Large Granule Fraction Research Articles

Functional Analysis of Tyrosinase Isozymes of Cultured Malignant Melanoma Cells During the Recovery Period Following Interrupted Melanogenesis Induced by Glycosylation Inhibitors

Multiple forms of tyrosinase, T1, T2, T3, have been shown to differ with respect to carbohydrate moieties of these isozymes. We demonstrated that, in cultured B-16 melanoma cells, melanization can be completely interrupted by glycosylation inhibitors, such as glucosamine and tunicamycin, and that these inhibitors cause a selective loss of membrane-bound T3. It is further found that inhibition of melanization induced by glucosamine occurs even in the presence of protease inhibitors, such as phenylmethylsulfonyl fluoride and leupeptin, and that melanization inhibition is reversible upon removal of the inhibitor. In this report we have also examined the process of development and recovery of the tyrosinase isozymes in cells in which the interruption of melanogenesis has been released by the removal of these glycosylation inhibitors. The recovery process, which occurs during the period after interrupted melanogenesis and is a process of remelanization, has been biochemically followed. Tyrosinases obtained from the deoxycholate-solubilized large-granule fraction of these melanoma cells have been analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immediately after removal (0 h recovery) of the glycosylation inhibitor, loss of melanization and T3 is accompanied by T1 heterogeneity which is visualized as two electrophoretically distinct species, T1' and T1''. At this time, T1' and T1'' do not have a concanavalin A affinitive carbohydrate moiety but do possess in vitro dopa reactivity. When recovery of melanization begins visibly 24 h later, T3 is re-formed with disappearance of T1 heterogeneity. By 48 h, the previous normal level of melanization is almost attained. These results suggest that maturation of tyrosinase may occur via T1' and T1'' as precursors of T3, or possibly T1 through the addition of N-glycosydically linked oligosaccharide moieties which can be interrupted by glucosamine and tunicamycin.

Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi

The degradation of glucose by Trypanosoma cruzi leads to the excretion of succinate. Malate dehydrogenase (MDH) participates in this process by reducing to malate the oxaloacetate synthesized by the glycosomal enzyme, phosphoenolpyruvate carboxykinase. The best coupling for these two sequential reactions would be attained if both enzymes were placed in the same subcellular compartment. The intracellular distribution of the MDH activity in epimastigotes of T. cruzi was studied by two methods. Selective disruption of cellular membranes with increasing concentrations of digitonin, indicated that trypanosomal MDH is particulate. Isopycnic centrifugation in a sucrose gradient of a large granule fraction, obtained by grinding the cells with silicon carbide, showed the presence of two MDH activities: one banding together with the glycosomal marker phosphoenolpyruvate carboxykinase, the other with the mitochondrial marker citrate synthase. Isoelectrofocusing of cell-free extracts led to the separation of two enzyme forms, with p I values of about 3.5 (MDH a) and 9.4 (MDH b). These forms had similar molecular weights (approx. 60 000) and apparent K m values, but showed a small but consistent difference in their pH optima (9.23 for MDH a and 9.05 for MDH b), and in their activation by inorganic phosphate (apparent K a values of 33 mM and 87 mM, for MDH a and MDH b, respectively). Determination of the pH optima of the enzyme forms separated by isopycnic centrifugation suggests that the glycosomal enzyme form is MDH a, and the mitochondrial one is MDH b.

