scholarly journals Identification and purification of the reconstitutively active glutamine carrier from rat kidney mitochondria

1998 ◽  
Vol 333 (2) ◽  
pp. 285-290 ◽  
Author(s):  
Cesare INDIVERI ◽  
Giuseppe ABRUZZO ◽  
Italo STIPANI ◽  
Ferdinando PALMIERI
Keyword(s):  
Rat Kidney ◽  
Thiol Group ◽  
Protein Band ◽  
Carrier Protein ◽  
Transport Activity ◽  
Kidney Mitochondria ◽  
Carrier Mediated Transport ◽  
Sds Page ◽  
Single Protein ◽  
Mitochondrial Extract

The glutamine carrier from rat kidney mitochondria, solubilized in dodecyl octaoxyethylene ether (C12E8) and partly purified on hydroxyapatite, was identified and completely purified by Celite chromatography. On SDS/PAGE, the purified glutamine carrier consisted of a single protein band with an apparent molecular mass of 41.5 kDa. When reconstituted into liposomes, the glutamine carrier catalysed both the unidirectional flux of glutamine and the glutamine/glutamine countertransport, which were completely inhibitable by a mixture of pyridoxal 5´-phosphate and N-ethylmaleimide. The carrier protein was purified 474-fold with a recovery of 58% and a protein yield of 0.12% with respect to the mitochondrial extract. The glutamine carrier-mediated transport is quite specific for l-glutamine. l-Asparagine is the only other amino acid that is efficiently transported by the reconstituted carrier protein. d-Glutamine, l-glutamate and l-aspartate are very poor substrates. The transport activity was inhibited by several thiol-group and amino-group reagents.

scholarly journals A murine hybridoma with large cytoplasmic inclusions of kappa light chains.

1987 ◽  
Vol 165 (2) ◽  
pp. 578-583 ◽  
Author(s):  
D C Reason
Keyword(s):  
Cell Line ◽  
Inclusion Bodies ◽  
Protein Band ◽  
Light Chains ◽  
Cytoplasmic Inclusions ◽  
Single Protein Band ◽  
Sds Page ◽  
Single Protein ◽  
Kappa Light Chains

A murine hybridoma cell line has been established that consistently forms large cytoplasmic inclusions. These structures bind antibody specific for mouse kappa L chain when stained in situ. SDS-PAGE analysis of isolated inclusion bodies produce a single protein band of approximately 26,000 Mr that reacts with anti-kappa antibody when transferred to nitrocellulose. No carbohydrate was detected in association with the purified protein. These data are consistent with the intracellular retention and deposition of complete kappa L chain protein.

scholarly journals Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3α-hydroxysteroid dehydrogenase

1994 ◽  
Vol 304 (2) ◽  
pp. 385-390 ◽  
Author(s):  
B Del Bello ◽  
E Maellaro ◽  
L Sugherini ◽  
A Santucci ◽  
M Comporti ◽  
...  
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Dehydroascorbic Acid ◽  
Hydroxysteroid Dehydrogenase ◽  
Protein Band ◽  
Sds Page ◽  
Single Protein ◽  
Ammonium Sulphate Fractionation ◽  
Inflammatory Drugs ◽  
Rat Liver Cytosol

Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.

scholarly journals Characterization of mitochondrial cytochromes P-450 from pig kidney and liver catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids

1991 ◽  
Vol 276 (2) ◽  
pp. 427-432 ◽  
Author(s):  
T Bergman ◽  
H Postlind
Keyword(s):  
Monoclonal Antibody ◽  
Protein Band ◽  
Similar Species ◽  
Mitochondrial Cytochrome ◽  
Pig Liver ◽  
Kidney Mitochondria ◽  
Pig Kidney ◽  
Sds Page ◽  
Liver And Kidney ◽  
Cytochrome P 450

