Aggregation States of a Regulatory Enzyme Complex Catalyzing the Early Steps of Pyrimidine Biosynthesis in Bakers' Yeast

1971 ◽  
Vol 49 (4) ◽  
pp. 403-411 ◽  
Author(s):  
P. F. Lue ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Density Gradient ◽  
Crude Extract ◽  
Analytical Ultracentrifugation ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Regulatory Site ◽  
Aspartate Transcarbamylase

Aspartate transcarbamylase (ATCase) of bakers' yeast has been purified 78-fold from a crude extract of a derepressed diploid strain; its specific activity was more than 300-fold that of a wild-type crude extract. During the last steps of the purification there was a parallel co-purification of carbamylphosphate synthetase (CPSase), and both activities retained full sensitivity to feedback inhibition by UTP; indeed the sensitivity of the ATCase to UTP increased during the purification doubtless due to discard of a feedback-insensitive ATCase subunit. The two enzyme activities co-eluted from gel filtration on Sepharose 6B together with the feedback site. Analytical ultracentrifugation revealed that the material was not homogeneous, showing two major peaks. Sucrose density gradient centrifugation in the presence of UTP, glutamine, and Mg2+ resulted in co-sedimentation of the two activities and the regulatory site, corresponding to a molecular weight of approximately 800 000 daltons. Omission of UTP from the gradient resulted in disappearance of the heavy peak and appearance of a new one, corresponding to a molecular weight of 380 000 and possessing both activities; the CPSase was still highly sensitive to UTP unlike the ATCase which was only slightly sensitive to retroinhibition. Omission of glutamine and Mg2+ from the sucrose density gradient caused a distinct CPSase peak to trail behind the ATCase; again, the CPSase (molecular weight 250 000) retained full sensitivity to feedback inhibition. This, together with genetic data, supports the view that the ura-2 gene which controls ATCase, CPSase, and the regulatory site is a polycistronic operon, coding for the production of two or three polypeptide chains; the CPSase subunit is inactive unless a regulatory site is present, whereas the ATCase subunit (molecular weight 140 000) is highly active but completely insensitive to feedback inhibition.

scholarly journals Crystallization and characterization of human liver kynurenine–glyoxylate aminotransferase. Identity with alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase

1980 ◽  
Vol 189 (3) ◽  
pp. 581-590 ◽  
Author(s):  
Etsuo Okuno ◽  
Yohsuke Minatogawa ◽  
Masayuki Nakamura ◽  
Naoki Kamoda ◽  
Junko Nakanishi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Density Gradient ◽  
Human Liver ◽  
Analytical Ultracentrifugation ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Glyoxylate Aminotransferase

Kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s20,w value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine–glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase, serine–pyruvate aminotransferase and alanine–hydroxypyruvate aminotransferase reactions of the enzyme are presented.

scholarly journals Purification and characterization of kynurenine-2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats

1975 ◽  
Vol 151 (2) ◽  
pp. 399-406 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
M Nakatani ◽  
M Morimoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Small Intestine ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Wide Range

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.

The combined effects of temperature and dilution on the activity and feedback inhibition of yeast aspartate transcarbamylase

1969 ◽  
Vol 47 (4) ◽  
pp. 477-479 ◽  
Author(s):  
J. G. Kaplan ◽  
Irmgard Messmer
Keyword(s):  
Low Temperature ◽  
Sharp Increase ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Combined Effects ◽  
Regulatory Site ◽  
Aspartate Transcarbamylase ◽  
Small Loss ◽  
Progressive Loss ◽  
Effects Of Temperature

Dilution of a partially purified preparation of yeast aspartate transcarbamylase caused only a small loss of feedback inhibition and no change in specific activity when the assay was carried out at low temperature (0–10 °C). As the temperature of assay was increased, there was in the case of the dilute preparation a progressive loss of feedback inhibition coupled with a sharp increase in specific activity, reaching a level 400–500% that of the concentrated preparations. The data suggest that this is due to a dissociation into subunits possessing high aspartate transcarbamylase activity but lacking the regulatory site.

scholarly journals Purification, properties and assay of d-ribulose 5-phosphate 3-epimerase from human erythrocytes

1983 ◽  
Vol 211 (3) ◽  
pp. 617-623 ◽  
Author(s):  
A Karmali ◽  
A F Drake ◽  
N Spencer
Keyword(s):  
Density Gradient ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Sucrose Density Gradient ◽  
Human Erythrocytes ◽  
Sucrose Density Gradient Centrifugation ◽  
Dimeric Form

A direct assay procedure is described for D-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) which exploits differences in the c.d. spectra of substrate and product. The enzyme has been purified from human erythrocytes and was resolved by gel filtration and sucrose-density-gradient centrifugation into a major component of apparent Mr 45 000 and a minor component of Mr 23 000. Electrophoresis in sodium dodecyl sulphate gave a single component corresponding to Mr 23 000. Kinetic and sucrose-density-gradient centrifugation data indicate dissociation of the dimeric form of the enzyme into monomers of low specific activity; substrate favours the active dimeric form of the enzyme. At concentrations of the enzyme where both forms of the enzyme are present initial velocity data yielded a Hill plot with an interaction coefficient of approx. 2.0, indicating co-operative binding of substrate under these conditions.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

1973 ◽  
Vol 135 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J. F. Giorgini ◽  
F. L. De Lucca
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Dimethyl Sulphoxide ◽  
Low Molecular Weight ◽  
28S Rrna ◽  
Sucrose Density Gradient Centrifugation ◽  
Crotalus Durissus Terrificus ◽  
Crotalus Durissus

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ‘6S’ and ‘8S’ low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0°C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.

scholarly journals Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins

1980 ◽  
Vol 185 (3) ◽  
pp. 667-677 ◽  
Author(s):  
J Elliott ◽  
S G Blanchard ◽  
W Wu ◽  
J Miller ◽  
C D Strader ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Selective Extraction ◽  
Synaptic Membranes ◽  
Detergent Solution ◽  
Major Step ◽  
Sucrose Density Gradient Centrifugation ◽  
Torpedo Californica

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

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