Enkephalins are associated with adrenergic granules in bovine adrenal medulla

The subcellular localization of enkephalins was studied in the bovine adrenal medulla. In the adrenal medulla enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg 6-Phe 7 and Met-enkephalin-Arg 6-Gly 7-Leu 8) are found free and in the form of cryptic peptides included in larger precursors. Total Met-enkephalin immunoreactivity, which includes free and cryptic peptides, was determined after a sequential enzymatic treatment with trypsin and carboxypeptidase B. Total Met-enkephalin immunoreactivity, dopamine beta-hydroxylase and catecholamines were found to have a parallel distribution in the various subcellular fractions. The bulk of the total Met-enkephalin immu noreactivity (42%) was recovered in the large granule fraction. The large granule fraction also contained 38% of the total dopamine beta-hydroxylase activity, and 42% of the total catecholamines. Enkephalins are thus concentrated in the chromaffin granules. Chromaffin granules were also separated according to the method of Terland & coworkers 17 into two fractions: one containing the dense noradrenergic vesicles and the other containing lighter adrenergic vesicles. Total Met-enkephalin immunoreactivity was restricted to the fractions containing the lighter adrenergic vesicles. In these fractions the molar ratio of adrenaline to total Met-enkephalin immu noreactivity was 97. This study is in accord with immunocytochemical observations which have indicated that enkephalins are located in adrenergic and not in the noradrenergic cells in the bovine adrenal medulla.

γ-Glutamyl Transpeptidase, Tyrosinase, and 5-S-Cysteinyldopa Production in Melanoma Cells

The distribution of gamma-glutamyl transpeptidase (gamma-GTP), tyrosinase, and 5-S-cysteinyldopa (5-S-CD) within melanoma cells has been studied in vitro as well as in vivo. Sodium periodate treatment of intact B-16 melanoma cells has been found to inhibit gamma-GTP present as an ectoenzyme. However, these periodate-treated cells in the presence of 10(-5) M dopa and glutathione have been found to continue to secrete large quantities of 5-S-CD in their medium. The large-granule fraction of Greene's melanotic melanoma contains substantial amounts of both tyrosinase and gamma-GTP. However, further separation of the large-granule fraction into sub-fractions indicates that tyrosinase and gamma-GTP seem to co-exist with premelanosome. It is suggested that glutathione-dependent t-S-CD genesis proceeds within premelanosomes through the formation of glutathione-dopa. The excess of glutathione-dopa and 5-S-CD, unutilized for pheomelanin formation overglow into the cytoplasm.

Cytoplasmic localization of the transforming protein of Fujinami sarcoma virus: salt-sensitive association with subcellular components.

Fujinami sarcoma virus (FSV) encodes a transforming protein of 130,000 daltons (P130) which is associated with a tyrosine-specific protein kinase activity. To elucidate mechanisms involved in cell transformation by FSV, we have studied the intracellular location of P130 in rat cells nonproductively infected with FSV. Immunofluorescent staining of several FSV-transformed rat cell lines with a tumor regressor antiserum specific against the fps sequences of P130 showed that the major staining was localized in the cytoplasm. Staining was also seen in cell ruffles and in some cases at areas of cell contact. The cytoplasmic location of P130 staining in cells infected with temperature-sensitive mutants of FSV was unchanged when they were grown at permissive or nonpermissive temperature. Cell fractionation of FSV-transformed cells under various conditions showed that the ionic strength used during cell fractionation had a striking effect on the distribution of P130. At 10 mM NaCl, 70% of P130 sedimented in the large granule fraction, whereas at 500 mM NaCl 70 to 90% of P130 was recovered in the cytosol fraction. Furthermore, a combination of ionic and nonionic detergents that effectively solubilized subcellular membranes was insufficient to solubilize P130 unless the salt concentration was raised. We conclude that the majority of P130 and its associated protein kinase activity are localized in the cytoplasm and that P130 is not an integral membrane protein.

Carboxypeptidase B activity from adrenal medulla — Is it involved in the processing of proenkephalin?

Lysed dialyzed bovine adrenal medulla chromaffin granules were used to study proenkephalin processing enzymes. With dynorphin (1–13) as substrate, carboxypeptidase-B activity was found although no trypsin-like activity was detectible. The granule preparation was capable of cleaving C-terminal amino acids from hippuryl-L-arginine and leu-enkephalin-arginine. Subcellular localization studies involving discontinuous sucrose gradient centrifugation of the large granule fraction showed that the leu-enkephalin-arginine cleaving activity coincided in the gradient with that of the lysosomal marker, cathepsin D and a minor peak of leu-enkephalin and was separate from the catecholamine and major enkephalin peaks.

