scholarly journals Studies on the structure-bound sedimentability of some rat liver lysosome hydrolases

1971 ◽  
Vol 122 (3) ◽  
pp. 363-371 ◽  
Author(s):  
F. M. Baccino ◽  
G. A. Rita ◽  
Maria Franca Zuretti
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
High Speed ◽  
Particulate Material ◽  
The Other ◽  
Acid Proteinase ◽  
Freezing And Thawing ◽  
Clear Cut ◽  
Almost All ◽  
Triton X

1. Lysosome-rich fractions from rat liver were subjected to several disruptive procedures: osmotic lysis or freezing and thawing in different media, shearing forces in a high-speed blender, treatment with Triton X-100. 2. The soluble and particulate phases were then separated by high-speed centrifugation and assayed for their content of acid phosphatase, β-galactosidase, β-N-acetylglucosaminidase, acid proteinase, acid ribonuclease, acid deoxyribonuclease and protein. 3. The degree of elution of these hydrolases appeared to depend on both the enzyme species and the treatment. The resulting patterns of solubilization were rather complex, so that a clear-cut discrimination between soluble and structure-bound enzymes could not always be traced. 4. Although only β-galactosidase was readily solubilizable after all treatments, acid proteinase could also be extensively eluted from the sedimentable material in the presence of EDTA and acid phosphatase was fully extracted by Triton X-100. On the other hand, considerable proportions of the other activities could not be solubilized by any of the procedures used. 5. In other experiments, the adsorbability of hydrolases on subcellular structures was investigated by measuring the partition between sedimentable particles and soluble fraction of solubilized enzymes added to ‘intact’ liver homogenates. 6. Large proportions of acid proteinase, ribonuclease and deoxyribonuclease, and almost all of β-N-acetylglucosaminidase, were found to be adsorbed on the particulate material.

scholarly journals Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver

1989 ◽  
Vol 257 (1) ◽  
pp. 221-229 ◽  
Author(s):  
L Schepers ◽  
M Casteels ◽  
K Verheyden ◽  
G Parmentier ◽  
S Asselberghs ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholic Acid ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
The Other ◽  
Acid Activation ◽  
Long Chain Fatty Acids ◽  
Microsomal Membranes ◽  
Triton X ◽  
Long Chain

The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.

The Demonstration of Lysosomes in Mouse Skin

1964 ◽  
Vol s3-105 (69) ◽  
pp. 73-78
Author(s):  
J. V. DIENGDOH
Keyword(s):  
Acid Phosphatase ◽  
Biochemical Characterization ◽  
Biochemical Study ◽  
Hydrolytic Enzymes ◽  
Mouse Skin ◽  
Freezing And Thawing ◽  
Sectioning Method ◽  
Repeated Freezing ◽  
Triton X

Lysosomes, as demonstrated biochemically in the liver, are subcellular particles containing a group of hydrolytic enzymes enclosed by a membrane-like barrier. They are apparently inactive in the normal state, but when subjected to various forms of injurious treatments the enzymes associated with them are released. The existence of lysosomes in skin is demonstrated in the present communication. The resistance of this tissue to homogenization makes the biochemical study of lysosomes very difficult. Yet, by the application of histochemical methods to sections of skin, it has been possible to use the same criteria as would be employed in the biochemical characterization of these organelles. By using the controlled-temperature freezing-sectioning method it has been possible to obtain frozen sections in which cytoplasmic particles could be demonstrated which were enzymically inactive for acid phosphatase until the sections were subjected to such injurious treatments as heat, repeated freezing and thawing, hypotonic solutions, distilled water, and ‘triton-X-100’. Since the subcellular particles demonstrated behaved in the same manner as lysosomes prepared biochemically from liver, it is concluded that the cytoplasmic organelles staining for acid phosphatase in mouse skin are lysosomes as biochemically defined.

scholarly journals On the presence of two non-specific nucleotide-sugar-hydrolysing enzymes in rat liver

1984 ◽  
Vol 220 (1) ◽  
pp. 95-103 ◽  
Author(s):  
H G Muilerman ◽  
A M Lasthuis ◽  
G J M Hooghwinkel ◽  
W Van Dijk
Keyword(s):  
Rat Liver ◽  
Sodium Dodecyl ◽  
Rat Tissues ◽  
Nucleotide Sugar ◽  
Nitrophenyl Phosphate ◽  
Molecular Masses ◽  
Almost All ◽  
Triton X ◽  
Enzyme I ◽  
Specific Nucleotide

