Tyrosine aminotransferase activity of frog (Rana esculenta) liver--II. Comparative aspects of intracellular distribution

1. A subcellular fractionation procedure for frog liver is reported and validated by the distribution pattern of several marker enzymes, also in comparison with rat liver. 2. The subcellular distribution of tyrosine aminotransferase was investigated in frog liver as compared to rat liver: a different distribution of the enzyme was observed, being the activity mostly recovered in mitochondrial and cytosolic compartments. 3. Results indicate that mitochondrial tyrosine aminotransferase of both frog and rat liver is a matrix enzyme, even if differences are observed concerning its release from the organelles upon detergent treatment.     

On the polydispersity of mitochondria in tissue homogenates and the determination of the average sedimentation coefficients of mixed populations of mitochondria

The sedimentation profile (sediterm) of subcellular particles in homogenous media depends on the average sedimentation coefficient ( value) and the size distribution. The present study has focused on the two common types of polydispersity, i.e., (i) a variable standard deviation in a normal (Gaussian) size distribution, and (ii) two populations of partieles with defined values and size distributions. Theoretical considerations and experimental data indicate that rat liver mitochondria have a normal size distribution, ( ) with much smaller standard deviation than previously assumed (σ = 0.118 μm) based on isokinetic gradient centrifugation and electron microscopy. Sedimentation of a mixture of rat liver and guinea pig ileal mitochondria having the values 17,040 S and 5640 S, respectively, gave the expected profile (sediterm) of two populations of particles. Their values were estimated to be identical to those obtained when the individual mitochondrial populations were sedimented. The ratio between the populations (based on the assay of marker enzyme) was found to be identical to the expected value.     

Immunological characterization of gamma-glutamyltransferases in human serum

The immunological properties of gamma-glutamyltransferases (gamma-GTs) from human serum, liver and tonsil were studied by using a monospecific antibody to human kidney gamma-GT for the purpose of elucidating their isozymic relationships. gamma-GTs partially purified from liver and tonsil were indistinguishable in this respect from kidney gamma-GT. gamma-GT in sera from patients with hepato-biliary diseases, on the other hand, was heterogeneous in molecular size as revealed by sucrose density gradient centrifugation and Sephadex G-150 gel filtration, and was inhibited and precipitated by the above antibody relatively poorly as compared with the kidney enzyme. When these sera were treated with bromelain, however, the molecular size of gamma-GT was reduced and the enzyme now reacted with the antibody as strongly as kidney gamma-GT. gamma-GT from bromelain-treated sera also exhibited a single immunoprecipitin line smoothly fusible with that from kidney gamma-GT; the enzyme-antibody complex still exhibited gamma-GT activity. The major form of gamma-GT partially purified from papain-treated sera, even though indistinguishable from kidney gamma-GT immunologically and in molecular size, exhibited a mobility on polyacrylamide gel electrophoresis which was higher than that of kidney gamma-GT but similar to that of liver gamma-GT. It is suggested that gamma-GT in human sera is heterogeneous in molecular size and electric charge but is composed of common peptide chains, probably identical to those of kidney gamma-GT.     

Subcellular distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase in bovine and murine adrenocortical tissue: species differences in the localization of activity and immunoreactivity

Key to the production of biologically active steroids is the enzyme 3 beta-hydroxysteroid dehydrogenase-isomerase. Some controversy has arisen concerning the subcellular distribution of this enzyme within steroidogenic cells. The distribution of 3 beta-hydroxysteroid dehydrogenase-isomerase was assessed in subcellular fractions obtained from homogenates of rat, bovine, and mouse adrenal glands in two ways. The activity of 3 beta-hydroxysteroid dehydrogenase-isomerase was quantitated by measuring the conversion of radiolabeled pregnenolone to radiolabeled progesterone in an aliquot of each of the fractions obtained. The presence of the enzyme was assessed by performing Western analyses on aliquots of each of the fractions obtained with the use of a specific polyclonal antiserum against 3 beta-hydroxysteroid dehydrogenase-isomerase, the characterization of which is described. In control experiments, the degree of contamination of the fractions was determined by assessing the presence of known subcellular fraction markers with Western analysis. In the bovine and mouse adrenal glands, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to be localized solely in the microsomal fraction, while in the rat, 3 beta-hydroxysteroid dehydrogenase-isomerase appears to have dual subcellular distribution: the microsomes and the inner mitochondrial membrane. We conclude that there is a species difference in the subcellular distribution of this important steroidogenic enzyme and that this species difference may be related to the steroidogenic pathway preferred in that species.     

