to the staining of I-band mitochondria. In cross-sections, an intense staining of the sarcolemma and of subsarcolemmal mitochondria was observed. Comparative analyses with polarized light revealed that the cytosolic enzymes under study are distributed in the relaxed muscle fibre predominantly within the isotropic zones. The same distribution holds also for heart. In contracting muscle a decrease in cross-striated fluorescence and a faint staining of the interfibrillar spaces suggests a location also within the interfibrillar space.     

The involvement of glutamate dehydrogenase in the adaptation of mitochondria to oxidize glutamate in sucrose starved pea embryos

Embryos of pea (Pisum sativum L. cv Sol) deprived of cotyledons were cultured for 3 days in medium with or without sucrose. Respiratory activity of embryos (intact) as well as the ability to oxidize glutamate by mitochondria isolated from embryos were studied. Respiration of intact embryos grown in sucrose supplemented medium was more intensive than in the starved ones. Transfer of the starved embryos to the sucrose-containing medium induced the increase in the intensity of O₂ consumption. Mitochondria isolated from both starved and control embryos exhibited respiratory control. Mitochondria isolated from embryos cultured in the absence of sucrose showed higher (about 60 %) ability to oxidize glutamate and α-ketoglutarate than mitochondria from embryos grown in sucrose containing medium. The absence of sucrose in the medium led to a rapid increase in the specific activity of glutamate dehydrogenase (NADH-GDH and NAD-GDH) and it was accompanied by changes in izoenzymatic pattern of enzyme. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase may be responsible for the increase of glutamate oxidation by mitochondria of pea embryos. Electrophoretic separation of glutamate dehydrogenase isolated from embryos cultured in medium without sucrose showed the presence of ca. 17 isoenzymes while in non-starved embryos only 7 isoenzymes were identified. However, the addition of sucrose to starved embryos after 24 hours of cultivation led to a decrease in glutamate dehydrogenase activity (up to 40 %) but it did not cause the changes in isoenzymatic pattern. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase maybe responsible for the increase of glutamate oxidation by mitochondria of pea embryos. The posibility of glutamate dehydrogenase regulation by sucrose is discussed.     

Differential expression and immunohistochemical localisation of the phenol and hydroxysteroid sulphotransferase enzyme families in the developing lung

Reversible sulphation, catalysed by sulphotransferases and sulphatases, of biologically active compounds such as androgens and oestrogens is a sensitive mechanism for regulating their bioavailabilty, and we have previously hypothesised that this process plays a significant role in the regulation of human fetal lung development. Sulphation is also a major detoxification reaction, contributing significantly to the body's chemical defence mechanism. We have used qualitative and semiquantitative immunological studies to determine the temporal expression and localisation of phenol and hydroxysteroid sulphotransferases during human lung development. Our results show that in the early fetal lung, phenol sulphotransferase expression is at its highest, and is most widely distributed throughout the developing respiratory epithelium. With later development, expression levels decrease and become predominantly restricted to the more proximal airways. In contrast, hydroxysteroid sulphotransferase is present only at very low levels in the early-gestation lung but expression increases rapidly through gestation to reach an apparent peak by 1 year postnatal age. The proximal-to-distal gradients of phenol and hydroxysteroid sulphotransferase expression were similar in mature respiratory epithelium, with immunoreactivity in ciliated cells, non-ciliated secretory cells and basal cells, but with no apparent expression in mucus-secreting cells. These studies provide supporting evidence for the hypothesis that hydroxysteroid sulphotransferase, an androgen-inactivating enzyme, contributes to the role of androgens in retarding the maturation of human lung in utero.     

The influence of fasting on chicken liver metabolites, enzymes and mitochondrial respiration

Chicken liver mitochondria were isolated in relatively pure form as indicated by electron microscopy and marker enzyme assay. The rate of respiration, respiratory control index and ADP/O ratios with several different substrates indicated that chicken liver mitochondria are more uncoupled than rat liver mitochondria. Chickens have ten-fold higher malate concentrations in liver than do rats, 2-oxoglutarate was also more abundant in chicken livers. Fasted birds had a five-fold increase in beta-hydroxybutyrate as compared with fed birds; whereas malate and lactate concentrations decreased. Fasted birds had increased levels of isocitrate dehydrogenase (NADP dependent) and lactate dehydrogenase in the cytosol, and increased malate dehydrogenase (NAD dependent), isocitrate dehydrogenase (NADP dependent) and malic enzyme activities in the mitochondria.     

Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5′-nucleotidase), endoplasmic reticulum (neutral α-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-β-glucosaminidase), peroxisomes (catalase). γ-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co²⁺, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and β-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.     

Subcellular localization of gamma-glutamyltransferase in calf thymocytes.