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid.

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.

Trypanosoma brucei: Preparation and some properties of a multienzyme complex catalysing part of the glycolytic pathway

After grinding Trypanosoma brucei with alumina or silicon carbide, it is possible to prepare a multienzyme complex which catalyses the breakdown of glucose to l-glycerol-3-phosphate and 3-phosphoglycerate. The complex sediments with the postnuclear large granule fraction which pellets at 14,500g; it is also eluted in the void volume during Bio-Gel A-5m column chromatography of a cell homogenate. During isopycnic sucrose gradient centrifugation, the multienzyme complex bands at a density of 1.22 g/ml. Because this is the density of T. brucei microbodies, and because Triton X-100 treatment of the material greatly enhances the activities of its component enzymes, we conclude that the multienzyme complex is probably located in the microbodies of the bloodstream long slender forms of T. brucei.

Dissociation of vasoconstrictor and platelet aggregatory activities of thromboxane by carbocyclic thromboxane A2, a stable analog of thromboxane A2.

Carbocyclic thromboxane A2 [2 beta (Z),3 alpha- (1E,3R*)-3-(3-hydroxy(1-octenyl)-bicyclo[3.1.1]hept-2-yl-5-heptenoic acid], a stable analog of thromboxane A2, has been tested for its physiologic properties. Carbocyclic thromboxane A2 is a potent coronary vasoconstrictor, stimulating cornonary vascular smooth muscle at concentrations as low as 29 pM. At 1-5 micro M it is also an inhibitor of arachidonic-acid- and endoperoxide-induced aggregation of platelets. At 200 nM it stimulated the release of lysosomal hydrolases from large granule fractions of liver homogenate. It inhibited thromboxane synthesis in platelets, although it did not inhibit synthesis of prostacyclin in ram seminal vesicles. Thus, carbocyclic thromboxane A2, a molecule closely related to thromboxane A2, separates coronary vasoconstrictor from platelet-aggregating activity. The constrictor activity predominates in vivo; carbocyclic thromboxane A2 induces coronary vasoconstriction leading to myocardial ischemia and sudden death in rabbits in the absence of pulmonary or coronary thrombosis.

The intracellular inactivation of catalase—I. Subcellular localization and inhibition in mouse liver

1. 1. The degradation of tritiated catalase. which had been purified from mouse liver, was studied under in vitro conditions by incubating the enzyme with subcellular fractions from the same tissue. 2. 2. The degradative process was monitored by following both the loss of catalase enzymatic activity and the formation of soluble radioactivity. 3. 3. Only the large granule fraction caused appreciable degradation of catalase at neutral pH and 37°C, but under conditions of lower pH the degradative rate due to this fraction increased markedly. 4. 4. Additionally, it was noticed that the cytosol fraction of liver slowed the rate of heat inactivation of the purified enzyme, and that a similar inhibition of inactivation by cytosol also extended to the tryptic inactivation of the enzyme. 5. 5. These results have been discussed in relation to the established characteristics of the degradation of mouse liver catalase in vivo, and possible means of regulation of this degradation

Intracellular distribution of dopa and 5-S-cysteinyldopa in pigment cells with minimal pigment formation

The hypothesis that only melanosomal catecholic amino acids contribute to melanin formation was tested by studying adult bovine eyes in which pigment synthesis is considered to be low or absent. Dopa and 5-S-cysteinyldopa were investigated in different cell fractions of the choroid and retinal pigment epithelium of cattle. Most of the dopa and 5-S-cysteinyldopa was found in the cytoplasm and very little in the large granule fraction. The presence of cysteinyldopa in the adult eye is evidence of tyrosinase activity, but the catechol amino acids in the cytoplasm probably do not give rise to melanin formation. It is assumed that they instead are excreted from the cells.