Developmental studies on gamma-glutamyl transferase in rat intestinal mucosa.

The activity of gamma-GT in rat intestinal mucosa has been studied during normal development. No significant differences in enzymatic activity have been recorded between 21-day-old fetuses, neonates tested at 3, 10, 20 and 30 days and older animals (65 days). Only in the period immediately prior to birth did the liver gamma-GT display activity levels similar to those of intestinal gamma-GT. In neonates and in adult rats, the intestinal gamma-GT activity was much higher as compared to the enzymatic activity in the liver, possibly revealing a species feature in rats. The results of the studies show that the rat is particularly suitable for experimental studies on intestinal gamma-GT in pathological conditions.     

Optimal assay and subcellular location of phosphatidylglycerol synthesis in lung

Synthesis of phosphatidylglycerol from CDPdiacylglycerol and glycerol 3-phosphate by membranous subcellular fractions of rat lung and liver was optimal when assayed in the presence of bovine serum albumin and Triton X-100. Specific activities of glycerolphosphate phosphatidyltransferase in all membranous subcellular fractions of lung were several times higher than the corresponding fractions from liver. Distribution of this enzyme in subcellular fractions of lung or liver closely parallel the activity of the mitochondrial enzymes monoamine oxidase and succinate cytochrome c reductase. The phosphatidylglycerol-synthesizing activity in microsomes of both lung and liver was a minor fraction of total tissue activity and could be interpreted as due either to contamination with outer mitochondrial membrane or to a small amount of activity innate to microsomes. These results suggest that phosphatidylglycerol, which is believed to be a component of pulmonary surfactant, is synthesized by lung at a rapid rate relative to liver and that the subcellular distribution of its synthesis is similar in both tissues, with mitochondria as the major site.     

Demonstration of argininosuccinate synthetase activity associated with mitochondrial membrane: Characterization and hormonal regulation

Argininosuccinate synthetase (AS) is the third enzyme in ureogenesis, it catalyses the reaction of condensation of citrulline and aspartate into argininosuccinate. In the present report, we described the first characterization of AS within the outer membrane of rat liver mitochondria. Mitochondria-associated AS displayed the same kinetic characteristics as the cytoplasmic enzyme, but was found to be thermostable while cytoplasmic AS was not. The evolution of the co-location of AS was analyzed during ontogenesis. Total AS activity increased throughout rat fetal development. Simultaneously, the subcellular distribution of the enzyme has changed. AS activity was mainly mitochondrial in fetal and new-born liver liver and cytoplasmic in adult rat liver. The variation in subcellular distribution of AS may be due to the dramatic changes in hormonal levels that occur during this period. The role of corticosteroid and pancreatic hormones was studied. During fetal period, corticosteroid hormones induced an increase in mitochondria-associated AS activity. This was prevented by insulin. Glucagon did not modify total AS activity but reduced mitochondrial AS activity, meanwhile, a comparable increase in cytoplasmic AS activity was observed. One may hypothesize that glucagon may participate in the transfer of mitochondrial enzyme into the cytosol.     

