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Biomedical subjects

O Touster

Publications and source records attributed to O Touster.

At least 55 records · Page 3Linked to original sources
PubMed1978

Rat liver microsomal and lysosomal beta-glucuronidases differ in both carbohydrate and amino acid compositions.

To investigate the chemical relationships between rat liver lysosomal and microsomal beta-D-glucuronidases (EC 3.2.1.31), which are essentially identical catalytically and in reactivity with antibody and similar in molecular weight, the two enzymes were isolated by procedures in which modifications of the proteins were avoided. The purified enzymes were found to differ in both sugar and amino acid compositions. The microsomal enzyme contained much more mannose and, in contrast to the lysosomal enzyme, contained sialic acid but no glucose. Moreover, although the amino acid compositions generally agreed closely, the microsomal enzyme contained much more serine and somewhat less arginine than the lysosomal form. These findings of specific differences in composition should have a bearing on the consideration of intracellular glycoprotein synthesis, translocation, and compartmentalization.

Amino Acids↗
PubMed1978

In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques.

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.

Amoeba↗
PubMed1977

The purification and characterization of rat liver lysosomal alpha-L-fucosidase.

The alpha-L-fucosidase from rat liver lysosomes was purified approximately 27,000-fold (from cytoplasmic extract) by a rapid procedure requiring only 7 h anf providing enzyme in a 20 per cent yield. The procedure is based upon affinity chromatography with agarose-epsilon-aminocaproyl-fucosamine. The isolated enzyme was found to be pure by a number of different analytical gel techniques and is essentially free of other lysosomal gylcosidases. The purified enzyme exhibits a positive periodic acid-Schiff stain, suggesting that it is a glycoprotein. The purified enzyme has a pH optimum of 5.7 to 5.9, a Vmax of 27 mumol/min/mg of protein, and a Km of 0.19 mM with p-nitrophenyl alpha-L-fucopyranoside as substrate. L-Fucose was the only possibly physiological effector of the enzyme which was identified; it exhibited a Ki of 1.6 mM, with p-nitrophenyl alpha-L-fucopyranoside as substrate. The enzyme has a subunit molecular weight of approximately 55,000 by Na dodecyl-SO4 electrophoresis in a variety of gel systems. The molecular weight of the native enzyme was indicated to be approximately 160,000 by sucrose density centrifugation, 300,000 by molecular sieve chromatography on Sephadex G-200, and 217,000 by sedimentation equilibrium centrifugation. The weight of evidence suggests that the enzyme is a tetramer. Incubation on the absence of sulfhydryl reagents under appropriate conditions generates a second alpha-L-fucosidase activity band on gels corresponding to a molecular weight of approximately 40,000 to 50,000. This result suggests that the subunit is relatively stable and may reassociate to form active enzyme. Alpha-L-Fucosidase requires a high concentration of protein and the presence of a sulfhydryl reagent for stabilization. It is rapidly inactivated by p-chloromercuriphenyl sulfonic acid, this inactivation being rapidly reversible by the addition of 10 mM 2-mercaptoethanol. The enzyme catalyzed the hydrolysis of 1 leads to 2, 1 leads to 3, and 1 leads to 4 fucosyl linkages and was found to be active on glycopeptides but not on native glycoproteins. The amino acid and carbohydrate composition of the enzyme was determined. The native enzyme contains the following sugars (residues per tetramer): fucose (3.5), mannose (32), galactose (8), glucose (9), glucosamine (32), and sialic acid (8). Rat liver lysosomal alpha-glucosidase, also produced in the rapid isolation procedure described herein, contained less than 0.1 residue of sialic acid per subunit.

Amino Acids↗
PubMed1976

Purification and characterization of the alpha-D-mannosidase of rat liver cytosol.

