scholarly journals Isolation, characterization and the role of rabbit testicular arysulphatase A in fertilization

1979 ◽  
Vol 181 (2) ◽  
pp. 331-337 ◽  
Author(s):  
A A Farooqui ◽  
P N Srivastava
Keyword(s):  
Specific Activity ◽  
Deae Cellulose ◽  
Cumulus Cells ◽  
Kinetic Properties ◽  
Neutral Sugar ◽  
Acetylneuraminic Acid ◽  
Crude Homogenate ◽  
Step Procedure ◽  
Arylsulphatase A

Arysulphatase A was purified from rabbit testis. The purification was accomplished by a four-step procedure involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, SP(sulphopropyl)-Sephadex and affinity chromatography on concanavalin A-Sepharose. The specific activity of purified preparation was 135 mumol/min per mg of protein, which represented an increase of 900-fold above that of the crude homogenate. The purified enzyme (20-50 micrograms) was found to move electrophoretically as a single band on polyacrylamide gel at pH 7.2 and 8.4. The homogeneous enzyme was shown to be a glycoprotein with 0.8% (w/w) of N-acetylneuraminic acid and 20% neutral sugar. The treatment of purified enzyme with bacterial neuraminidase had no effect on enzyme activity or kinetic properties, but it changed the elution prolife of rabbit testis arylsulphatase A through DEAE-Sephadex. The purified enzyme was strongly inhibited by Cu2+, Fe3+ and Ag+. It hydrolysed several sulphate esters including cerebroside 3-sulphate, ascorbic acid 2-sulphate and steroid sulphates. Pure arysulphatase was effective in dispersing the cumulus cells of rabbit ova.

scholarly journals Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties

1978 ◽  
Vol 175 (2) ◽  
pp. 391-406 ◽  
Author(s):  
R Jones ◽  
M B Wilkins ◽  
J R Coggins ◽  
C A Fewson ◽  
A D B Malcolm
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Maximum Velocity ◽  
Single Band ◽  
Kinetic Properties ◽  
Subunit Structure

Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

scholarly journals Cerebral-cortex hexokinase. Comparison of properties of solubilized mitochondrial and cytoplasmic activities

1970 ◽  
Vol 118 (1) ◽  
pp. 25-34 ◽  
Author(s):  
M. F. Thompson ◽  
H. S. Bachelard
Keyword(s):  
Cerebral Cortex ◽  
Deae Cellulose ◽  
Kinetic Properties ◽  
Sucrose Solutions ◽  
Michaelis Constants ◽  
Ammonium Sulphate Fractionation ◽  
Triton X ◽  
Two Peaks ◽  
Basic Similarity

1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50–80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, Km(ATP) 0.60mm, Km(glucose) 0.042mm; peak II, Km(ATP) 0.66mm, Km(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, Km(ATP) 0.56mm, Km(glucose) 0.048mm; peak II, Km(ATP) 0.68mm, Km(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.

scholarly journals Isolation of β-N-acetylhexosaminidase from rabbit semen and its role in fertilization

1980 ◽  
Vol 191 (3) ◽  
pp. 827-834 ◽  
Author(s):  
A A Farooqui ◽  
P N Srivastava
Keyword(s):  
Enzyme Activity ◽  
Metal Ions ◽  
Seminal Plasma ◽  
Concanavalin A ◽  
Zona Pellucida ◽  
Specific Activity ◽  
Optimal Activity ◽  
Step Procedure ◽  
Km Value ◽  
Arylsulphatase A

Beta-N-Acetylhexosaminidase was purified from the rabbit seminal plasma by a three-step procedure involving hydroxyapatite, Sephadex G-200 and concanavalin A–Sepharose chromatography. The specific activity of the purified preparation was 56mu mol/min per mg of protein, which represented a 226-fold purification and a 54% yield of the enzyme activity. The purified enzyme was electrophoretically homogeneous. The homogeneous enzyme showed optimal activity at pH4.0. The apparent Km value and Vmax. were 1.4 mM and 56mu mol/min per mg of protein respectively. Metal ions such as Ag + and Hg2+ and p-chloromercuribenzoate strongly inhibited the enzyme activity. The treatment of rabbit ova with a mixture of Beta-N-acetylhexosaminidase and arylsulphatase A results in the swelling of the zona pellucida.