The localization in rat liver of alkaline phosphodiesterase to a discrete organelle implicated in ligand internalization

The subcellular distribution of alkaline phosphodiesterase and NADH pyrophosphatase, two activities thought to be expressed by the same enzyme, was investigated. Although the two activities share a localization to a low-density vesicular membrane (equilibrium density = 1.12 g.cm-3), little NADH pyrophosphatase activity, in contrast to alkaline phosphodiesterase, was found in plasma membrane (equilibrium density = 1.18 g.cm-3), as reflected by the distribution of 5'nucleotidase. The binding and uptake of 125I-labelled insulin in perfused rat liver was also investigated. This ligand was found to bind to sinusoidal plasma membrane at 4 degrees C, but was rapidly internalized at 37 degrees C to the low-density membrane, which is rich in alkaline phosphodiesterase and NADH pyrophosphatase. These vesicular membranes were shown to belong to none of the enzymatically characterized subcellular bodies, and it is proposed that they represent discrete organelles participating in the subcellular processing of receptor-ligand complexes.     

Subcellular distribution and properties of acyl/alkyl dihydroxyacetone phosphate reductase in rodent livers

On subcellular fractionation, the enzyme acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) in guinea pig and rat liver was found to be present in both the light mitochondrial (L) and microsomal fractions. By using metrizamide density gradient centrifugation, it was shown that the alkyl DHAP reductase activity in the "L" fraction is localized mainly in peroxisomes. From the distribution of the marker enzymes it was calculated that about two-thirds of the liver reductase activity is in the peroxisomes and the rest in the microsomes. The properties of this enzyme in peroxisomes and microsomes are similar with respect to heat inactivation, pH optima, sensitivity to trypsin, and inhibition by NADP+ and acyl CoA. The enzyme activity in the peroxisomes and microsomes from mouse liver is increased to the same extent by chronically feeding the animals clofibrate, a hypolipidemic drug. The kinetic properties of this enzyme in these two different organelles are also similar. From these results it is concluded that the same enzyme is present in two different subcellular compartments of liver.     

Isolation of gamma-glutamyl transpeptidase from human primary hepatoma and comparison of its kinetic and catalytic properties with the enzyme from normal adult and fetal liver

gamma-Glutamyl transpeptidase (gamma-GT) from human primary hepatoma was solubilised and purified 290-fold with 25% recovery. The kinetic and catalytic properties were compared with those purified from human fetal and normal adult liver. Hepatoma gamma-GT did not differ from the fetal and adult liver gamma-GT in its pH optima for transpeptidation and auto-transfer reaction, heat stability, Km for the two substrates and inhibition by L-serine + borate. Enzyme from the three sources behaved in a similar manner towards various cations, sulphhydryl reagents, amino acid dipeptides. Adult liver enzyme showed a 4 time higher Ki value for anthglutin than hepatoma and fetal liver. The hepatoma gamma-GT could not be differentiated from that of adult and fetal liver by concanavalin-A Sepharose 4B column chromatography. The tissue concentration of gamma-GT was 3 to 13 times higher in hepatoma and fetal liver than in adult liver.     

Fluorometric assay for rat liver peroxisomal fatty acyl-coenzyme A oxidase activity

These studies report the development of a simple, specific, and highly sensitive fluorometric assay for rat liver peroxisomal fatty acyl-CoA oxidase activity. In this in vitro procedure fatty acyl-CoA-dependent H2O2 production was coupled in a peroxidase-catalyzed reaction to the oxidation of scopoletin (6-methoxy-7-hydroxycoumarin), a highly fluorescent compound, to a nonfluorescent product. Enzyme-catalyzed reaction rates as low as 5 pmol of H2O2 produced per minute could readily be detected. The reaction was studied in liver homogenates from normal rats with respect to absolute activity, time course, protein concentration dependence, substrate concentration dependence, pH optimum, substrate specificity, and cofactor requirements. The properties of the enzyme activity as assessed by the fluorometric assay agree well with those determined by other investigators using other assay methods. After subcellular fractionation of liver homogenates by differential centrifugation, the fatty acyl-CoA oxidase activity distributed like known peroxisomal marker enzymes. These results demonstrate that the fluorometric assay of fatty acyl-CoA oxidase should be useful in studying the distribution, properties, and subcellular localization of the enzyme, particularly in enzyme sources of low activity or in situations when only small amounts of material are available.     

