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1.
Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.  相似文献   

2.
Previous studies in our laboratory have shown that rat heart glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) separates into two forms upon ion-exchange chromatography. Both forms could be shown to have the same subunit Mr and to incorporate one molecule of phosphate per subunit. The studies reported here were done to check whether both forms are native isoenzymes and, further, which form might represent the heart-specific phosphorylase. Firstly, the iso-electric points of the purified enzymes are compared with those associated with phosphorylase activity in crude extracts from rat heart. Two out of four major bands coincided with the bands of purified phosphorylase Ib and IIb (isoelectric points: 5.5 and 6.25), indicating apparent identity. Secondly, antibodies to rat skeletal muscle phosphorylase reacted with rat heart phosphorylase I, whereas phosphorylase II was neither inhibited nor precipitated by the antibody. Thirdly, peptide maps obtained after proteolytic digestion of SDS-denatured phosphorylase I and II showed different patterns. In addition to the kinetic differences between these two forms reported earlier, phosphorylase IIa was inhibited by glucose 6-phosphate, whereas phosphorylase Ia was not. These results suggest that phosphorylase II is a heart-specific isoenzyme which is presumably encoded by a different gene.  相似文献   

3.
Bovine liver mitochondria contain about 10% of the total glyoxalase II activity in the homogenate. Electrophoresis and isoelectric focussing of either crude mitochondrial extract or the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms (pl 6.3, 6.7, 7.1, 7.7, and 7.9). Since bovine liver cytosol contains a single form of glyoxalase II (pl 7.5), at least four forms are exclusively mitochondrial with no counterpart in the cytosol. The relative molecular mass of mitochondrial glyoxalase II is about 23-24 kDa, similar to the cytosolic form. The kinetic constants obtained using S-D-lactoyl, S-acetyl-, S-acetoacetyl-, and S-succinyl-glutathione as substrates are similar to those reported for glyoxalase II from rat liver mitochondria. S-D-Lactoyl- and S-acetoacetyl-glutathione are the best substrates. S-Acetylglutathione is the poorest substrate with respect to both Vmax and Km values.  相似文献   

4.
1. The presence of beta-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of beta-acetylglucosaminase (EC 3.2.1.30) and of beta-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the beta-acetylglucosaminase 35 times and for the beta-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated.  相似文献   

5.
Aldehyde oxidase (AO) and xanthine oxidase (XO) are cytosolic enzymes that have been involved in some pathological conditions and play an important role in the biotransformation of drugs and xenobiotics. The increasing interest in these enzymes demands for a simple and rapid procedure for their purification. This paper describes for the first time a method that allows simultaneous purification of both enzymes from the same batch of rat livers. It involves few steps, is reproducible and offers high enzyme yields with high specific activities. The rat liver homogenate was fractionated by heat denaturation and by ammonium sulphate precipitation to give a crude extract containing both enzymes. This extract was chromatographed on an Hydroxyapatite column that completely separated AO from XO. Further purification of XO by anion exchange chromatography on a Q-Sepharose Fast Flow column resulted in a highly purified (1200-fold) preparation, with a specific activity of 3.64 U/mg and with a 20% yield. AO was purified about 1000-fold at a yield of 15%, with a specific activity of 3.48 U/mg, by affinity chromatography on Benzamidine-Sepharose 6B. The purified enzymes gave single bands of approximately 300 kDa on a polyacrylamide gel gradient electrophoresis and displayed the characteristic absorption spectra of highly purified enzymes.  相似文献   

6.
Bovine heart mitochondrial matrix contains two proteins possessing the oxaloacetate keto-enol tautomerase (EC 5.3.2.2) activity. A procedure for the isolation and purification of the enzymes to an electrophoretically homogeneous state has been developed. The purified proteins have molecular masses of 37 kD and 80 kD and catalyze the keto-enol oxaloacetate tautomerization reaction with the turnover numbers of approximately 3000 and approximately 2000 min-1. The both enzymes were found to differ significantly in all their physicochemical and kinetic properties. Fractionation of rat liver mitochondria revealed that the oxaloacetate keto-enol tautomerase activity is predominantly localized in the mitochondrial matrix. The essential role of oxaloacetate keto-enol tautomerase in the operation of the Krebs cycle is discussed.  相似文献   

7.
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.  相似文献   

8.
An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones, D. H. and Matus, A. I. (1974) Biochim. Biophys. Acta 356, 276-287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organelles. Fraction 2 was recovered from the 28.5-34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na+ +K+)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5'-nucleotidase (EC 3.1.3.5) were, respectively, 4.5, 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.  相似文献   