The isolation, properties, and physiological role of lactic dehydrogenase from soybean cotyledons

1970 ◽  
Vol 48 (3) ◽  
pp. 533-540 ◽  
Author(s):  
J. King
Keyword(s):  
Specific Activity ◽  
Sulfhydryl Group ◽  
Physiological Role ◽  
Lactic Dehydrogenase ◽  
Reverse Direction ◽  
Kinetic Properties ◽  
Soybean Seeds ◽  
Time Period ◽  
Glycine Max L

L-Lactate:NAD oxidoreductase (EC. 1.1.1.27) was purified 110-fold from non-green soybean (Glycine max L. var. Canadian No. 1) cotyledons, and some of its kinetic properties were studied and compared to the properties of lactic dehydrogenases isolated from animals and microorganisms. The soybean enzyme was specific for L-lactate and NAD+ but in the reverse direction reduced not only pyruvate but also hydroxypyruvate and glyoxylate in the presence of NADH, although pyruvate was shown to be the preferred substrate. Optimum activity occurred at pH 9.2 in the direction of pyruvate formation and at pH 7.0 in the reverse direction. In its response to the use of coenzyme analogues and to heat treatment it resembled closely the L-lactic dehydrogenase from Lactobacillus plantarum. Its responses to acrylamide gel electrophoresis and to sulfhydryl group inhibitors were comparable to those of similar enzymes from animal sources.The physiological role of the enzyme in germinating soybean seeds, especially during the first 30 h when anaerobic conditions obtain within the seed, was assessed by measuring its specific activity and also by measuring the rise and fall of lactic acid concentration in cotyledons over the same time period. Various aspects of the metabolism of germinating fatty seeds are discussed in relation to this and other work recently reported.

scholarly journals Alkaline phosphatase from pig kidney. Microheterogeneity and the role of neuraminic acid

1974 ◽  
Vol 141 (1) ◽  
pp. 293-298 ◽  
Author(s):  
Kunio Hiwada ◽  
Ernst D. Wachsmuth
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border Membrane ◽  
Deae Cellulose ◽  
Multiple Forms ◽  
Alkaline Phosphatases ◽  
Acetylneuraminic Acid ◽  
Pig Kidney ◽  
Ph Dependency ◽  
Polypeptide Chains

Several alkaline phosphatases (EC 3.1.3.1) could be obtained from pig kidney brush-border membrane on extraction with butan-1-ol. Three of the multiple forms were separated by DEAE-cellulose chromatography and further purified. They form a regular series with different degrees of glycosylation (mainly owing to N-acetylneuraminic acid), of charge, of molecular weight, of stability to temperature, to pH and to urea, of minimal requirement for Mg2+ and of extractability by butan-1-ol. In contrast, the detectable antigenic sites, the inhibition by amino acids and the pH-dependency of Km and Vmax. were identical for these multiple forms. On treatment with neuraminidase, the multiple forms became identical in all their properties. It was therefore concluded that the microheterogeneity of alkaline phosphatase is due to different degrees of glycosylation at polypeptide chains which appear to be otherwise identical.

scholarly journals Thrombokinase of the Blood as Trypsin-Like Enzyme

1962 ◽  
Vol 45 (4) ◽  
pp. 103-113 ◽  
Author(s):  
J. H. Milstone
Keyword(s):  
Methyl Ester ◽  
Trypsin Inhibitor ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Protein Band ◽  
Soybean Trypsin Inhibitor ◽  
Major Protein ◽  
Bovine Plasma ◽  
Major Protein Band

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity.

scholarly journals Kinetic properties of the acylneuraminate cytidylyltransferase from Pasteurella haemolytica A2

2001 ◽  
Vol 358 (3) ◽  
pp. 585-598 ◽  
Author(s):  
Ignacio G. BRAVO ◽  
Sofía BARRALLO ◽  
Miguel A. FERRERO ◽  
Leandro B. RODRÍGUEZ-APARICIO ◽  
Honorina MARTÍNEZ-BLANCO ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Specific Activity ◽  
Polysialic Acid ◽  
Kinetic Mechanism ◽  
Pasteurella Haemolytica ◽  
Kinetic Properties ◽  
Acid Activation ◽  
Acetylneuraminic Acid ◽  
Apparent Homogeneity ◽  
Encapsulated Bacteria

Neuroinvasive and septicaemia-causing pathogens often display a polysialic acid capsule that is involved in invasive behaviour. N-Acetylneuraminic acid (NeuAc) is the basic monomer of polysialic acid. The activated form, CMP-Neu5Ac, is synthesized by the acylneuraminate cytidylyltransferase (ACT; EC 2.7.7.43). We have purified this enzyme from Pasteurella haemolytica A2 to apparent homogeneity (522-fold). The protein behaved homogeneously on SDS/PAGE as a 43kDa band, a size similar to that of Escherichia coli, calf, mouse and rat. Specific activity in crude lysate displayed one of the highest values cited in the literature (153m-units/mg). We have studied the steady-state kinetic mechanism of the enzyme by using normalized plot premises. The catalysis proceeds through a Ping Pong Bi Bi mechanism, with CTP as the first substrate and CMP-NeuAc as the last product. The true Km values were 1.77mM for CTP and 1.82mM for NeuAc. The nucleotides CDP, UTP, UDP and TTP, and the modified sialic acid N-glycolylneuraminic acid were also substrates of the ACT activity. The enzyme is inhibited by cytidine nucleotides through binding to a second cytidyl-binding site. This inhibition is greater with nucleotides that display a long phosphate tail, and the genuine inhibitor is the substrate CTP. At physiological concentrations, ATP is an activator, and AMP an inhibitor, of the ACT activity. The activated sugar UDP-N-acetylglucosamine acts as an inhibitor, thus suggesting cross-regulation of the peptidoglycan and polysialic acid pathways. Our findings provide new mechanistic insights into the nature of sialic acid activation and suggest new targets for the approach to the pathogenesis of encapsulated bacteria.