Three forms of alpha-D-mannosidase have previously been identified in rat liver, and each is localized in a different subcellular fraction: lysosomes, Golgi membranes, and cytosol. This communication reports the purification and characterization the cytosolic form. The enzyme was purified 12,000-fold in good yield to approximately 90% purity with the aid of the competitive inhibitor mannosylamine and dithioerythritol as stabilizers. The molecular weight of the enzyme is in the range of 372,000 to 490,000 depending on the method used. Since the subunit molecular weight is 110,000 by sodium dodecyl sulfate polyacrylamide electrophoresis, the enzyme is probably a tetramer. The pH optimum was shown to be between 5.5 and 5.9 (in the presence of 1 mM CoCl2) with the substrate p-nitrophenyl-alpha-D-mannoside. Normal Michaelis-Menten kinetics were observed with a Km of 0.14 mM. Mannosylamine was a competitive inhibitor with a Ki of 0.007 mM. The purified enzyme, stabilized by Co2+, Mn2+, and Fe2+ under some conditions, was unstable at low protein concentrations. Since an electrophoresed sample showed a positive periodic acid-Schiff stain, the enzyme may contain carbohydrate. The availability of purified cytosolic alpha-D-mannosidase should now make it possible to carry out substrate specificity, immunological, and structural studies which may shed light on the biological role of this enzyme.

Animals↗
PubMed1976

Evidence for specific recognition sites mediating clearance of lysosomal enzymes in vivo.

A study of the clearance of liver lysosomal enzymes was carried out in the rat. Purified rat liver lysosomal beta-D-glucuronidase (EC 3.2.1.31), N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30), alpha-L-fucosidase (EC 3.2.1.51), and alpha-D-mannosidase (EC 3.2.1.24), as well as rat preputial gland beta-glucuronidase, were infused intravenously into anesthetized rats. All of the enzymes were rapidly cleared from the circulation. Sodium periodate oxidation of lysosomal beta-glucuronidase resulted in a near abolition of rapid clearance, a reduction in concanavilin-A-Sepharose binding, and a reduction in neutral sugar content, accompanied by alteration in isoelectric focusing properties. Similarly, periodate oxidation of lysosomal N-acetyl-beta-D-glucosaminidase resulted in a loss of the rapid clearance property. These results suggest that specific recognition sites occur on lysosomal hydrolases which mediate clearance following intravenous injection, and that these sites involve the carbohydrate portions of the enzymes.

Acetylglucosaminidase↗
PubMed1976

In vitro fusion of Acanthamoeba phagolysosomes. I. Demonstration and quantitation of vacuole fusion in Acanthamoeba homogenates.

Fusion of phagolysosomes (PLs) has been demonstrated to occur in vitro. Two separate cell homogenates of the ameba Acanthamoeba sp. (Neff) were prepared, each rich in PLs labeled with distinctive particulate markers. Portions of each were incubated together in vitro and fusion occurred as evidenced by the appearance of PLs containing both types of markers. Fusion was confirmed by electron microscopy, including serial sectioning. The membranes of fused vacuoles excluded the dye eosin Y. Surviving cells in the homogenates were not responsible for the observed fusion. Fusion was obtained using either synthetic markers (polystyrene and polyvinyltoluene latex) or biological markers (autoclaved yeast cells and glutaraldehyde-fixed goat red blood cells), or a combination of both. The specificity of PL fusion in vivo appeared to be maintained in vitro. As determined by light and electron microscopy, the fusion reaction was dependent on time and temperature, and on the initial presence of membrane around both marker particles. A minimum of 10% of the vacuoles fused by 10 min of incubation at 30 degrees C, and no rupture of the vacuoles was detected during this time. After 10 min of incubation, vacuole rupture began and fusion ceased. At a constant initial vacuole concentration, the extent of PL fusion in vitro was quantitatively reproducible. This appears to be a promising system for further investigation of membrane fusion in the lysosomal system.

Amoeba↗
PubMed1975

Purification and chemical properities of mouse liver lysosomal (L form) beta-glucuronidase.