Effect of triton WR-1339 on peroxisomal enzymes of rat liver

The effect of Triton WR-1339 on peroxisomal enzymes of rat liver was studied. The dose vs. response relationships of peroxisomal enzyme activities to Triton WR-1339 were first examined 3.5 days after injection. Catalase activity was reduced to 50% of that of the control at a dose of 200 mg per 100 g body weight; it was found that the decrease depended on the dose of this compound. Urate oxidase activity was not significantly affected. D-Amino acid oxidase activity showed intermediate behavior. The activities of these enzymes were found to be reduced more markedly at 2 days than at 3.5 days after injection, and subsequently the levels of the activities recovered. At 2 days after injection of a dose of 200 mg per 100 g body weight, the activities of catalase, D-amino acid oxidase and urate oxidase had decreased to 40, 60 and 60%,respectively, of the control values. It was found that the decreases in the activities of these enzymes caused by Triton WR-1339 had occurred in the large granule fraction, but not in the cytoplasm. Measurement of the specific activity, Ouchterlony gel diffusion and quantitative immunoprecipitation suggested that there was a similarity between the Triton WR-1339-treated and untreated rats in the nature of purified catalases. These results suggest that Triton WR-1339 depresses the activities of liver peroxisomal enzymes, especially the catalase activity.

On the polydispersity of bovine adrenal chromaffin granules Analytical differential centrifugation in isotonic homogeneous medium

The polydispersity of chromaffin granules of bovine adrenal medulla homogenates has been analyzed by analytical differential centrifugation in homogeneous media. Adrenaline and noradrenaline were determined by automated ion-exchange column chromatographic preseparation using ninhydrin as the subsequent detection reagent. 1. 1. Two populations of storage granules were detected in the large granule fraction, containing about 95% of the catecholamines in the homogenate, by sedimentation in a medium containing 0.25 M sucrose whether adrenaline or noradrenaline was used as the specific marker. The average sedimentation coefficents at 4°C ( s 4, B ) were estimated to s H = 12 440 ± 1850 S and s L = 3910 ± 280 S for the heavy and light population, respectively. The relative proportion of the two populations was estimated to be 65 ± 9% ( s L and 35 ± 8% ( s H ) . 2. 2. Several lines of the chromaffin granules in both types of adrenal medullary cells, i.e., their degree of maturation.

The Biosynthesis of Rat-Liver Cytochrome c. 1. Subcellular Distribution of Cytochrome c

A radioimmunoassay for cytochrome c is described. It has been applied successfully to crude subcellular preparations with a very low content in cytochrome c. Quantitative fractionation of rat liver homogenates by differential centrifugation has shown that, on the basis of the distribution patterns of cytochrome oxidase and cytochrome c, the overwhelming bulk of the cytochrome c recovered in the nuclear and large granule fractions may be assigned to mitochondria. In contrast, the cytochrome c of the microsomal and the supernatant fractions, which amounts to about 6% of the total cytochrome c content of liver, cannot be accounted for only by mitochondrial contamination. Analysis of microsomes by isopycnic centrifugation in sucrose gradient indicates that this subcellular fraction contains three pools of cytochrome c. Part of the microsomal cytochrome c belongs to contaminating mitochondria; another part is redistributed cytochrome c adsorbed to the ribosomes; a third part is associated with small vesicles of low density, enzymically characterized by monoamine oxidase activity, and presumably derived from, or related to, the outer mitochondrial membranes.

Effects of chloroquine and colchicine on the degradation of chyle cholesteryl ester and phospholipids in vivo.