scholarly journals Role of neuraminic acid in the heterogeneity of alkaline phosphatase in sheep brain

1968 ◽  
Vol 107 (2) ◽  
pp. 185-190 ◽  
Author(s):  
S. Saraswathi ◽  
B. K. Bachhawat
Keyword(s):  
Alkaline Phosphatase ◽  
Deae Cellulose ◽  
Acetylneuraminic Acid ◽  
Deae Cellulose Column ◽  
Elution Pattern ◽  
Sheep Brain ◽  
Substrate Affinities ◽  
Enzyme I ◽  
Cellulose Column

1. Two alkaline phosphatase fractions from sheep brain obtained by DEAE-cellulose column chromatography were shown to be associated with different concentrations of NANA (N-acetylneuraminic acid). Enzyme II contains nearly three times as much NANA as does enzyme I. 2. Partial removal of NANA by neuraminidase digestion from these alkaline phosphatase fractions has different effects on their chromatographic properties. Though the enzymic release of NANA has no effect on the elution pattern of enzyme I from a DEAE-cellulose column, such a treatment shifts the elution pattern of enzyme II towards that of enzyme I. 3. However, this change in the elution pattern of enzyme II as a result of the removal of NANA does not produce any change in the kinetics of this fraction, and the differences between enzyme I and enzyme II with respect to their substrate affinities and Ki for phosphate inhibition are maintained even after the removal of NANA. 4. Results indicate that NANA is not the only factor responsible for the heterogeneity of alkaline phosphatase in sheep brain and enzyme I is not the result of the removal of NANA from enzyme II.

Molecular and kinetic characterisation of sugarcane pyrophosphate: fructose-6-phosphate 1-phosphotransferase and its possible role in the sucrose accumulation phenotype

2007 ◽  
Vol 34 (6) ◽  
pp. 517 ◽  
Author(s):  
Jan-Hendrik Groenewald ◽  
Frederik Coenraad Botha
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Kinetic Properties ◽  
Sucrose Accumulation ◽  
Forward Reaction ◽  
Ph Optimum ◽  
Aggregation State ◽  
Inorganic Pyrophosphate ◽  
Fructose 1,6 Bisphosphate

The amount of pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in sugarcane internodal tissue is inversely correlated with sucrose content. To help elucidate this apparent role of PFP in sucrose accumulation in sugarcane we have determined its molecular and kinetic properties. Sugarcane PFP was purified 285-fold to a final specific activity of 4.23 µmol min–1 mg–1 protein. It contained two polypeptides of 63.2 and 58.0 kDa respectively, at near equal amounts that cross-reacted with potato PFP-α and –β antiserum. In gel filtration analyses the native enzyme eluted in three peaks of 129, 245 and 511 kDa, corresponding to dimeric, tetrameric and octameric forms, respectively and fructose 2,6-bisphosphate (Fru 2,6-P2) influenced this aggregation state. Both the glycolytic (forward) and gluconeogenic (reverse) reactions had relative broad pH optima between pH 6.7 and 8.0. The Fru 2,6-P2 saturation curves were hyperbolic with approximate Ka values of 69 and 82 nm for the forward and reverse reactions, respectively. The enzyme showed hyperbolic saturation curves for all its substrates with Km values comparable with that of other plant PFP, i.e. 150, 37, 39 and 460 µM for fructose 6-phosphate, inorganic pyrophosphate, fructose 1,6-bisphosphate and inorganic phosphate, respectively. Sugarcane PFP’s molecular and kinetic characteristics differed slightly from that of other plant PFP in that: (i) Fru 2,6-P2 directly induced the octameric state from the dimeric state; (ii) Fru 2,6-P2 shifted the pH optimum for the forward reaction to a slightly more basic pH; and (iii) Fru 2,6-P2 increased the Vmax for the forward and reverse reactions by similar amounts.

Sign in / Sign up

Export Citation Format

Share Document