9.
Glycerate kinase (ATP : D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of gluconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serine. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase.  相似文献   

10.
Glycerate kinase (ATP: D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of glyconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serin. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase.  相似文献   

11.
Abstract: NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3) were isolated from various subcellular organelles as well as from different regions of rat brain. The mitochondrial, microsomal, and cytosolic fractions were found to contain 40%, 28%, and 12%, respectively, of the total aldehyde dehydrogenase (5.28 ± 0.44 nmol NADH/min/g tissue) found in rat brain homogenate when assayed with 70 μ. M propionaldehyde at pH 7.5. The total activity increased to 17.3 ± 2.7 nmol NADH/min/g tissue when assayed with 5 m M propionaldehyde. Under these conditions the three organelles contained 49%, 23%, and 9%, respectively, of the activity. The enzyme isolated from cytosol possessed the lowest K m. The molecular weight of the enzyme isolated from all three subcellular organelles was ∼100,000. Four activity bands were found by electrophoresis of crude homogenates, isolated mitochondria, or microsomes on cellulose acetate strips. Cytosol possessed just two of the forms. The total activity was essentially the same in homogenates obtained from cortex, subcortex, pons-medulla, or cerebellum. Further, the enzyme had the same molecular distribution and total activity in each of these four brain regions. Disulfiram was found to be an in vivo and in vitro inhibitor of the enzymes obtained from these brain regions. Mercaptoethanol, required for the stability of the enzyme, reversed the inhibition produced by disulfiram. The effect was greater for enzyme isolated from cytosol than from mitochondria. Calculations led to the prediction that aldehydes such as acetaldehyde are oxidized in cytosol.  相似文献   

12.
The very high affinity for GTP of glutamate dehydrogenase was used to purify this enzyme by affinity chromatography. After periodic acid oxidation, GTP was covalently bound to an activated Sepharose. When crude mitochondrial extracts were applied on a column of this GTP-Sepharose, glutamate dehydrogenase was retained with very few other proteins. Glutamate dehydrogenase from rat liver was eluted with a KCl gradient with only one contaminating protein. From a pig heart mitochondrial extract the enzyme was purified 300-fold in one step. A chromatography on hydroxyapatite was sufficient to achieve the purification. This very simple technique avoids the long and troublesome crystallization steps generally involved in glutamate dehydrogenase purification.  相似文献   

13.
Glutamine synthetase isozymes in elasmobranch brain and liver tissues   总被引:1,自引:0,他引:1  
Glutamine synthetase is present as isozymic forms in the elasmobranchs Squalus acanthias (dogfish shark) and Dasyatis sabina (stingray). Subcellular fractionation of elasmobranch brain and liver tissue shows the enzyme to be predominantly cytosolic in the former tissue and mitochondrial in the latter. For the cytosolic brain enzyme, the subunit Mr equals 42,000 in the stingray and 45,000 in the shark, as determined by sodium dodecyl sulfate-gel electrophoresis/Western blotting. The subunit Mr = 45,000 and 47,000, respectively, for stingray and dogfish mitochondrial liver enzymes. Translation of total brain RNA from both species gives immunoprecipitable nascent peptides of the same size as their respective mature enzymes. However, in liver tissue, translation of glutamine synthetase mRNA yields peptides of higher Mr than that of the mature enzymes. In dogfish liver, Mr = 50,000 for the translation product and, in stingray liver, Mr = 48,000. This suggests that the translocation of the enzyme into liver mitochondria may be via a signal or leader sequence mechanism. The larger liver isozyme of elasmobranch glutamine synthetase is found in kidney where it is also known to be mitochondrial. The smaller cytosolic isozyme occurs in retina, heart, gill, and rectal gland tissue as well as in brain.  相似文献   

14.
Induction of the activity of aconitate hydratase (AH) was observed in rat hepatocytes under the conditions of food deprivation. The increase in AH activity after 4 days of starvation in the studied tissues was from 0.57 to 2.05 U/g crude liver weight. The induction of aconitase was associated both with the cytoplasmic and mitochondrial AH isoforms. The activities of cytosolic and mitochondrial AH isoforms in starving animals consisted of 83 and 17% of the total activity, respectively. The cytoplasmic and mitochondrial isoforms of the enzyme with specific activities 11.1 and 6.13 U/mg protein, respectively, were obtained by a five-step purification procedure that included fractionation with ammonium sulfate, ion-exchanging chromatography on DEAE-Toyopearl and gel filtration. The purified preparations of these AH isoforms were electrophoretically homogenous. The molecular weights of these isoforms were estimated and several kinetic and regulatory properties were studied.  相似文献   