1. Both chloroquine and cholchicine given intraperitoneally delayed the hydrolysis of chyle cholesteryl ester after the uptake by the liver. The cholesteryl ester radioactivity per mg protein of hepatocytes isolated from the drug‐treated rats was 97.2 and 96.5%, respectively, of that in the intact liver. The decrease could be accounted for by hydrolysis of cholesteryl ester during cell isolation. Most of the unhydrolyzed cholesteryl ester was thus intracellular or firmly bound to the cells. Also in perfused rat livers chloroquine inhibited the hydrolysis of chyle cholesteryl ester that had been taken up in vivo.2. After subcellular fractionation of livers from chloroquine‐treated rats, that had been injected with cholesterol‐labelled chyle 60 min earlier, 55% of the radioactive cholesteryl ester was in the large granule fractions (mitochondrial plus light mitochondrial pellet). The 600 ×g and 100000 ×g pellets contained a higher proportion of the unesterified radioactive cholesterol. Similar findings were made with colchicine‐treated animals.3. 60 min after injection of chyle labelled with 32P and [3H]cholesterol only 6% of the injected [32P]phospholipid radioactivity was in the liver in normal rats and 7.3% and 7.8% in the liver of rats treated with chloroquine and colchicine, respectively. Whereas the cholesteryl ester radioactivity in the large granule fractions in rats treated with chloroquine and colchicine was increased by 2.4 and 3.1 times, there was no such increase in the 32P radioactivity of any of the phospholipid classes.4. Three conclusions are drawn. Chylomicron remnant cholesteryl esters are likely to be interiorized by the hepatocytes before their degradation. The data are compatible with a role of the acid cholesterol esterase in their hydrolysis. There was no positive evidence that any significant part of the chylomicron phospholipids are taken up by the liver and metabolized by lysosomal enzymes.

Intracellular distribution of dopa and 5-S-cysteinyldopa in Harding-Passey melanoma

The concentrations of dopa and 5-S-cysteinyldopa were determined in the various cell fractions of Harding-Passey melanomas. Dopa was present in larger amounts than was 5-S-cysteinyldopa in all cell fractions, but the dopa/5-S-cysteinyldopa ratio was lower in the soluble fraction and in the small-granule fraction than in the large-granule fraction. The soluble fraction contained the greatest amount of catechols. These findings are compatible with high tyrosinase activity not only in the melanosomes but also in the small-granule and soluble fractions.

The Stichosome and Its Secretion Granules in the Mature Muscle Larva of Trichinella spiralis

The stichosome of the mature muscle larva of Trichinella spiralis consists of a single row of 45 to 55 stichocytes. Each stichocyte is about 25 mum in diameter and possesses a single nucleus. A duct leads from each stichocyte to the lumen of the esophagus. The stichocyte cytoplasm contains mitochondria, structures resembling Golgi-complexes, rough endoplasmic reticulum, and usually 1 of 2 types of secretory granules. The alpha-granule measures about 800 mn in diameter, contains a prominent inclusion, and has a granular matrix. The beta-granule is about 600 mn in diameter and is homogeneous in appearance. Both granule types are surrounded by a single membrane. Ten to thirteen stichocytes containing alpha-granules are confined to the posterior portion of the stichosome. After isopycnic centrifugation in sucrose gradient of large granule fractions obtained from cell-free homogenates, the alpha- and beta-granules show characteristic distribution patterns as revealed by the morphology of the fractions. The median equilibrium density of the alpha-granules is 1.245, while that of the beta-granules is 1.230. There is a correlation between the distribution of the granules and of antigens reacting with hyperimmune antitrichinella seruma. At least 4 unique antigens can be attributed to each of the granule types. Fractions enriched in mitochondria do not contain specific antigens. Antigens from both types of secretory granules cross react totally with those present in the excretion-secretion products of living muscle larvae. Cytoimmunochemical data show that antigens are distributed in a patchy fashion throughout the stichocyte cytoplasm. This finding is consistent with the distribution of the secretory granules in the intact stichocyte.

Hepatic nucleases. Extrahepatic origin and association of neutral liver ribonuclease with lysosomes.