The subcellular localization of gamma-glutamyltransferase in calf thymocytes was investigated and compared with that of alkaline phosphodiesterase I, alkaline nitrophenyl phosphatase, succinate-tetrazolium oxidoreductase (succinate-INT reductase) and lactate dehydrogenase after two different methods of cell disruption and differential centrifugation. Most of the activity was recovered in the crude membrane fractions (43.0%), but significant amounts co-pelleted with the large-granule (mitochondria) fractions (31%). The specific activity of the gamma-glutamyltransferase in the purified plasma membrane was 30-50 times that of the enzyme in the cell homogenate and had a similar subcellular distribution to the plasma-membrane markers, alkaline phosphodiesterase I and alkaline nitrophenyl phosphatase. It was concluded that gamma-glutamyltransferase was primary a plasma-membrane-bound enzyme, and that its location in other subcellular fractions was probably due to their contamination with plasma-membrane vesicles.     

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.     

Ultrathin-layer microelectrophoretic determination of lactate dehydrogenase isoenzymes in corneal and conjunctival epithelium of the cow

In order to evaluate the impact of tissue oxygenation on the distribution pattern of lactate dehydrogenase isoenzymes, activities of the isoenzymes were measured in microdissected samples of bovine tissue. A highly sensitive ultrathin-layer electrophoretic technique was used to determine the distribution pattern of lactate dehydrogenase isoenzymes in basal, intermediate and superficial layers of the epithelium of central and peripheral cornea and in the epithelium of the bulbar conjunctiva. Measurements revealed almost homogeneous intraepithelial distribution patterns of lactate dehydrogenase isoenzymes in both tissues. In the cornea the lactate dehydrogenase isoenzymes 4 and 5, which are regarded to be specialized for anaerobic glucose metabolism, were found to predominate. In the well-oxygenated conjunctival epithelium most of the activity could be ascribed to the lactate dehydrogenase isoenzyme 3. In contrast to the isoenzymatic activities, total activity of lactate dehydrogenase was inhomogeneously distributed; maximum activities were found in the basal layer of corneal epithelium and in the intermediate layer of conjunctival epithelium. The results indicate that oxygen supply is relevant rather for the intraepithelial distribution of total enzyme activity than for the expression of lactate dehydrogenase isoenzymes.Parts of this study were presented as an inaugural dissertation to the Medical Faculty of the University of Basel by K. Krieger     

Succinate dehydrogenase localization in the mitochondria of the renal tubules of cold- and warm-blooded vertebrates

Using electron microscopic histochemical technique, studies have been made on the activity of succinic dehydrogenase in the kidneys of the cod Gadus morrhua and dog. It was shown that chelate granules indicating localization of the enzyme in the mitochondria of nephronal cells, concentrate mainly in two zones -- between the membranes and inside the cristae. This distribution of the enzyme implies the presence of two pools of succinic dehydrogenase in the mitochondria which are utilized at different stages of oxidative phosphorylation. Succinic dehydrogenase content of the cristae is lower in cod than in dog.     

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.     

Free Mitochondria and Synaptosomes from Single Rat Forebrain. A Comparison Between Two Known Subfractionation Techniques

Two published subcellular subfractionation techniques employing Ficoll-sucrose or sucrose-density gradient centrifugation, respectively, are evaluated for their capacity to yield fractions containing free mitochondria and synaptosomes from a single rat forebrain. The enzymes lactate dehydrogenase, acetylcholinesterase, NAD(P)H-cytochrome c reductase, and citrate synthase, markers of different subcellular components, were used to assess the purity and integrity of the fractions. Judged by the distribution of these specific enzymatic markers, the free mitochondria obtained by the Ficoll-sucrose gradient technique were less contaminated by synaptosomes and had greater biochemical integrity than those obtained by the sucrose-gradient technique. By contrast, the synaptosomes obtained by the Ficoll-sucrose gradient technique resulted in more contamination by microsomes than those prepared in a sucrose gradient.     

Localization of dihydroorotate oxidase in myocardium and kidney cortex of the rat

Biochemical studies have demonstrated that dihydroorotate dehydrogenase (DHOdehase; EC 1.3.3.1 or 1.3.99.11) is the sole enzyme of de novo pyrimidine synthesis in mitochondria, whereas the rest of the pathway takes place in the cytosol. The dehydrogenation of dihydroorotate to orotate is linked to the respiratory chain via ubiquinone. In this study, we show for the first time the ultrastructural localization of DHOdehase. Since the purified enzyme was found to act both as dehydrogenase and as oxidase, the cerium capture technique for detecting enzymatically generated hydrogen peroxide could be applied to pin-point the in situ activity of DHOdehase oxidase in mitochondria of rat heart and kidney cortex. Cerium perhydroxide as the final reaction product was detected predominantly in the matrix with some focal condensation along the inner membrane, but not in the intermembrane space. From this pattern of localization, it is concluded that the active site of the membrane-bound enzyme could face the mitochondrial matrix similar to succinate dehydrogenase. The reliability of the applied method for the demonstration of DHOdehase oxidase was demonstrated by the addition of Brequinar sodium to the incubation medium. This quinoline-carboxylic acid derivative is a potent inhibitor of DHOdehase and has proven anti-proliferative activity. The present observations do not ascertain whether the oxidase is permanently active as a constant portion of the enzyme in vivo, similar to xanthine oxidase/dehydrogenase. However, DHOdehase should be considered as a source of radical oxygen species under pathophysiological conditions.     