Evidence is presented for the occurrence of two different non-specific nucleotide-sugar hydrolases in rat liver and other rat tissues. These two enzymes (I and II) were separated by chromatography on a 5′-AMP-aminohexyl-Sepharose column. Enzyme I is most probably identical with phosphodiesterase I (EC 3.1.4.1). Enzyme II appeared to be identical with an enzyme described in literature as ‘CMP-sialic acid hydrolase’ [Kean & Bighouse (1974) J. Biol. Chem. 249, 7813-7823], since almost all activity with CMP-N-acetylneuraminate as substrate was recovered in this enzyme fraction. CMP-N-acetylneuraminate was a poor substrate for Enzyme I, whereas deoxythymidine-5′-p-nitrophenyl phosphate and all nucleoside-diphosphosugars tested were good substrates for both Enzyme I and II. Therefore it is suggested that CMP-N-acetylneuraminate is used as an additional substrate to discriminate between the activities of Enzyme I and II in homogenates or membrane preparations. The various substrates appeared to be competitive inhibitors of each other, suggesting that, in each enzyme preparation, only one enzyme is responsible for the hydrolysis of the various substrates. The dissimilar properties of the two enzymes are substantiated by studying the subunit molecular masses (Enzyme I, 125 kDa; Enzyme II, 50-55 kDa), the sensitivity towards Triton X-100, Sarkosyl and sodium dodecyl sulphate and towards trypsin treatment. It is discussed whether the alpha-N-acetylglucosamine phosphodiesterase described by Varki & Kornfeld [(1981) J. Biol. Chem. 256, 9937-9943] is identical with one of the nucleotide-sugar hydrolases described here.

scholarly journals Effect of cations on structure-linked sedimentability of lysosomal hydrolases

1968 ◽  
Vol 109 (1) ◽  
pp. 149-154 ◽  
Author(s):  
M. Anthony Verity ◽  
R. Caper ◽  
W. Jann Brown
Keyword(s):  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Lysosomal Hydrolases ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Bivalent Cation ◽  
Repeated Freezing ◽  
Triton X ◽  
Total Enzyme

1. A partially purified lysosomal preparation was obtained from mouse liver sucrose homogenates by differential and discontinuous gradient centrifugation. 2. Triton X-100 or repeated freezing and thawing of the lysosomal suspension (subfraction B) allowed comparison of free and activated values for acid phosphohydrolase, β-glucuronidase and N-acetylglucosaminidase in the presence and absence of ascorbate. 3. The distribution of hydrolase activities between supernatant and pellet after high-speed centrifugation was measured and the percentages of total enzyme found in the supernatant were: acid phosphohydrolase, 40·7; β-glucuronidase, 51; N-acetylglucosaminidase, 39·4. 4. Differential rates of elution of the three hydrolases from the membrane fraction occurred with increasing Na+ and K+ concentrations, whereas complex biphasic elution curves were obtained as a function of bivalent cation concentration with Ca2+ and Mg2+. 5. Sucrose-density-gradient centrifugation of frozen-and-thawed subfraction B demonstrated highly significant changes in the protein gradient profile in the presence of a low concentration of bivalent cation, indicating membrane aggregation and enzyme–membrane association. 6. The data provide further evidence for the nature of lysosomal enzyme binding and indicate the presence of different enzyme–membrane bonds conferring structure-linked latency upon individual lysosomal enzymes.

scholarly journals The topographical location and unique nature of a glucokinase associated with the Golgi apparatus of rat liver

1976 ◽  
Vol 154 (1) ◽  
pp. 193-201 ◽  
Author(s):  
G Berthillier ◽  
R Coleman ◽  
D G Walker
Keyword(s):  
Golgi Apparatus ◽  
Rat Liver ◽  
Freezing And Thawing ◽  
Golgi Membranes ◽  
Unique Nature ◽  
Triton X