15.
The purification of human placenta and rat liver protein disulphide-isomerase (PDI, EC 5.3.4.1) and the production of a panel of monoclonal antibodies against these proteins are described. The physical and enzymic properties of human PDI and rat PDI were similar; immunological characterization revealed the presence of unique, as well as shared, antigenic determinants. Although purified rat liver PDI was present as three forms differing slightly in Mr value, evidence was presented that the multiple forms represent proteolytic degradation products of a single 59,000-Mr species. Purified human PDI had an apparent Mr of 61,200. Two of the monoclonal antibodies against human PDI partially inactivated the enzyme, and one of these in indirect immunoprecipitation led to the precipitation of all glutathione:insulin transhydrogenase activity from a crude extract of human placenta. Results of immunofluorescence experiments with HT-29 human colon carcinoma cells were consistent with localization of PDI in the nuclear membrane and cell cytoplasm.  相似文献   

16.
The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD(+)-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD(+)-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously "dribbled" off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5'-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD(+)-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (l-LDH, EC 1.1.1.27).  相似文献   

17.
The efflux of [14C]citrate from rat heart mitochondria was significantly greater with L-malate as the extramitochondrial substrate as compared with [12C]citrate, isocitrate or phosphoenolpyruvate. The concentration of L-malate required for half-maximal rate of efflux of citrate was 0.45 mM and the maximum velocity was 0.36 nmol min-1 mg-1 mitochondrial protein at 23 degrees C. This citrate transporter was inhibited by 1,2,3-benzenetricarboxylate and palmitoyl-CoA but not to the same extent as these compounds inhibit the tricarboxylate carrier in rat liver mitochondria. The apparent inability of these mitochondria to transport citrate in the inward direction necessitates the presence of a cytosolic citrate removal pathway. We propose that the enzymes of this pathway in rat heart could be ATP citrate (pro-3S)-lyase (EC 4.1.3.a) and carnitine acetyltransferase (EC 2.3.1.7), both of which we demonstrate to have adequate activity in both the fed and fasted state. An hypothesis has been put forward to account for the inhibition of rat heart phosphofructokinase by citrate in the fasted state incorporating these properties of the citrate transporter and ATP citrate (pro-3S)-lyase.  相似文献   

18.
An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones. D. H. and Matus. A. I. (1974) Biochim. Biophys. Acta 356, 276–287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organcelles. Fraction 2 was recovered from the 28.5–34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na++K+)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5′-nucleotidase (EC 3.1.3.5) were, respectively, 4.5. 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.  相似文献   

19.
The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD+-dependent dehydrogenases on their complementary immobilized cofactor derivative. Application of this strategy to the purification of NAD+-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100%. However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously “dribbled” off the matrix during the chromatographic procedure. The fact that this problem can be overcome by including a short pulse of 5′-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report. The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract. The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD+-dependent dehydrogenases when using the locking-on strategy. These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart -lactate dehydrogenase ( -LDH, EC 1.1.1.27).  相似文献   

20.
The primary structure of the mitochondrial form of horse liver aldehyde dehydrogenase has been determined, utilizing peptide analyses and homology with other enzyme forms. The subunit exhibits N-terminal heterogeneity in size similar to that for the corresponding human mitochondrial protein, the longest form having 500 residues. Catalase was identified as a contaminant of the preparations. All four pairs within a set of aldehyde dehydrogenases can now be compared, including the same two species variants (horse and human) for both the cytosolic and mitochondrial enzyme, revealing characteristic differences although Cys-302 and other segments of presumed functional importance are unchanged. The cytosolic and mitochondrial enzymes are clearly different (172 exchanges in the horse pair; 160 exchanges in the human pair) and the mitochondrial forms are more conserved (28 exchanges of 500 residues) than the cytosolic ones (43 exchanges). Distributions of the residue substitutions also differ between the two enzyme types. These results suggest a comparatively distant separation of the cytosolic and mitochondrial enzymes into forms with separate functional constraints that are more strict on the mitochondrial than the cytosolic enzyme. Unexpectedly, positions with residues unique to one of the four enzymes are about twice as common in both of the horse proteins than in either of the human proteins. This difference may reflect a general pattern for human/non-human proteins, showing that not only functional properties of the protein, but also other factors, such as generation time (longer in man than in horse), are important for enzyme divergence.  相似文献   

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