β-Galactosidase from Bacillus stearothermophilus

1976 ◽  
Vol 22 (6) ◽  
pp. 817-825 ◽  
Author(s):  
Richard E. Goodman ◽  
Dennis M. Pederson
Keyword(s):  
Activation Energy ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Thermal Inactivation ◽  
Ph Dependence ◽  
Temperature Sensitive ◽  
Optimum Temperature ◽  
Arrhenius Activation Energy ◽  
Ph Optimum

Several strains of thermophilic aerobic spore-forming bacilli synthesize β-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The β-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 °C and the Arrhenius activation energy is about 24 kcal/mol below 47 °C and 16 kcal/mol above that temperature. At 55 °C the Km is 0.11 M for lactose and 9.8 × 10−3 M for o-nitrophenyl-β-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki = 2.5 × 10−3 M). It is relatively stable at 50 °C, losing only half of its activity after 20 days at this temperature. At 60 °C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 °C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Characterization and distribution of arginine kinase in the tissues of the scorpion, Palamneus phipsoni

1985 ◽  
Vol 63 (10) ◽  
pp. 2262-2266 ◽  
Author(s):  
A. V. Arjunwadkar ◽  
S. Raghupathi Rami Reddy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Alimentary Canal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Arginine Kinase ◽  
High Specificity ◽  
Ph Optimum ◽  
High Concentrations ◽  
Claw Muscle ◽  
Slight Inhibition

Arginine kinase in claw muscle extracts of the scorpion, Palamneus phipsoni, was characterized. The enzyme, with a pH optimum of 8.5 in the direction of phosphoarginine synthesis, showed activation by Mg2+, high specificity towards L-arginine as the guanidino substrate, slight inhibition by high concentrations of L-arginine and ATP, and a molecular weight of 33 500. On polyacrylamide gel electrophoresis at pH 8.3 the enzyme migrated to the anode as a single molecular species. In addition to the claw muscle, the enzyme activity was also found to be present in the heart, alimentary canal, hepatopancreas, and nervous system. In general, scorpion muscle arginine kinase appears to be similar in its properties to the enzyme from other arthropods.

Isozymes of α-galactosidase from Bacillus stearothermophilus

1980 ◽  
Vol 26 (8) ◽  
pp. 978-984 ◽  
Author(s):  
Dennis M. Pederson ◽  
Richard E. Goodman
Keyword(s):  
Gel Electrophoresis ◽  
Half Life ◽  
Protein Concentration ◽  
Mixed Type ◽  
Bacillus Stearothermophilus ◽  
Single Band ◽  
Molecular Forms ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Low Protein

Two molecular forms of α-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. α-Galactosidase I (with the substrate p-nitrophenyl α-D-galactopyranoside (PNPG)) has a pH optimum of 6 and a half-life at 65 °C of > 2 h at low protein concentration. α-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 °C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the β-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are α-galactosidase I, 280 000 ± 30 000 and α-galactosidase II, 325 000 ± 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 + 500 for α-galactosidase I and 84 000 ± 500 for α-galactosidase II, suggest that both enzymes consist of four subunits.

Extracellular endodextranase from the yeast Lipomyces starkeyi

1983 ◽  
Vol 29 (9) ◽  
pp. 1092-1095 ◽  
Author(s):  
E. Webb ◽  
I. Spencer-Martins
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Minimal Medium ◽  
Enzyme Preparation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Extracellular Fluid ◽  
Sole Source ◽  
Optimum Temperature ◽  
Lipomyces Starkeyi ◽  
High Concentrations

Strain IGC 4047 of the yeast Lipomyces starkeyi grew well with dextran as sole source of carbon and energy, and was able to hydrolyse blue dextran and Sephadex G-100. The enzyme was partially purified by fractionated isopropanol precipitation from the extracellular fluid of cultures grown in a minimal medium with dextran. The enzyme preparation showed only one band by polyacrylamide gel electrophoresis. The enzyme had the following properties: molecular weight, 23 000; optimum temperature and pH for activity, around 50 °C and pH 5.0, respectively; pH stability, pH 3.5–7.5; after 2 h at 50 °C and pH 5.0, 30% reduction in activity; isoelectric point, pI = 5.4; final products of dextran hydrolysis, isomaltooligosaccharides from glucose up to isomaltohexaose, with high concentrations of isomaltose and isomaltotriose. These results suggest that the enzyme is an endodextranase.