Modulation of the activity of hepatic gamma-glutamyl transpeptidase, adenosine triphosphatase, placental glutathione S-transferase and adenylate cyclase by acute administration of lead nitrate

The effect of a single administration of lead nitrate on the activity of gamma-glutamyltranspeptidase (gamma-GT), adenosine triphosphatase (ATPase), the placental form of glutathione S-transferase (GST-P) and adenylate cyclase (AC), four enzymes widely used as phenotypic markers for preneoplasia, was investigated in the liver of male Wistar rats. The results of the histochemical enzymatic staining indicated that an acute treatment with lead nitrate induces the activity of gamma-GT, mainly in the hepatocytes located around zone I of the liver acinus, with a maximum seen between 72-96 hours. On the other hand, the activity of ATPase was found to be severely inhibited at 2-3 days after treatment, as shown by a strong decrease in the staining of the bile canaliculi of zones II and III. Immunohistochemical analysis revealed that lead nitrate administration also resulted in the appearance in most of the hepatocytes of GST-P, an enzyme whose activity is almost undetectable in normal rat liver, but is elevated in preneoplastic liver lesions. Finally, lead nitrate treatment resulted in an inhibition of AC activity which was maximal after 24 hours.     

Partial purification, properties, and subcellulsr distribution of rat liver phosphatidate phosphatase.

Phosphatidate phosphatase (EC 3.1.3.4Y was purified 15- to 20-fold from the soluble fraction of rat liver. The purification procedure involved calcium phosphate gel adsorption and elution, ammonium sulfact precipitation, and molecular-sieve chromatography. For the enzyme assay, and aqueous dispersion of phosphatidate, rather than "membrane-bound" phosphatidate, was used as substrate. The partially purified enzyme depends almost entirely on the presence of Mg2+ for its activity. Morover, the activity of the enzyme is stimulated by phosphatidylcholine. The enzyme exhibits a high substrate specificity for phosphatidate. The apparent Km for phosphatidate is approximately 0.05 mM. The optimum pH is between 7.4 and 7.6. The enzyme is inhibited by fluoride and by p-chloromercuribenzoate. The subcellular distribution of phosphatidate phosphatase in rat liver was studied by assaying the activity of the enzyme in the presence of Mg2+ and phosphatidylcholine. In contrast ot the results of previous studies, most of the enzyme activity was found in the soluble fraction.     

Thyroxine 5'-monodeiodinase activity in regenerating liver of triiodothyronine-treated rats

The effect of triiodothyronine (T3) on hepatic thyroxine (T4) 5'-monodeiodinase and the subcellular localization of the enzyme were examined in regenerating rat liver, because it seemed likely that the effect of T3 might be accentuated during liver regeneration. Five days after T3 treatment, the specific activity of the monodeiodinase in the microsomal fraction (105,000 X g pellet) of regenerating liver was increased to 207% of the control value. Lineweaver-Burk plots showed that the Vmax for T4 5'-monodeiodination was about 3 times greater in T3-treated rats than in controls, but that there was no difference between the two groups in the apparent Km value for T4. About 55% of the total enzyme activity was found in the endoplasmic reticulum (ER) of the liver of both controls and T3-treated rats. The subcellular distribution of the enzyme was similar to that of NADPH-cytochrome c reductase (NADPH-cyt c reductase), a marker of the ER, but different from that of Na+,K+-ATPase, a marker of plasma membranes (PM).     

