scholarly journals Characterization of Activity of a Potential Food-Grade Leucine Aminopeptidase from Kiwifruit

2010 ◽  
Vol 2010 ◽  
pp. 1-5 ◽  
Author(s):  
A. A. A. Premarathne ◽  
David W. M. Leung
Keyword(s):  
Enzyme Activity ◽  
Leucine Aminopeptidase ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Food Grade ◽  
The Core ◽  
Alkaline Conditions ◽  
Potential Food ◽  
Cellulose Column

Aminopeptidase (AP) activity in ripe but firm fruit of Actinidia deliciosa was characterized using L-leucine-p-nitroanilide as a substrate. The enzyme activity was the highest under alkaline conditions and was thermolabile. EDTA, 1,10-phenanthroline, iodoacetamide, and had inhibitory effect while a low concentration of dithiothreitol (DTT) had stimulatory effect on kiwifruit AP activity. However, DTT was not essential for the enzyme activity. The results obtained indicated that the kiwifruit AP was a thiol-dependent metalloprotease. Its activity was the highest in the seeds, followed by the core and pericarp tissues of the fruit. The elution profile of the AP activity from a DEAE-cellulose column suggested that there were at least two AP isozymes in kiwifruit: one unadsorbed and one adsorbed fractions. It is concluded that useful food-grade aminopeptidases from kiwifruit could be revealed using more specific substrates.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

Sinapine Esterase I. Characterization of Sinapine Esterase from Cotyledons of Raphanus sativus

1979 ◽  
Vol 34 (9-10) ◽  
pp. 715-720 ◽  
Author(s):  
Gerhild Nurmann ◽  
Dieter Strack
Keyword(s):  
Enzyme Activity ◽  
Esterase Activity ◽  
Raphanus Sativus ◽  
Sinapic Acid ◽  
Inhibitory Effect ◽  
Ph Optimum ◽  
Diisopropyl Fluorophosphate ◽  
E 600 ◽  
Red Radish

Abstract From cotyledons of Raphanus sativus (red radish) an esterase activity which catalyzes the hy­drolysis of sinapine into sinapic acid and choline has been isolated. The enzyme, which has a near absolute specificity, is not analogous with any esterase described in the literature. The reaction has a pH optimum of 8.5 and the apparent Km is 1.95 × 10-5 m. The enzyme is relatively insensi­tive to both physostigmine (eserine) {Ki = 1.73 × 10-4 m) and neostigmine (Ki = 2 .1 3 × 10-4 ᴍ). Diisopropyl fluorophosphate (DFP) showed no inhibition and diethyl p-nitrophenylphosphate (E 600) only a slight inhibitory effect at 10-5 ᴍ, respectively. Choline (10-2 ᴍ) was inhibitory but acetylcholine (10-2 ᴍ) stimulated the enzyme activity.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

Extracellular nuclease produced by a marine bacterium. II. Purification and properties of extracellular nuclease from a marine Vibrio sp.

1976 ◽  
Vol 22 (10) ◽  
pp. 1443-1452 ◽  
Author(s):  
M. Maeda ◽  
N. Taga
Keyword(s):  
Enzyme Activity ◽  
Deae Cellulose ◽  
Strong Stability ◽  
Rnase Activity ◽  
Salting Out ◽  
Deae Cellulose Column ◽  
Marine Vibrio ◽  
Extracellular Nuclease ◽  
Marine Vibrio Sp ◽  
Cellulose Column

Extracellular nuclease produced by a marine Vibrio sp., strain No. 2, was purified by salting out with ammonium sulfate and by chromatography on a DEAE-cellulose column and twice on a Sephadex G-200 column. The nuclease was eluted as a single peak in which the deoxyribonuclease (DNase) activity and ribonuclease (RNase) activity appeared together. Polyacrylamide disc gel electrophoresis showed a single band of stained protein which had both DNase and RNase activity. The molecular weight of the enzyme was estimated to be 100 000 daltons. When using partially purified enzyme from the DEAE-cellulose column, the optimum pH for activity was 8.0, and the enzyme was activated strongly by 0.05 M Mg2+ ion and stabilized by 0.01 M Ca2+ ion. These concentrations of Mg2+ and Ca2+ ions are similar to those of the two cations in seawater. Indeed, the enzyme revealed high activity and strong stability when kept in seawater. The presence of particulate matter, such as cellulose powder, chitin powder, Hyflosupercel, Kaolin, and marine mud increased the stability of the enzyme. When the hydrostatic pressure was increased from 1 to 1000 atmospheres, the decrements of the enzyme activity were more pronounced at 30 and 40 °C than at 25 or 50 °C. The enzyme activity was restored after decompression to 1 atm at 30 °C.

scholarly journals Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa
Keyword(s):  
Phospholipase C ◽  
Column Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Isolation And Characterization ◽  
Delta Type ◽  
Hydrolysis Of ◽  
Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

scholarly journals Detection, purification and characterization of a human cancer-associated galactosyltransferase acceptor

1979 ◽  
Vol 178 (2) ◽  
pp. 279-287 ◽  
Author(s):  
D K Podolsky ◽  
M M Weiser
Keyword(s):  
Molecular Weight ◽  
Human Cancer ◽  
Deae Cellulose ◽  
Structural Data ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Purification And Characterization ◽  
Peptide Moiety ◽  
Cellulose Column