scholarly journals Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

1981 ◽  
Vol 195 (3) ◽  
pp. 545-560 ◽  
Author(s):  
Heinz Fankhauser ◽  
Jerome A. Schiff ◽  
Leonard J. Garber
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Low Ph ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Unknown Compound ◽  
Reactive Blue 2

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH4)2SO4 precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent Km for the last-mentioned compound is 0.82mm. The apparent Km for ammonia (assuming NH3 to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.

Incidence of plasmid DNA in Salmonella strains isolated from clinical sources in Ontario, Canada, during 1979 and 1980

1982 ◽  
Vol 28 (10) ◽  
pp. 1150-1157 ◽  
Author(s):  
Diane E. Taylor ◽  
Jessie G. Levine ◽  
Kallie L. Kouvelos
Keyword(s):  
Molecular Weight ◽  
Plasmid Dna ◽  
Gel Electrophoresis ◽  
Temperature Sensitive ◽  
Large Plasmid ◽  
Incompatibility Group ◽  
Transfer Resistance ◽  
Antibiotic Resistant ◽  
Molecular Weights ◽  
Resistant Strains

Gel electrophoresis of DNA from 70 clinical strains of Salmonella revealed a heterogeneous plasmid population. Plasmid DNA, ranging in molecular weight from 1.4 × 106 to 145 × 106, was demonstrated in 26 of 32 antibiotic-resistant strains. Several resistant strains carried up to six plasmids; however, of these, five strains which were multiply resistant contained a single plasmid of molecular weight 54 × 106 to 145 × 106. Only one incompatibility group H2 (IncH2) plasmid (pDT28) was detected in a strain of S. heidelberg; thus, this represents a reduction in the prevalence of these plasmids in Ontario Salmonella strains since 1974. The pDT28 plasmid resembled other IncH2 plasmids by its high molecular weight (145 × 106) and by virtue of its temperature-sensitive mode of transfer, resistance to tellurium, and inhibition of coliphage development. Of the 38 antibiotic-susceptible Salmonella strains, approximately half contained plasmids, ranging in molecular weight from 1.4 × 106 to 60 × 106. The plasmid-containing antibiotic-susceptible strains carried either a group of two to four small plasmids, with molecular weights less than 4.5 × 106, or a single large plasmid of molecular weight 23 × 106 or 60 × 106.

scholarly journals Human placental cathepsin B1. Isolation and some physical properties

1974 ◽  
Vol 137 (2) ◽  
pp. 223-228 ◽  
Author(s):  
Arnold A. Swanson ◽  
Bill J. Martin ◽  
Sam S. Spicer
Keyword(s):  
Heavy Metals ◽  
Molecular Weight ◽  
Physical Properties ◽  
Human Placenta ◽  
Homogeneous State ◽  
Optimum Temperature ◽  
Thiol Groups ◽  
Ph Optimum

A reproducible procedure for the isolation, from human placenta, of a cathepsin B1 in a homogeneous state, demonstrated by electrophoretic, ultracentrifugal and enzymic criteria, was carried out. The pH optimum was near pH5.5. The placental enzyme catalysed the release of acid-soluble u.v.-dense products from haemoglobin and myoglobin. It was inhibited by heavy metals and several compounds which react with the thiol groups. The optimum temperature was between 37° and 42°C. The molecular weight of the enzyme was calculated to be 24250.

