scholarly journals Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1965 ◽  
Vol 97 (1) ◽  
pp. 112-124 ◽  
Author(s):  
PJ Peterson ◽  
L Fowden
Keyword(s):  
Carboxylic Acid ◽  
Enzyme Preparation ◽  
Enzymic Activity ◽  
The Other ◽  
Asparagus Officinalis ◽  
Acid Activation ◽  
Phaseolus Aureus ◽  
Substrate Specificities ◽  
Imino Acids ◽  
Ammonium Sulphate Fractionation

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.

scholarly journals Interaction of soluble glucosyl- and mannosyl-transferase enzyme activities in the synthesis of a glucomannan

1972 ◽  
Vol 129 (3) ◽  
pp. 645-655 ◽  
Author(s):  
J. S. Heller ◽  
C. L. Villemez
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Enzyme Preparation ◽  
The Other ◽  
Acceptor Molecule ◽  
Soluble Enzyme ◽  
Phaseolus Aureus ◽  
Polymeric Product ◽  
Sugar Nucleotide ◽  
Solubilized Enzyme

A neutral-detergent-solubilized-enzyme preparation derived from Phaseolus aureus hypocotyls contains two types of glycosyltransferase activity. One, mannosyltransferase enzyme activity, utilizes GDP-α-d-mannose as the sugar nucleotide substrate. The other, glucosyltransferase enzyme activity, utilizes GDP-α-d-glucose as the sugar nucleotide substrate. The soluble enzyme preparation catalyses the formation of what appears to be a homopolysaccharide when either sugar nucleotide is the only substrate present. A β-(1→4)-linked mannan is the only polymeric product when only GDP-α-d-mannose is added. A β-(1→4)-linked glucan is the only polymeric product when only GDP-α-d-glucose is added. In the presence of both sugar nucleotides, however, a β-(1→4)-linked glucomannan is formed. There are indications that endogenous sugar donors may be present in the enzyme preparation. There appear to be only two glycosyltransferases in the enzyme preparation, each catalysing the transfer of a different sugar to the same type of acceptor molecule. The glucosyltransferase requires the continual production of mannose-containing acceptor molecules for maintenance of enzyme activity, and is thereby dependent upon the activity of the mannosyltransferase. The mannosyltransferase, on the other hand, does not require the continual production of glucose-containing acceptors for maintenance of enzyme activity, but is severely inhibited by GDP-α-P-glucose. These properties promote the synthesis of β-(1→4)-linked glucomannan rather than β-(1→4)-linked glucan plus β-(1→4)-linked mannan when both sugar nucleotide substrates are present.

Carbocation catalyzed carboxylic acid activation in Staudinger reaction for stereoselective synthesis of β-lactams

2016 ◽  
Vol 57 (46) ◽  
pp. 5084-5088 ◽  
Author(s):  
Ankita Rai ◽  
Puneet K. Singh ◽  
Prashant Shukla ◽  
Vijai K. Rai
Keyword(s):  
Carboxylic Acid ◽  
Stereoselective Synthesis ◽  
Acid Activation ◽  
Staudinger Reaction

scholarly journals Proline Control of the Feeding Reaction of Cordylophora

1963 ◽  
Vol 46 (4) ◽  
pp. 823-837 ◽  
Author(s):  
Chandler Fulton
Keyword(s):  
Carboxylic Acid ◽  
Brine Shrimp ◽  
Reduced Glutathione ◽  
Significant Activity ◽  
Magnesium Ions ◽  
Active Molecule ◽  
Imino Acid ◽  
Phylogenetic Implications ◽  
Imino Acids ◽  
Colonial Hydroid

The colonial hydroid Cordylophora is a carnivore whose feeding is induced by substances released from captured prey. An active molecule, probably the only one, has been isolated from a fraction of the laboratory food of Cordylophora, brine shrimp larvae, and identified on paper chromatograms as the imino acid proline. Reagent proline induces the feeding reaction at 10-5 M. The reaction is specific in that only two α-imino acids very closely related to proline were found to possess significant activity: azetidine-2-carboxylic acid and pipecolic acid. The response to proline is inhibited by magnesium ions and enhanced by phosphate. Since previous studies have shown that the feeding reactions of Hydra, Physalia, and Campanularia are controlled by reduced glutathione, the phylogenetic implications of the proline control of feeding in Cordylophora are discussed. The feeding reactions of both Cordylophora and Hydra are also induced by proteases, suggesting similar mechanisms of induction in the two hydroids.

scholarly journals Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver

1989 ◽  
Vol 257 (1) ◽  
pp. 221-229 ◽  
Author(s):  
L Schepers ◽  
M Casteels ◽  
K Verheyden ◽  
G Parmentier ◽  
S Asselberghs ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholic Acid ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
The Other ◽  
Acid Activation ◽  
Long Chain Fatty Acids ◽  
Microsomal Membranes ◽  
Triton X ◽  
Long Chain

The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.