The properties of cytochrome P-450 from pig kidney mitochondria, catalysing 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids [Postlind & Wikvall (1989) Biochem. Biophys. Res. Commun. 159, 1135-1140; Postlind (1990) Biochem. Biophys. Res. Commun. 168, 261-266], were compared with those of a 26-hydroxylating cytochrome P-450 from pig liver mitochondria. The liver enzyme was purified to a cytochrome P-450 content of 7.4 nmol/mg of protein and showed only one protein band with an apparent Mr of 53,000 upon SDS/PAGE. The cytochrome P-450 catalysed 26-hydroxylation of 25-hydroxyvitamin D3, cholesterol and 5 beta-cholestane-3 alpha, 7 alpha-diol at rates of 361, 1090 and 2065 pmol/min per nmol of cytochrome P-450. A monoclonal antibody against the purified liver mitochondrial cytochrome P-450 26-hydroxylase (cytochrome P-450(26] was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(26) from liver as well as from kidney mitochondria and to immunoprecipitate the 26-hydroxylase activity towards 25-hydroxyvitamin D3 and cholesterol when assayed in a reconstituted system. After SDS/PAGE and immunoblotting with the antibody, the cytochrome P-450(26) was detected in the purified liver and kidney preparations. These results indicate that similar species of cytochrome P-450 catalyse 26-hydroxylation of 25-hydroxyvitamin D3 and C27 steroids in liver and kidney mitochondria. The results with the monoclonal antibody together with the finding that cholesterol competitively inhibits the 26-hydroxylation of 25-hydroxyvitamin D3 further indicate that 26-hydroxylation of 25-hydroxyvitamin D3 and cholesterol is catalysed by the same species of cytochrome P-450 in each tissue. The N-terminal amino acid sequence of cytochrome P-450(26) in kidney mitochondria resembled that of pig kidney microsomal 25-hydroxylase active in 25-hydroxylation of vitamin D3 and C27 steroids, whereas the sequence of pig liver mitochondrial cytochrome P-450(26) differed from those of rabbit and rat liver mitochondrial 26-hydroxylases as well as from those of other hitherto isolated mammalian cytochromes P-450.

scholarly journals Purification of Catalase by Modified Procedure and some Properties of Enzyme

2018 ◽  
Vol 40 (2) ◽  
pp. 214-222
Author(s):  
Nguyen Hoang An ◽  
Godagama Gamarchchige Dinesh Suminda ◽  
Nguyen Thi Thu Phuong ◽  
Dinh Nho Thai ◽  
Nguyen Thi Hong Loan
Keyword(s):  
Specific Activity ◽  
Bovine Liver ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Optimal Conditions ◽  
Sds Page ◽  
Single Protein ◽  
Bovine Liver Catalase ◽  
Acetone Fractionation ◽  
Oxidative Effect

Catalase (EC 1.11.1.6) plays an important role in protecting organism from oxidative effect by breaking down H2O2 into H2O and O2 molecules. In this study, a modified procedure for catalase from bovine liver and its properties were reported. Bovine liver catalase was purified to electrophoretic homogeneity as a single protein band around 60 kDa on SDS-PAGE by acetone fractionation, followed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose columns. The specific activity of the purified catalase was 79,277 units per mg of protein (U/mg) with 1.87% recovery and purification fold was roughly 60 times. The catalase was a homo-tetramer with a molecular mass of about 240.987 kDa as determined by native gel electrophoresis. The purified enzyme showed the highest activity at pH 7, 37°C, and remained active over a broad range of pH from 5 to 10 and range of temperature from 4°C to 40°C. Its activity was inactivated by incubating in 60°C for 30 min. The activity of the enzyme was induced by Ca2+ and inhibited by Na+, Ni2+, Cu2+, Zn2+, Fe2+, Fe3+ and NaN3. Under the optimal conditions, Km and Kcat/Km values of the catalasewas found to be 23,69 mM and Kcat/Km = 5.106 (M.s)-1, respectively

scholarly journals Isolation and identification of a strain producing cold-adapted β galactosidase, and purification and characterisation of the enzyme