The lysosomal form (L form) of beta-glucuronidase was purified 6,500-fold from the liver of C57BL/6J mice with high yield. Purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence or absence of sodium dodetcyl sulfate. The microsomal forms of beta-glucuronidase were spontaneously converted to the L form. The purified L form is a tetramer of molecular weight of 280,000 to 300,000, composedd of four identical subunits of 75,000 molecular weight. The enzyme contains a high content of arginine and glutamic acid and a very low content of sulfur-containing amino acids. Approximately 7% of the enzyme molecule is compose of carbohydrate. Sugars in the L form are glucosamine, mannose, galactose, and glucose. Sialic acid and fucose are absent in the enzyme.

Amino Acids↗
PubMed1975

Turnover studies on proteins of rat liver lysosomes.

The turnover of rat liver lysosomal proteins was studied by a double isotope-labeling technique. The cellular fractions investigated included soluble lysosomal proteins, lysosomal membrane proteins, highly purified lysosomal beta-glucuronidase, and for comparison, microsomal proteins and soluble cytoplasmic proteins. Both "normal" lysosomes and Triton WR-1339-filled lysosomes (tritosomes) were studied, with similar results. It was found that (a) the turnover rate of lysosomal proteins, of both the soluble and membranous compartments, was very similar to that of the proteins of the microsomal and soluble cytoplasmic fractions, and (b) the turnover rate of lysosomal proteins was asynchronous. The latter conclusion was based on two lines of evidence: (a) lysosomal beta-glucuronidase had a distinctly slower turnover rate than the average rate of the soluble lysosomal proteins, and (b) subunits of the proteins of the soluble lysosomal fraction as separated by sodium dodecyl sulfate. Sephadex G-200 gel filtration showed different rates of degradation.

Animals↗
PubMed1975

Physical and chemical properties of beta-glucuronidase from the preputial gland of the female rat.

In order to obtain sufficient quantities of beta-glucuronidase for use in structural studies, the enzyme was purified from its richest known source, the female rat preputial gland, by a method similar to that of Ohtsuka and Wakabayashi (1969) (Enzymologia 12, 109). The purified enzyme has an S-o20, w of 12.5 S and a D-o20, w of 4.3 times 10- minus 7 cm-2 S-minus 1. Sedimentation diffusion and sedimentation equilibrium yielded molecular weights of 267,000 and 283,000, respectively. The limiting viscosity (3.6 ml/g) and the f/fo (1.08 at sigma equals to 0.2 g of H2O/g of protein) indicate that the enzyme is a typical globular protein possessing little asymmetry. The circular dichroism spectrum indicates approximately 14% alpha-helix and a far greater amount of random coli than beta structure. The enzyme is acidic, having an isoelectric point of 6.15. In electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate the enzyme exhibits a single band at molecular weight 72,000, a result indicating that the enzyme consists of four subunits of similar molecular weight. Tryptic peptide mapping suggests that the subunits are identical.

Animals↗
PubMed1975

The preparation and chemical composition of the multiple forms of beta-glucuronidase from the female rat preputial gland.

Beta-Glucuronidase isolated from the preputial gland of the female rat has previously been shown to be a tetrameric glycoprotein. We have now separated the enzyme into several molecular forms by chromatography on hydroxylapatite columns. The three major forms (A, B, and C) have a very similar or identical amino acid composition, and kinetic and stability studies on forms B and C disclosed no differences between these two forms. However, from C contained much more carbohydrate than forms A and B, which were very similar in carbohydrate composition. The sugars in forms A and B are mannose (2.8%), glucosamine (1.9%), fucose (0.2%), galactose (0.16%), and glucose (0.17%). Form C is a little higher in mannose content, but, more distinctively, is much richer in fucose (0.6%), galactose (1.1%), and glucose (1.5%). The presence of glucose was established by paper chromatography as well as by gas-liquid chromatography, and several special experiments were performed to rule out the possibility that this hexose was present in a persistent contaminant. Direct chemical analysis for sialic acid consistently showed the absence of this sugar in the enzyme. The fact that the carbohydrate-protein linkage is alkali-stable suggests that the linkage involves an asparaginyl-N-acetylglucosamine residue. The NH2-terminal amino acid in the polypeptide chain is leucine.

Amino Acid Sequence↗