A particulate glucokinase was recovered in the Golgi-rich fraction of rat liver prepared by the method of Morré [Methods Enzymol. (1971) 22, 130-148], thus extending the demonstration by Berthillier et al. [Biochim. Biophys. Acta (1973), 293, 370-378] of particulate glucokinase activity in a microsomal subfraction that showed enrichment in Golgi characteristics. The purity of this fraction was examined and it was then subjected to several treatments, the action of Triton X-100, freezing and thawing, and sonication to establish the topographical location of the glucokinase activity thus solubilized. The evidence suggests that the glucokinase activity is either soluble in the lumen of the Golgi apparatus or loosely associated with the inside of the Golgi membranes.

scholarly journals Studies on the purification and properties of UDP-galactose glycoprotein galactosyltransferase from rat liver and serum

1976 ◽  
Vol 156 (2) ◽  
pp. 347-355 ◽  
Author(s):  
I H Fraser ◽  
S Mookerjea
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
High Speed ◽  
Broad Band ◽  
Chromatographic Technique ◽  
Appreciable Effect ◽  
Serum Enzyme ◽  
Membrane Bound ◽  
Triton X ◽  
High Speed Supernatant

1. Rat liver microsomal preparations incubated with 200mM-NaCl at either 0 or 30 degrees C released about 20-30% of the membrane-bound UDP-galactose-glycoprotein galactosyl-transferase (EC 2.4.1.22) into a ‘high-speed’ supernatant. The ‘high-speed’ supernatant was designated the ‘saline wash’ and the galactosyltransferase released into this fraction required Triton X-100 for activation. It was purified sixfold by chromatography on Sephadex G-200, and appeared to have a higher molecular weight than the soluble serum enzyme. 2. Rat serum galactosyltransferase was purified 6000-7000-fold by an affinity-chromatographic technique using a column of activated Sepharose 4B coupled with α-lactalbumin. The purified enzyme ran as a single broad band on polacrylamide gels and contained no sialytransferase, N-acetylglucosaminyltransferase and UDP-galactose pyrophosphatase activities. 3. The highly purified enzyme had properties similar to those of both soluble and membrane-bound galactosyltransferase. It required 0.1% Triton X-100 for stabilization, but lost activity on freezing. The enzyme had an absolute requirement for Mn2+, not replaceable by Ca2+, Mg2+, Zn2+ or Co2+. It was active over a wide pH range (6-8) and had a pH optimum of 6.8. The apparent Km for UDP-galactose was 12.5 × 10(-6) M. α-Lactalbumin had no appreciable effect on UDP-galactose-glycoprotein galactosyltransferase, but it increased the specificity for glucose rather than for N-acetylglucosamine, thus modifying the enzyme to a lactose synthetase. 4. The possibility of a conversion of higher-molecular-weight liver enzyme into soluble serum enzyme is discussed, especially in relation to the elevated activities of this and other glycosyltransferases in patients with liver diseases.

scholarly journals EFFECTS OF TRITON X-100 UPON THE ACTIVITY OF SOME ELECTROPHORETICALLY SEPARATED ACID PHOSPHATASES AND ESTERASES

1965 ◽  
Vol 13 (6) ◽  
pp. 434-440 ◽  
Author(s):  
SALLY LYMAN ALLEN ◽  
JOHN M. ALLEN ◽  
BARBARA MORRISON LICHT
Keyword(s):  
Acid Phosphatase ◽  
Enzymatic Activity ◽  
Rat Liver ◽  
Tetrahymena Pyriformis ◽  
Fatty Acid Esters ◽  
Acid Phosphatases ◽  
Ionic Detergent ◽  
Starch Gels ◽  
Triton X ◽  
Acid Esters

Triton X-100, a non-ionic detergent, was incorporated into reaction mixtures used for the visualization of esterases and acid phosphatases separated by electrophoresis in starch gels. Its effects were tested, in combination with 12 different substrates, on enzymes derived from Tetrahymena pyriformis and rat liver. The effects of Triton X-100 were complex. It promoted the solubilization of some substrates, notably the α-naphthyl fatty acid esters. It also altered the color of the enzymatically produced end product. The net effect was apparent enhancement of enzymatic activity with certain substrates and apparent inhibition of enzymatic activity with other substrates. Differential activation and inhibition of some of the electrophoretically resolved enzymes was observed. Both quantitative and electrophoretic studies indicated that Triton X-100 is an activator of certain esterases. A cathodally migrating acid phosphatase of rat liver was activated by Triton X-100 in the presence of naphthol AS, naphthol AS-BI, or naphthol AS-MX phosphates.