2008 ◽  
Vol 26 (No. 4) ◽  
pp. 284-290 ◽  
Author(s):  
W.Y. Liu ◽  
Y.W. Shi ◽  
X.Q. Wang ◽  
K. Lou
Keyword(s):  
Food Industry ◽  
Protein Band ◽  
Lactose Hydrolysis ◽  
Isolation And Identification ◽  
Cold Adapted ◽  
Rdna Sequencing ◽  
Sds Page ◽  
Single Protein ◽  
Potential Applications ◽  
Beta Galactosidase

Enzymes with high specific activities at low temperatures have potential uses in the food industry. Cold-adapted microorganisms are potentially useful sources of cold-active enzyme. To find cold-adapted &beta;-galactosidase, we isolated several cold-adapted microorganisms from glacier zone soil. One cold-adapted &beta;-galactosidase producing strain was obtained. The biochemical characteristics and the results of 16S rDNA sequencing identified the strain as <I>Rahnella aquatilis</I>. The enzyme was purified by column chromatography after which a single protein band migrating near 60 kDa was observed by means of SDS-PAGE. The &beta;-galactosidase was optimally active at 35°C and at pH 6.5 when assayed with <I>o</I>-nitrophenyl-&beta;-D-galactopyrano-side as substrate. The enzyme activity was sensitive to temperatures above 40°C and was undetectable at 45°C. Metal ions Mn<sup>2+</sup>and K<sup>+</sup> activated the enzyme while Cu<sup>2+</sup>, Zn<sup>2+</sup>, Fe<sup>3+</sup>, and Al<sup>3+</sup> inhibited the activity. The enzyme was also assayed for lactose hydrolysis. When milk is treated with the enzyme at 30°C for 2 h, the degree of lactose hydrolysis can reach 80%. It has, thus, potential applications in the food industry.

scholarly journals Purification and properties of kynurenine aminotransferase from rat kidney

1991 ◽  
Vol 279 (2) ◽  
pp. 595-599 ◽  
Author(s):  
M R Mawal ◽  
A Mukhopadhyay ◽  
D R Deshmukh
Keyword(s):  
Rat Kidney ◽  
Dicarboxylic Acids ◽  
Supernatant Fraction ◽  
Subunit Structure ◽  
Mitochondrial Enzyme ◽  
Kynurenine Aminotransferase ◽  
Kidney Mitochondria ◽  
Purification And Properties ◽  
Single Protein ◽  
Degree Of Purification

Previous reports indicated that a single protein exhibits kynurenine aminotransferase (KAT) and alpha-aminoadipate aminotransferase (AadAT) activities. However, recently we discovered that KAT and AadAT activities are associated with two different proteins. KAT from rat kidney supernatant fraction was purified to electrophoretic homogeneity by (NH4)2SO4 fractionation, DEAE-Sephacel and hydroxyapatite chromatography. This procedure separated KAT from AadAT and improved the overall yield and the degree of purification over previously published methods. Some of the properties of purified KAT, such as Mr, subunit structure and the inhibition by dicarboxylic acids, were identical with those reported previously. However, the substrate specificity and pI of purified KAT were different from earlier reports. The same procedure can also be used to purify KAT from rat kidney mitochondria. These results support our earlier observation that KAT and AadAT activities are associated with two proteins and suggest that cytosolic KAT may be structurally similar to the mitochondrial enzyme.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

scholarly journals Glutamate transprot in rat kidney mitochondria.

1983 ◽  
Vol 258 (3) ◽  
pp. 1735-1739
Author(s):  
A C Schoolwerth ◽  
K F LaNoue ◽  
W J Hoover
Keyword(s):  
Rat Kidney ◽  
Kidney Mitochondria
Sign in / Sign up

Export Citation Format

Share Document