scholarly journals Characterization of proteins structurally related to human N-acetyl-β-d-glucosaminidase

1978 ◽  
Vol 173 (1) ◽  
pp. 191-196 ◽  
Author(s):  
M Carroll
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Hexosaminidase A ◽  
Cellulose Column

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000–200000) were present both in the unadsorbed fraction and in the 0.05–0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000–70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.

scholarly journals Detection, purification and characterization of a human cancer-associated galactosyltransferase acceptor

1979 ◽  
Vol 178 (2) ◽  
pp. 279-287 ◽  
Author(s):  
D K Podolsky ◽  
M M Weiser
Keyword(s):  
Molecular Weight ◽  
Human Cancer ◽  
Deae Cellulose ◽  
Structural Data ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Purification And Characterization ◽  
Peptide Moiety ◽  
Cellulose Column

A low-molecular-weight acceptor of galactosyltransferase activity was detected in sera and effusions of patients with extensive maligant disease. This substance was purified to homogeneity from both human serum and effusion by using sequential charcoal/Celite and DEAE-cellulose column chromatography. The purified acceptor was shown to act as substrate for both purified normal and cancer-associated human galactosyltransferase (EC 2.4.1.22) isoenzymes, but had a higher affinity for the cancer-associated isoenzyme (Km = 20 microM) than for the normal isoenzyme (Km = 500 microM). The substrate was found to be a glycopeptide with mol.wt. approx. 3600 determined by polyacrylamide-gel chromatography. Carbohyydate analysis demonstrated only the presence of glucosamine and mannose. Amino acid analysis revealed that the peptide moiety consisted of eight different amino acids, including two residues of asparagine and one residue of serine, but no threonine. These structural data suggest that the acceptor is a fraction of an asparagine-glucosamine type of glycoprotein.

scholarly journals The separation and characterization of the methylumbelliferyl β-galactosidases of human liver

1976 ◽  
Vol 157 (1) ◽  
pp. 189-195 ◽  
Author(s):  
P S Cheetham ◽  
N E Dance
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Neuraminidase Treatment ◽  
High Molecular Weight Component ◽  
Electrophoretic Procedure ◽  
Beta Galactosidase

1. A previously uncharacterized form of human liver acid beta-galactosidase (EC 3.2.1.23), possibly a dimer of molecular weight 160 000, was resolved by gel filtration. It has the same ability to hydrolyse GM1 ganglioside as the two other acid beta-galactosidase forms. 2. The low-molecular-weight forms of acid beta-galactosidase undergo salt-dependent aggregation. 3. The high-molecular-weight component may consist of the low-molecular-weight forms bound to membrane fragments. It can be converted completely into a mixture of these forms. 4. The neutral beta-galactosidase activity can be resolved into two forms by DEAE-cellulose chromatography. They differ in their response to Cl-ions. 5. A new nomenclature is suggested for the six beta-galactosidases so far found in human liver. 6. The enzymic constituents of the beta-galactosidase bands resolved by electrophoresis were re-examined. The A band contains three components. A two-dimensional electrophoretic procedure for resolving the A band is described. 7. The effect of neuraminidase treatment on the behaviour of beta-galactosidases in various separation systems is examined.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

scholarly journals N-Acetyl-β-d-hexosaminidase component A. Different forms in human tissues and fluids

1973 ◽  
Vol 135 (3) ◽  
pp. 457-462 ◽  
Author(s):  
J. U. Ikonne ◽  
R. B. Ellis
Keyword(s):  
Salt Concentration ◽  
Human Liver ◽  
Deae Cellulose ◽  
Minor Component ◽  
Human Tissues ◽  
Serum Enzyme ◽  
Hexosaminidase A ◽  
Minor Form ◽  
A Minor ◽  
Cellulose Column

1. Hexosaminidase A of human serum was resolved into two components, a minor form with properties identical with those of the single hexosaminidase A component of human liver, and a major form with significantly different properties. 2. The major serum hexosaminidase A form was eluted from a DEAE-cellulose column at a lower salt concentration than that required to elute the liver form. 3. A multiple-pass technique was used to elute the major serum enzyme A from a Sephadex G-150 column before that of liver enzyme A. 4. Clostridium perfringens neuraminidase converted the major component of serum hexosaminidase A into a form that was held less tightly by DEAE-cellulose, but the minor component of the A enzyme of serum, and the A enzyme of liver were not affected. 5. The hexosaminidase A from tears was similar to the A enzyme from serum, whereas those from several human tissues and from urine and lymph were similar to the liver form. 6. The A enzyme from serum may be derived from the A enzyme from liver by glycosylation before secretion.

scholarly journals Purification and characterization of three forms of glutathione S-transferase A. A comparative study of the major YaYa-, YbYb- and YcYc-containing glutathione S-transferases

1982 ◽  
Vol 207 (3) ◽  
pp. 459-470 ◽  
Author(s):  
J D Hayes ◽  
G H D Clarkson
Keyword(s):  
Single Gene ◽  
Deae Cellulose ◽  
Transferase Activity ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Glutathione S Transferases ◽  
Peptide Maps

Rat liver glutathione S-transferases have previously been defined by their elution behaviour from DEAE-cellulose and CM-cellulose as M, E, D, C, B, A and AA. These enzymes are dimeric proteins which comprise subunits of mol.wt. 22 000 (Ya), 23 500 (Yb) or 25 000 (Yc). Evidence is presented that YaYa protein, one of two previously described lithocholate-binding proteins which exhibit transferase activity, is an additional enzyme which is not included in the M, E, D, C, B, A and AA nomenclature. We therefore propose that this enzyme is designated transferase YaYa. Transferases YaYa, C, A and AA have molecular weights of 44 000, 47 000, 47 000 and 50 000 respectively and each comprises two subunits of identical size. These enzymes were purified to allow a study of their structural and functional relationships. In addition, transferase A was further resolved into three forms (A1, A2 and A3) which possess identical activities and structures and appear to be the product of a single gene. Transferases YaYa, C, A and AA each had distinct enzymic properties and were inhibited by cholate. The recently proposed proteolytic model, which attributes the presence of multiple forms of glutathione S-transferase activity to partial proteolysis of transferase AA, was tested and shown to be highly improbable. Peptide maps showed significant differences between transferases YaYa, C, A and AA. Immunotitration studies demonstrated that antisera raised against transferases YaYa and C did not precipitate transferase AA.

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