Separation and characterization of an α-glucosidase and maltase from Bacillus amyloliquefaciens

1985 ◽  
Vol 31 (8) ◽  
pp. 670-674 ◽  
Author(s):  
William M. Fogarty ◽  
Catherine T. Kelly ◽  
Sunil K. Kadam
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Growth Medium ◽  
Isoelectric Point ◽  
Bacillus Amyloliquefaciens ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Ph Optimum

A novel α-glucosidase and a maltase were isolated from Bacillus amyloliquefaciens. The formation of both enzymes was induced by trehalose, sucrose, or lactose in the growth medium. Trehalose is by far the most efficient inducer of both systems. The α-glucosidase and maltase were separated and purified by ion-exchange chromatography on DEAE Bio-Gel A. Purified α-glucosidase hydrolysed p-nitrophenyl-α-D-glucoside, isomaltose, and isomaltotriose but sucrose, maltose, or related saccharides were not attacked. β-Glucosides and polymeric glucosides were not degraded. The optimum temperature for α-glucosidase activity was 40 °C and its pH optimum was 5.3. The molecular weight and isoelectric point (pI) of the enzyme were 27 000 and 4.6, respectively. Purified maltase attacked maltose and sucrose, while maltotriose and melezitose were hydrolysed at slower rates and p-nitrophenyl-α-D-glucoside was not degraded. Other properties of the maltase were as follows: optimum temperature for activity, 30 °C; pH optimum, 6.5; molecular weight, 64 000; and pI, 4.7.

AMP metabolism by the marine bacterium Vibrio (Beneckea) natriegens: purification and properties of adenylate kinase

1981 ◽  
Vol 27 (10) ◽  
pp. 1053-1059 ◽  
Author(s):  
Karamchand Ramotar ◽  
Michael A. Pickard
Keyword(s):  
Molecular Weight ◽  
Adenylate Kinase ◽  
Gel Electrophoresis ◽  
Marine Bacterium ◽  
Specific Activity ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Atp Formation ◽  
Purification And Properties ◽  
Beneckea Natriegens

Adenylate kinase (EC 2.7.4.3) has been purified 484-fold from extracts of Vibrio natriegens to a specific activity of 1350 μmol ADP formed∙min−1∙mg protein−1. The preparation was 97% pure as judged by gel electrophoresis and exhibited molecular weight values of 29 000 by gel filtration and 32 000 by SDS–gel electrophoresis. The isoelectric point was at pH 4.7. Only ATP (Km 0.067 mM), ADP (Km 0.45 mM), and AMP (Km 0.12 mM) exhibited high activity as substrates, though dATP or dAMP could serve as cosubstrates with AMP or ATP, respectively, at reduced rates. The equilibrium constant in the direction of ATP formation was 1.09, and the pH optimum in both directions was broad, from pH 7.2 to pH 7.6. Enzyme activity was sensitive to the thiolalkylating agents iodacetamide and p-chloromercuriphenyl sulfonate.

scholarly journals d-Xylose (d-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Purification and properties

1991 ◽  
Vol 277 (1) ◽  
pp. 255-261 ◽  
Author(s):  
C A Smith ◽  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Activation Energy ◽  
Soluble Protein ◽  
Protein Sequences ◽  
Glucose Isomerase ◽  
Competitive Inhibitor ◽  
Total Soluble Protein ◽  
Arrhenius Activation Energy ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Arthrobacter Strain

D-Xylose (D-glucose) isomerase was purified to homogeneity in yields of approx. 1 g/kg of wet cells from a strain of Arthrobacter that produces it as about 10% of total soluble protein. It is a tetramer of identical 43,114 Da subunits containing a preponderance of acidic residues and no cysteine. Partial protein sequences were determined as a step to gene cloning. It requires Mg2+, Co2+ or Mn2+ for activity, Mg2+ being best; Ca2+ is an inhibitor, competitive with Mg2+. It is a good D-glucose isomerase with kcat. 1200 min-1 at pH 8 at 60 degrees C, which is higher than that of any other enzyme of this class. L-Arabinose, D-ribose and D-lyxose are poor substrates, with kcat. 78, 31 and 3.7 min-1 respectively at pH 8 at 30 degrees C, compared with 533 min-1 for D-xylose. Xylitol is a true competitive inhibitor for D-xylose (Ki 0.3 mM), but D-sorbitol shows mixed inhibition (Ki 6.5 mM). For D-fructose the pH optimum at 60 degrees C is 8, and at pH 7 the Arrhenius activation energy is 75 kJ/mol over the range 30-70 degrees C.

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