scholarly journals Partial purification and properties of carbamoyl phosphate synthetase of Alaska pea (Pisum sativum L. cultivar Alaska). Purification and general properties

1969 ◽  
Vol 113 (2) ◽  
pp. 271-279 ◽  
Author(s):  
T. Denny O'neal ◽  
Aubrey W. Naylor
Keyword(s):  
Pisum Sativum ◽  
Ammonium Sulphate ◽  
Partial Purification ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Pisum Sativum L ◽  
Low Concentrations ◽  
Purification And Properties ◽  
Phosphate Donor ◽  
Increased Activity

1. Carbamoyl phosphate synthetase was purified up to 45-fold from Alaska pea seedling (Pisum sativum L. cultivar Alaska). 2. The enzyme was most active with and had the lowest Km for l-glutamine as compared with NH4+. 3. The purest preparations utilized very poorly or not at all l-asparagine and urea as nitrogen donors. 4. At saturating concentrations of components of the reaction, the Km for l-glutamine was 1·2×10−4m, and the Km for ATP was approx. 3·9×10−4m. 5. Although the enzyme was very labile, stability was improved by glutamine, asparagine, ammonium sulphate, dithiothreitol and especially l-ornithine. 6. Free ATP was markedly inhibitory, and MgATP2− and Mg2+ appeared to be the actual substrates utilized. 7. Fe2+ and Mn2+ were also utilized, but not as readily as Mg2+ except at low concentrations. K+ increased activity significantly. 8. Of the four nucleotides tested (ITP, ATP, GTP and UTP) only ATP served as an effective phosphate donor.

Human liver aldehyde dehydrogenase: partial purification and properties

1969 ◽  
Vol 47 (3) ◽  
pp. 265-272 ◽  
Author(s):  
A. H. Blair ◽  
F. H. Bodley
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Human Liver ◽  
Deae Cellulose ◽  
Maximal Activity ◽  
Partial Purification ◽  
Aliphatic Aldehydes ◽  
Low Concentrations ◽  
Purification And Properties ◽  
Liver Aldehyde Dehydrogenase ◽  
Metabolic Reduction

Aldehyde dehydrogenase was partially purified from human liver. During purification, activity was resolved into one major and one minor species by DEAE-cellulose column chromatography; the properties of the predominant form were investigated.Aldehydes are oxidized when NAD+, but not NADP+, is the electron acceptor, maximal activity occurring between pH 9 and 10. Several aliphatic aldehydes and hydroxyaldehydes served as substrates for the enzyme. Benzaldehyde also was oxidized, but at a comparatively low rate. Aliphatic aldehydes carrying negatively charged groups are not oxidized. The enzyme is sensitive to low concentrations of two sulfhydryl reagents, p-chloromercuribenzoate and mercuric ions; this inhibition was reversed with sulfhydryl compounds. Like other aldehyde dehydrogenases, the human liver enzyme is inhibited by arsenite and the inhibition is potentiated by mercaptoethanol. Only 35% inhibition was produced by disulfiram at 40 μM; and diethyldithiocarbamate, its metabolic reduction product, had no effect on activity below 10 mM.

scholarly journals Purification and properties of asparaginase from the testa of immature seeds of pea (Pisum sativum L.)

2001 ◽  
Vol 44 (3) ◽  
pp. 239-245 ◽  
Author(s):  
Eliana P. Chagas ◽  
Ladaslav Sodek
Keyword(s):  
Pisum Sativum ◽  
Competitive Inhibition ◽  
Native Page ◽  
Protein Affinity ◽  
Pisum Sativum L ◽  
Crude Extracts ◽  
Purification And Properties ◽  
Sds Page ◽  
Immature Seeds ◽  
Pea Seeds

A K+-dependent asparaginase (E.C. 3.5.1.1.) was purified 1328-fold from the testas of immature pea seeds (Pisum sativum L., var. Bolero) and characterized. Antibodies raised against purified asparaginase cross-reacted with the putative asparaginase band in Western blot analyses of semi-purified extracts. However, for crude extracts of pea testas, a cross-reaction was obtained with at least four protein bands, one of which was asparaginase protein. Affinity-purified antibodies to the four strongest bands of crude extracts were fairly specific for the bands from which they were purified, suggesting a mixture of specific antibodies. The Mr of asparaginase was 69,000 by Sephacryl S200 chromatography and also by mobility on native PAGE relative to BSA. There was no evidence for dissociation into subunits on SDS-PAGE, suggesting a monomeric protein of Mr 69,000. Other properties include an apparent Km of 2.4 mM, pI between 4.5 and 5, and competitive inhibition by aspartate and glycine.

scholarly journals Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis

1984 ◽  
Vol 217 (2) ◽  
pp. 435-440 ◽  
Author(s):  
P C Rumsby ◽  
P C Campbell ◽  
L A Niswander ◽  
J N Davidson
Keyword(s):  
Pyrimidine Biosynthesis ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Multifunctional Protein ◽  
Aggregation Site ◽  
Aspartate Carbamoyltransferase ◽  
Low Concentrations ◽  
Proteinase A

When the multifunctional protein that catalyses the first three steps of pyrimidine biosynthesis in hamster cells is treated with staphylococcal V8 proteinase, a single cleavage takes place. The activities of carbamoyl-phosphate synthetase (EC 6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3) and the allosteric inhibition by UTP are unaffected. One fragment, of Mr 182000, has the first and third enzyme activities, whereas the other fragment, of Mr 42000, has aspartate carbamoyltransferase activity and an aggregation site. A similar small fragment is observed in protein digested with low concentrations of trypsin. A similar large fragment is seen after digestion with trypsin and as the predominating form of this protein in certain mutants defective in pyrimidine biosynthesis. These results indicate that a region located adjacent to the aspartate carbamoyltransferase domain is hypersensitive to proteinase action in vitro and may also be sensitive to proteolysis in vivo.

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