In the large granule fraction of rat liver, the density distribution of inhibitor-sensitive neutral ribonuclease is similar to that for acid hydrolases and its density distribution is similarly modified by Triton WR-1339 accumulation in lysosomes. Particulate neutral ribonuclease is latent; the enzyme is unmasked by very low digitonin concentrations or hypoosmotic shock. These observations demonstrate that the bulk of liver neutral ribonuclease is associated with the lysosomal system. In view of the neutral pH optimum of the enzyme and of some particularities of its distribution in fractionation experiments, the possiblilty of an extrahepatic origin of neutral ribonuclease has been investigated. After partial pancreatectomy, a significant decrease is observed in both plasma and liver neutral ribonuclease. The effect is specific, for it does not occur for other lysosomal enzymes. Also, labelled bovine pancreatic ribonuclease, when injected intravenously, is taken up by the liver. The sedimentable labelled enzyme has a density distribution similar to the distribution of other foreign proteins, horseradish peroxidase or yeast invertase. These results are explained by the uptake of plasmatic neutral ribonuclease from pancreatic origin by the liver.

Interaction of glycine with brain-cortex membrane fragments: Kinetics, ionic requirements and amino acid specificity

The binding of [ 14]glycine to rat brain-cortex membrane fragments, incubated in artificial cerebrospinal fluid, was studied in vitro by means of a nitrocellulose filter assay. The membranes were obtained from the large granule fraction (P 2) of a brain-cortex homogenate, which was osmotically shocked and the larger membrane fractions isolated by centrifugation. Initial binding velocity lasts for about 2 min and equilibrium is reached in 10 min. The binding reaction is reversible, and [ 14C]glycine can be displaced by an excess of [ 12C]glycine or by dilution. Binding is strongly dependent on temperature and on sodium ions. The latter activate the binding process in a cooperative manner. Two binding components may be discerned: one with high affinity for glycine ( K m = 40 ± 8 μM) and one with lower affinity. Lowering the sodium concentration to 60 m M increases the K m of the high-affinity component to 59 μ M, with no change in V max. The bound product is, after incubating the membranes at 37 °C for 10 min, 85% glycine. A large fraction of it may be released by hypoosmotic media. The affinity of the receptor for different amino acids is in the order: glycine, alanine, serine, leucine. Negligible affinity is shown towards ß-alanine, lysine or arginine. γ-Aminobutyric acid (GABA), glutamate and aspartate inhibit glycine binding by a mechanism which is probably non-competitive. This affinity pattern is the same as that of the receptor involved in the transport of small neutral amino acids in brain slices. The sodium and temperature dependence are also characteristic of transport phenomena. The main conclusion of this work is that in brain-cortex only transport receptors, as opposed to synaptic receptors, can be demonstrated for glycine.

Hepatic Nucleases. 2. Association of Polyadenylase, Alkaline Ribonuclease and Deoxyribonuclease with Rat-Liver Mitochondria

Six types of nuclease activities were found to be concentrated in the large granule fraction isolated from rat liver homogenastes by differential centrifugation. Analysis by density equilibration shows that three nucleases are associated with mitochondria: an alkaline ribonulcease (pH optimum 8.8), an alkaline deoxyribonuclease (pH optimum 7.6) and an enzyme acting on polyriboadenylate (pH optimum 7.5). When the outer mitochondrial membrane is ruptured in hypotonic medium, the three mitochondrial nucleases are partially solubilized. Solubilization is however obtained by addition of KCL to the suspension medium. It is concluded that mitochondrial nucleases are localized in the intermembrane space but that an adsorption to the outer face of the inner mitochondrial membrane occurs in sucrose 0.25 M. The mitochondrial localization of alkaline ribonuclease, alkaline deoxyribonuclease and polyadenylate accounts for at least 80% of the activity of liver homogenate; nevertheless, an excess of these enzymes is present in the microsomal fraction. Although no definite conculusion can be reached for the significance of this observation, it is shown by density equilibration analysis that these nuclease are not associated either with ribosomes or with the membranes which are the major component of the microsomal fraction.