Lactate dehydrogenase isoenzyme 1 with the centrifugal analyzer after immunochemical removal of other isoenzymes.

1981 ◽  
Vol 27 (6) ◽  
pp. 922-923 ◽  
Author(s):  
D E Bruns ◽  
J C Emerson ◽  
R L Bertholf ◽  
K E Hill ◽  
J Savory
Keyword(s):  
Lactate Dehydrogenase ◽  
Reference Interval ◽  
Enzymic Activity ◽  
The Other ◽  
Dehydrogenase Isoenzyme

Abstract We describe a centrifugal analyzer method for measuring the LD-1 isoenzyme of lactate dehydrogenase (EC 1.1.1.27) in serum, after immunochemical precipitation of the other four isoenzymes. Enzymic activity was measured kinetically at 30 degrees C with the pyruvate-to-lactate assay. The method for LD-1 was linear to 1000 U/L. The precision (CV) of the assay was 1.0--2.2% within-run and 3.1--4.5% day-to-day. The reference interval was 26--73 U/L (n = 51), corresponding to 21--35% of total LD activity.

scholarly journals Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents

1988 ◽  
Vol 250 (1) ◽  
pp. 53-58 ◽  
Author(s):  
F Flamigni ◽  
C Guarnieri ◽  
C M Caldarera
Keyword(s):  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Effective Concentration ◽  
The Other ◽  
Limited Effect ◽  
Liver Cytosol ◽  
Rat Liver Cytosol ◽  
Dependent Mechanism

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated ‘A’, eluted near the void volume (Mr greater than or equal to 60,000), and the other designated ‘B’, eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.

THE ROLE OF ADSORPTION IN CONTROLLING THE RATE OF REACTION OF CHLORAMBUCIL WITH PROTEIN

1963 ◽  
Vol 41 (4) ◽  
pp. 931-939 ◽  
Author(s):  
J. H. Linford
Keyword(s):  
Molecular Weight ◽  
Carboxylic Acid ◽  
Bovine Serum Albumin ◽  
Inverse Relationship ◽  
Reaction Rate ◽  
The Other ◽  
Alkylation Reaction ◽  
Rate Of Reaction

Two proteins, haemoglobin and bovine serum albumin, have been studied with respect to their rates of alkylation by chlorambucil in vitro at 37 °C and pH 8.4. The proteins are of nearly the same molecular weight and free carboxylic acid content, but the alkylation reaction is 30 times faster with haemoglobin. On the other hand, the adsorption of chlorambucil by albumin is 20 times greater than that exhibited by haemoglobin. This inverse relationship between extent of adsorption and reaction rate suggests that adsorption protects the chlorambucil from activation in the solvent.

IV. Cysteine proteinases

1970 ◽  
Vol 257 (813) ◽  
pp. 231-236 ◽  
Keyword(s):  
Oxidation State ◽  
Proteolytic Enzyme ◽  
Enzyme Preparation ◽  
Chemical Nature ◽  
Enzymic Activity ◽  
Active Enzyme ◽  
Tropical Tree ◽  
Cysteine Proteinases ◽  
Sulphydryl Group ◽  
Sh Group

Papain (EC 3.4.4.10) is a proteolytic enzyme which is isolated from the Papaya, a common tropical tree. It is a sulphydryl enzyme and its SH group is required for enzymic activity. Papain as usually prepared (Kimmel & Smith 1954) contains only a small portion of active molecules. The majority of the molecules are inactive because their sulphydryl group is blocked. Part of the blocking is caused by disulphide formation with cysteine (Sluyterman 1967). This disulphide can be reduced by an excess of cysteine resulting in an active enzyme preparation. The free SH content never reaches 100% and is often not more than about 50% , so that we must distinguish between papain molecules with a reversibly and an irreversibly blocked SH group. The chemical nature of the irreversible blocking is not yet known. It might well be due to a higher oxidation state of the sulphur which cannot be reduced by an excess of cysteine (Glazer & Smith 1965).

ISOLATION AND IDENTIFICATION OF TWO PHENAZINES FROM A STRAIN OF PSEUDOMONAS AUREOFACIENS

1965 ◽  
Vol 43 (9) ◽  
pp. 1055-1062 ◽  
Author(s):  
J. I. Toohey ◽  
C. D. Nelson ◽  
G. Krotkov
Keyword(s):  
Carboxylic Acid ◽  
Absorption Spectra ◽  
Infrared Absorption ◽  
Chemical Properties ◽  
The Other ◽  
Isolation And Identification ◽  
Pseudomonas Aureofaciens ◽  
Infrared Absorption Spectra

A method is described for the purification of two phenazine compounds from cultures of a strain of Pseudomonas aureofaciens. The chemical properties of the compounds are described and the ultraviolet, visible, and infrared absorption spectra are shown. One compound is identified as phenazine-1-carboxylic acid and the other is probably 2-hydroxyphenazine-1-carboxylic acid.

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