scholarly journals The effect of anoxia on cerebral acid hydrolases in the five-day-old rat

1972 ◽  
Vol 129 (5) ◽  
pp. 1079-1084 ◽  
Author(s):  
B. P. F. Adlard ◽  
S. W. De Souza
Keyword(s):  
Acid Phosphatase ◽  
High Speed ◽  
Recovery Period ◽  
Brain Homogenate ◽  
Acid Hydrolases ◽  
Whole Brain ◽  
Immature Rat ◽  
Glucuronidase Activity ◽  
Anaesthetized Rats ◽  
Triton X

1. Five-day-old anaesthetized rats subjected to slow, prolonged asphyxia (50–55 min) were either allowed to die or resuscitated when at the point of death. Activities of various cerebral acid hydrolases known to be associated with lysosomes were determined in these animals and in littermate controls. 2. Asphyxia to death resulted in a significant increase in the activities of acid phosphatase, cathepsin (pH5.0) and β-glucuronidase in whole-brain homogenates. 3. The effect of asphyxia on β-glucuronidase activity was not apparent when the assay was performed in the presence of Triton X-100 (0.1%, v/v). 4. In resuscitated animals whole-brain-homogenate β-glucuronidase activity showed the greatest increase (31%) 15 min after recovery. After a 60 min recovery period differences between control and asphyxiated animals were no longer apparent. 5. In animals anoxiated to death activities of acid phosphatase and β-N-acetylglucosaminidase in brain high-speed supernatants were significantly higher than in controls. Acid phosphatase activity was similarly increased in asphyxiated animals resuscitated for 5 or 60 min. 6. It is suggested that the response of the immature rat brain to asphyxia involves a disruption or increased fragility of lysosomal particles.

scholarly journals Strain differences in rat liver (UDP-glucuronyltransferase activity towards androsterone

1979 ◽  
Vol 179 (3) ◽  
pp. 483-487 ◽  
Author(s):  
M Matsui ◽  
F Nagai ◽  
S Aoyagi
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Strain Differences ◽  
Transferase Activity ◽  
Sprague Dawley ◽  
Freezing And Thawing ◽  
Sprague Dawley Rats ◽  
Long Evans ◽  
Triton X

Male Donryu, Wistar King rats showed discontinuous variations in hepatic microsomal UDP-glucuronyltransferase activities towards androsterone, but not towards testosterone, bilirubin, phenolphthalein and 4-nitrophenol. Fresh microsomal fraction with a low transferase activity towards androsterone formed 0.049–0.080 nmole of glucuronide/min per mg of protein, whereas fresh microsomal fraction with a high transferase activity towards androsterone formed 0.335–0.557 nmol of glucuronide/min per mg of protein. The microsomal fraction with low enzyme activity towards androsterone was not stimulated by treatment with Triton X-100 or freezing and thawing. In contrast, male Long Evans and Sprague-Dawley rats did not exhibit such diversity.

scholarly journals Heterogeneity of liver acid phosphatases in developing chick embryo

1969 ◽  
Vol 115 (2) ◽  
pp. 191-197 ◽  
Author(s):  
K.-M. Wang
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Soluble Fraction ◽  
The Other ◽  
Acid Phosphatases ◽  
Peak Activity ◽  
Ph Optimum ◽  
Triton X ◽  
Soluble Fractions

1. The development, localization and heterogeneity of acid phosphatase and a Zn2+-activated acid phosphatase in cellular fractions of developing chick liver were studied. 2. Acid phosphatase is distributed abundantly in the particulate and soluble fractions. The soluble fraction is rich in Zn2+-activated acid phosphatase, which attains its peak activity at about 15 days of incubation. 3. The particulate acid phosphatase activity is inhibited by fluoride but not by sodium l(+)-tartrate or cysteine. On the other hand, the soluble Zn2+-activated acid phosphatase activity is inhibited by sodium l(+)-tartrate and cysteine but not by fluoride. 4. The pH optimum of these two enzymes is similar at about 5·6. 5. The soluble Zn2+-activated acid phosphatase activity appears to be thermally stabilized by the treatment with Triton X-100 or bovine serum albumin.

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