A low-molecular-weight acceptor of galactosyltransferase activity was detected in sera and effusions of patients with extensive maligant disease. This substance was purified to homogeneity from both human serum and effusion by using sequential charcoal/Celite and DEAE-cellulose column chromatography. The purified acceptor was shown to act as substrate for both purified normal and cancer-associated human galactosyltransferase (EC 2.4.1.22) isoenzymes, but had a higher affinity for the cancer-associated isoenzyme (Km = 20 microM) than for the normal isoenzyme (Km = 500 microM). The substrate was found to be a glycopeptide with mol.wt. approx. 3600 determined by polyacrylamide-gel chromatography. Carbohyydate analysis demonstrated only the presence of glucosamine and mannose. Amino acid analysis revealed that the peptide moiety consisted of eight different amino acids, including two residues of asparagine and one residue of serine, but no threonine. These structural data suggest that the acceptor is a fraction of an asparagine-glucosamine type of glycoprotein.

scholarly journals Characterization of proteins structurally related to human N-acetyl-β-d-glucosaminidase

1978 ◽  
Vol 173 (1) ◽  
pp. 191-196 ◽  
Author(s):  
M Carroll
Keyword(s):  
Molecular Weight ◽  
Human Liver ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Enzyme Preparations ◽  
Molecular Weights ◽  
Functional Relationships ◽  
Hexosaminidase A ◽  
Cellulose Column

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000–200000) were present both in the unadsorbed fraction and in the 0.05–0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000–70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.

Effect of Streptococcal Extracellular Nuclease on the Carrier Activity of RNA for Streptolysin S

1983 ◽  
Vol 38 (1-2) ◽  
pp. 107-111 ◽  
Author(s):  
Akira Taketo ◽  
Yoriko Taketo
Keyword(s):  
Hemolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nucleotide Composition ◽  
Active Fraction ◽  
Streptolysin S ◽  
The Core ◽  
Extracellular Nuclease ◽  
Effective Component ◽  
Cellulose Column

Upon digestion with a streptococcal extracellular nuclease, yeast RNA yielded acid-insoluble core having increased carrier activity for streptolysin S. The carrier activity was found in minor fractions of the core which were eluted from a DEAE-cellulose column at higher salt concentrations. Upon gel filtration through a Sephadex G-75 column, the effective component (Fr. I) was eluted earlier than bulk oligonucleotides (Fr. II). Nucleotide composition (in mol %) of Fr. I was AMP: 21.8; GMP: 55.1; CMP: 8.2; UMP: 14.9, whereas that of Fr. II was AMP: 38.0; GMP: 33.1; CMP: 8.0; UMP: 20.9. Chromatographic patterns of SLS complex induced by Fr. I were similar to those of the toxin formed in the presence of active fraction prepared from RNase I core. Hemolytic activity of the latter complex was, like the former, unaffected by streptococcal nuclease treatment. The carrier activity of DNA digested with the nuclease was also investigated.

scholarly journals Purification and characterization of amylase from local isolate Pseudomonas sp.SPH4

2012 ◽  
Vol 6 (1) ◽  
pp. 69-79
Author(s):  
Hala M. Ali ◽  
Ghazi M. Aziz
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Silver Ions ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Mercury Ions ◽  
Local Isolate ◽  
Optimum Ph

The amylase produced from local isolate Pseudomonas sp. SPH4 was purified by precipitation with 30% saturation ammonium sulphate, followed by ion-exchange chromotography using DEAE-cellulose column, and Gel filtration using Sephacryl S-300 column.The two iso-enzymes (a, b) were purified to (2.83, 3.47) times in the last step with an enzymes yields of (32.36, 76.34)% respectively. Enzyme characterization of the two iso-enzymes indicated that the optimum pH for the two iso-enzymes a and b were (7, 7.5) respectively, while the optimum pH for the iso-enzymes stability were (6.5, 7) respectively. The maximum activity for iso-enzymes (a, b) appeared at 45ºC and stable for 15 min at 30-50ºC and lost approximately 50% of it's activity at rang above 75ºC. Enzyme characterization results showed that the chlorides of silver and mercury had inhibitory effect on enzyme activity, the remaining enzyme activity for the iso-enzymes (a, b) were (46.66, 36.36)% for silver ions and (41.33, 33.63)% for mercury ions at 5 mM respectively, and (28, 28.18)% for silver ions and (25.33, 19.09)% for mercury ions at 10 mM respectively. The iso-enzymes a and b were affected by chelating agent ethylene diamine tetra acetic acid (EDTA) at concentration 2mM the remaining activity (45.33, 43.63)% respectively, and 5mM the remaining activity (28, 28.18)% respectivily, and these iso-enzymes (a, b) refered to metalloenzymes. The iso-enzymes (a, b) were kept their activity when treated by reducing agent (2-mercaptoethanol) at 2 mM the remaining activity (92, 92.72)% respectively, and 5 mM the remaining activity (85.3, 89.09)% respectivily. The iso-enzymes (a, b) were kept their activity when treated by phenyl methyl sulphonyl fluoride (PMSF) at concentration 1mM the remaining activity (93.33, 90.90)% respectivily,and 5 mM the remaining activity (90.66, 87.27)% respectivily, and these indicated that these iso-enzymes didnot referred to serineamylases group.

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