scholarly journals Affinity-chromatographic purification of S-adenosyl-l-homocysteine hydrolase. Some properties of the enzyme from rat liver

1981 ◽  
Vol 193 (2) ◽  
pp. 503-512 ◽  
Author(s):  
E O Kajander ◽  
A M Raina
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Adenosine Kinase ◽  
Native Enzyme ◽  
Apparent Homogeneity ◽  
A Subunit ◽  
Adenosine Derivatives ◽  
Pore Gradient

S-Adenosyl-L-homocysteine hydrolase has been purified to apparent homogeneity from rat liver by means of affinity chromatography on 8-(3-aminopropylamino)adenosine linked to Sepharose. The purified enzyme was free from adenosine kinase and adenosine deaminase activities and was homogeneous on SDS/polyacrylamide-gel electrophoresis which gave a subunit mol.wt. of 47 000. The native enzyme showed some microheterogeneity on polyacrylamide-gel electrophoresis under increased-resolution conditions but was homogeneous on isoelectric focusing (pI 5.6). The molecular weight of the native enzyme was about 220 000 as judged by pore-gradient electrophoresis. The native enzyme bound adenosine tightly and showed Km values of 0.6 microM, 0.9 microM and 60 microM for adenosine, S-adenosyl-L-homocysteine and L-homocysteine respectively. The enzyme was rapidly inactivated when incubated in the presence of adenosine, S-adenosyl-L-homocysteine or several adenosine derivatives or analogues. Inactivation took place both at 0 and 37 degrees C. Freezing in the absence of glycerol resulted in the appearance of dissociation products of the oligomeric protein. Multimer formation was observed at low thiol concentrations.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

scholarly journals Purification, characterization and radioimmunoassay of adenosine deaminase from human leukaemic granulocytes

1981 ◽  
Vol 195 (2) ◽  
pp. 389-397 ◽  
Author(s):  
D A Wiginton ◽  
M S Coleman ◽  
J J Hutton
Keyword(s):  
Molecular Weight ◽  
Adenosine Deaminase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Periodic Acid Schiff ◽  
Apparent Homogeneity

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.

Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits

1982 ◽  
Vol 60 (4) ◽  
pp. 463-470 ◽  
Author(s):  
T. Youdale ◽  
J. P. MacManus ◽  
J. F. Whitfield
Keyword(s):  
Rat Liver ◽  
Ribonucleotide Reductase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Relative Mass ◽  
Purification And Properties ◽  
Exclusion Chromatography

Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 × 10−4 for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS–polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.

scholarly journals Purification and partial characterization of human polyamine synthases

1989 ◽  
Vol 259 (3) ◽  
pp. 879-886 ◽  
Author(s):  
E O Kajander ◽  
L I Kauppinen ◽  
R L Pajula ◽  
K Karkola ◽  
T O Eloranta
Keyword(s):  
Gel Electrophoresis ◽  
Cross Reactivity ◽  
Specific Pattern ◽  
Native Enzyme ◽  
Spermidine Synthase ◽  
Enzyme Preparations ◽  
Spermine Synthase ◽  
Apparent Homogeneity ◽  
Gradient Gel Electrophoresis ◽  
Pore Gradient

Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins.

scholarly journals Purification and characterization of rat liver glycosylasparaginase

1989 ◽  
Vol 260 (1) ◽  
pp. 101-108 ◽  
Author(s):  
O K Tollersrud ◽  
N N Aronson
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Oligosaccharide Chain ◽  
Molecular Masses ◽  
High Mannose

1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.

Isolation and Analysis of Non-Histone Chromatin Proteins from Rat-Liver Nuclei by Three Different Methods

1974 ◽  
Vol 52 (12) ◽  
pp. 1143-1153 ◽  
Author(s):  
D. Suria ◽  
C. C. Liew
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Soluble Proteins ◽  
Polyacrylamide Gel ◽  
Pulse Labelling ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Chromatin Proteins

Non-histone chromatin proteins were isolated from rat-liver nuclei by three different methods, and defined as (I) phenol-soluble proteins, (II) SDS-soluble proteins and (III) proteins not adsorbed by cation-exchange chromatography. About 62–70% of chromatin proteins were recovered from the total nuclear proteins. The yield of non-histone chromatin proteins varied from 17 to 26% of chromatin proteins, depending on the method used. The amino-acid composition of these proteins showed that they are acidic in nature. Their phosphorus content was found to be 0.9, 1.1, and 1.4%, respectively, according to method I, II, or III. In-vivo pulse-labelling experiments indicated that chromatin proteins were highly labelled with 3H-acetate and 32P-phosphoric acid. In particular, the specific activities of 32P incorporation were higher in all non-histone chromatin proteins isolated as compared with histones. One-dimensional SDS–polyacrylamide gel electrophoresis showed that at least 26 similar fractions can be detected in the samples prepared by these three methods.The similarity of some of the proteins obtained from methods I and III was further confirmed by fractionation of the non-histone chromatin proteins in an isoelectro-focusing system followed by a second-dimensional SDS–polyacrylamide gel electrophoresis. It was found that more than 100 components could be identified. However, some minor variations of the non-histone chromatin proteins were detected by this system. The differences in proteins isolated by these methods are mainly quantitative rather than qualitative. The methods examined are not specific for the fractionation of a certain class of non-histone chromatin proteins.

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