Characterization of tyrosylprotein sulfotransferase from rat liver and other tissues

The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active peptides and proteins. In this paper we describe the characterization of a rat liver tyrosylprotein sulfotransferase that is capable of catalyzing the transfer of a sulfate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the synthetic polymer, poly-(Glu6,Ala3,Tyr1) (EAY; Mr 47,000) using a simple filter paper assay. Following sucrose density gradient centrifugation and comparison with known subcellular marker enzyme activities, rat liver tyrosylprotein sulfotransferase activity was shown to have a distribution similar to the Golgi enzyme, galactosyltransferase. Using the enriched Golgi preparation, rat liver tyrosylprotein sulfotransferase displayed a pH optimum of 6.7 and required the presence of 20 mM Mn2+ for maximal activity. Co2+ (20 mM) was able to produce 26% of the maximal stimulation observed with Mn2+, whereas other metal ions, such as Mg2+, Ca2+, and Co2+, were not effective in stimulating tyrosylprotein sulfotransferase activity. Whereas tyrosylprotein sulfotransferase activity was observed in the native membrane-bound state, EAY sulfation was maximally enhanced 3-fold when assayed in the presence of Lubrol Px. Under the optimal conditions for assaying the sulfation of EAY by a rat liver enriched Golgi fraction, significant degradation of the sulfate donor, PAPS, was observed. The addition of both NaF and 5'-AMP to the incubation mixture was found to effectively prevent PAPS degradation and increase the amount of product formed in the assay by 10-fold. Using the optimized conditions for the sulfation of EAY by rat liver tyrosylprotein sulfotransferase, membrane-bound sulfotransferase activity was also observed in the crude microsomal pellets of a variety of rat tissues, including lung, pituitary, and cerebellum, as well as in livers from different species.     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.     

Gamma-glutamyl transpeptidase, glutathione, and L-glutamic acid in the rat epididymis during postnatal development

During postnatal development, gamma-glutamyl transpeptidase (gamma-GT), reduced glutathione (GSH), and L-glutamic acid (L-Glu) were assayed in the epididymides of rats at 5-day intervals between 10 and 60 days of age and compared to adult levels. gamma-GT activity (with gamma-glutamyl-p-nitroanilide as substrate) and L-Glu (nicotinamide adenine dinucleotide conversion-dependent assay) were measured photometrically, while GSH (o-phthalaldehyde reaction) was quantified with a fluorometric assay. In immature rats, the epididymal gamma-GT was very low but increased after 25 days of age in the caput and after 50 days of age in the cauda. The enzyme level in the epididymal caput was by far the highest in the adult rat reproductive tissues. The postnatal increase of gamma-GT in epididymal caput and cauda was associated with a decline of its substrate GSH and an accumulation of the product L-Glu. These observations provide evidence for the in vivo hydrolytic activity of gamma-GT and explain the high levels of L-Glu found in the epididymis of rats and other mammals.     

Intracellular distribution of fumarase in various animals

The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.     

Subcellular localization and enzymatic properties of rat liver phosphatidylinositol-4-phosphate kinase

The phosphatidylinositol-4-phosphate kinase activity in rat liver showed a subcellular distribution different from that of phosphatidylinositol kinase. It was preferentially associated with plasma membrane-rich subcellular fractions, while no or minimal activity could be ascribed to mitochondria, lysosomes, Golgi membranes or the endoplasmic reticulum. The plasma membrane enzyme phosphorylated endogenous and exogenously added phosphatidylinositol 4-phosphate at comparable initial rates. The phosphorylation of endogenous substrate was strongly inhibited by Triton X-100, while the phosphorylation of added substrate was enhanced, suggesting that endogenous phosphatidylinositol 4-phosphate was readily available to the enzyme in unperturbed plasma membranes. The total activity of phosphatidylinositol-4-phosphate kinase in rat liver was only 1/20 that of phosphatidylinositol kinase. The enzyme activity showed an unusually broad pH-optimum in the neutral range. Mg2+ was the preferred divalent cation and Km towards ATP was about 3-fold higher than the corresponding value for phosphatidylinositol kinase.