The effect of rotenone on respiration in pea cotyledon mitochondria

Respiration utilizing NAD-linked substrates in mitochondria isolated from cotyledons of etiolated peas (Pisum sativum L. var. Homesteader) by sucrose density gradient centrifugation exhibited resistance to rotenone. The inhibited rate of α-ketoglutarate oxidation was equivalent to the recovered rate of malate oxidation. (The recovered rate is the rate following the transient inhibition by rotenone.) The inhibitory effect of rotenone on malate oxidation increased with increasing respiratory control ratios as the mitochondria developed. The cyanide-resistant and rotenone-resistant pathways followed different courses of development as cotyledons aged. The rotenone-resistant pathway transferred reducing equivalents to the cyanide-sensitive pathway. Malic enzyme was found to be inhibited competitively with respect to NAD by rotenone concentrations as low as 1.67 micromolar. In pea cotyledon mitochondria, rotenone was transformed into elliptone. This reduced its inhibitory effect on intact mitochondria. Malate dehydrogenase was not affected by rotenone or elliptone. However, elliptone inhibited malic enzyme to the same extent that rotenone did when NAD was the cofactor. The products of malate oxidation reflected the interaction between malic enzyme and malate dehydrogenase. Rotenone also inhibited the NADH dehydrogenase associated with malate dehydrogenase. Thus, rotenone seemed to exert its inhibitory effect on two enzymes of the electron transport chain of pea cotyledon mitochondria.     

Regulators of 3-hydroxybutyrate dehydrogenase activity in rat liver mitochondria

Effects of numerous organic acids on the 3-hydroxybutyrate dehydrogenase activity were studied in isolated rat liver mitochondria with nonspecific permeability. Amino acids, most of citric acid cycle intermediates, lactate, maleate, acetate, glycerol-3-phosphate, urea, palmitate, and phosphoenolpyruvate plus ADP were shown to modify the enzyme activity insignificantly. The inhibitory effect of pyruvate seems to be a result of the concomitant cytosolic lactate dehydrogenase activity, and the effect of oxaloacetate is that of the mitochondrial matrix malate dehydrogenase activity. Malonate proves to be a competitive inhibitor of the 3-hydroxybutyrate dehydrogenase activity, enzyme affinity for malonate being the same irrespective of the source or purification of the preparation.     

Fluorescence immunohistochemical localization of malate dehydrogenase isoenzymes in watermelon cotyledons : a developmental study of glyoxysomes and mitochondria

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination.     

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.     

Protein synthesis in mitochondria. 4. Preparation and properties of mitochondria from Krebs II mouse ascites-tumour cells

1. Methods of disrupting Krebs II mouse ascites-tumour cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. 2. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0·3 m-sucrose. 3. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succin-oxidase was measured in the various cell fractions after separation by differential centrifuging. 4. The mitochondrial fraction sedimented at 9500g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500g pellet contains other material besides mitochondria. 5. Krebs-cell mitochondria contain up to five times as much RNA as do liver mitochondria. 6. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.     

Demonstration of a heterogeneous distribution of glycolytic enzymes and of pyruvate kinase isoenzymes types M1 and M2 in unfertilized hen eggs

The intracellular distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and the pyruvate kinase isoenzymes type M1 and type M2 within unfertilized hen eggs was studied. Most of glycolytic enzyme activities were found in the yolk fraction; 8-24% of total glycolytic enzyme activities were found in the vitelline membrane fraction. However, the specific activities of these enzymes in the vitelline membrane fraction are 19-72-fold higher (U/mg protein) and 45-178-fold more concentrated (U/g wet weight) than in the yolk fraction. The study of intracellular localization of pyruvate kinase isoenzymes shows that the blastodisc, latebra and vitelline membrane contain only pyruvate kinase type M2, whereas pyruvate kinase types M1 and M2 are found in the egg yolk. The exclusive occurrence of pyruvate kinase type M2 in the blastodisc is consistent with the concept that this isoenzyme is involved in the cell proliferation. The heterogeneous distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and the heterogeneous localization of the pyruvate kinase isoenzymes types M1 and M2 indicate that glycolysis is distributed heterogeneously within the unfertilized hen egg cell.     

Characterization and localization of human type10 17beta-hydroxysteroid dehydrogenase.

The tissue distribution, subcellular localization, and metabolic functions of human 17beta-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase have been investigated. Human liver and gonads are abundant in this enzyme, but it is present in only negligible amounts in skeletal muscle. Its N-terminal sequence is a mitochondrial targeting sequence, but is not required for directing this protein to mitochondria. Immunocytochemical studies demonstrate that this protein, which has been referred to as ER-associated amyloid beta-binding protein (ERAB), is not detectable in the ER of normal tissues. We have established that protocols employed to investigate the subcellular distribution of ERAB yield ER fractions rich in mitochondria. Mitochondria-associated membrane fractions believed to be ER fractions were employed in ERAB/Abeta-binding alcohol dehydrogenase studies. The present studies establish that in normal tissues this protein is located in mitochondria. This feature distinguishes it from all known 17beta-hydroxysteroid dehydrogenases, and endows mitochondria with the capability of modulating intracellular levels of the active forms of sex steroids.