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.

A Comparative Study of the Distribution of Soluble and Particulate Glycyl-l-Leucine Hydrolase in the Small Intestine

1974 ◽  
Vol 46 (4) ◽  
pp. 501-510
Author(s):  
Manjusri Das ◽  
A. N. Radhakrishnan
Keyword(s):  
Enzyme Activity ◽  
Guinea Pig ◽  
Comparative Study ◽  
Specific Activity ◽  
Maximal Activity ◽  
Total Activity ◽  
Soluble Enzyme ◽  
Km Value ◽  
Specific Activities ◽  
Very High

1. A comparative study has been made of glycyl-l-leucine hydrolase activity in the soluble and particulate fractions of intestinal mucosa from monkey, guinea-pig, rabbit and rat. The specific activity of the soluble enzyme is very high in monkey and guinea-pig, and lower in rabbit and rat. The particulate enzymes from all the four species show low specific activities and form 1–10% of the total activity. 2. The pH optima in all cases lie in the range 7·6–7·8. The Km values of the substrate were similar for both soluble and particulate enzyme from monkey and guinea-pig, but in the rabbit and rat the Km value with the particulate enzyme was higher than with the soluble enzyme. 3. The particulate enzyme activity in all cases was the highest in the distal regions of the intestine, whereas the soluble enzyme showed maximal activity in the proximal and middle regions.

Isolation and characterization of electrophoretic variants of human prostatic acid phosphatase.

1983 ◽  
Vol 29 (11) ◽  
pp. 1886-1889 ◽  
Author(s):  
M D Hibbard ◽  
R C McCarthy ◽  
H Markowitz
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Specific Activity ◽  
Prostatic Acid Phosphatase ◽  
Ph Gradient ◽  
Electrophoretic Variants ◽  
Isolation And Characterization ◽  
Immunochemical Determination ◽  
Km Value ◽  
Specific Activities

Abstract Prostatic acid phosphatase (EC 3.1.3.2) purified from benign hypertrophic prostate tissue was fractionated by preparative slab isoelectric focusing over a pH gradient of 3.16 to 7.16. Twenty-two of 29 fractions contained enzyme activity. We further examined each active fraction by determining the Michaelis-Menten constant and specific activity. The protein concentration used in the latter determination was estimated either spectrophotometrically or immunochemically by three different radioimmunoassays for the enzyme. Determination of specific activities for each fraction directly correlated enzyme activity with an immunochemical determination, which indicated the immunochemical relationships among different molecular species of the enzyme. We found that the Michaelis-Menten constants for the isolated fractions were similar to the Km value for purified, unfractionated enzyme. Most fractions analyzed by each immunoassay had similar specific activities; the few fractions with discrepant specific activities were found at either end of the pH gradient. The similarity in specific activities among the fractions indicates that RIAs involving polyclonal antisera detect all of the electrophoretic variants of the enzyme.

A simple procedure for partial purification of an RNAase of Entamoeba invadens

1981 ◽  
Vol 27 (8) ◽  
pp. 856-859
Author(s):  
David L. Weller ◽  
Andrea Richman
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Sucrose Gradient ◽  
Specific Activity ◽  
Simple Procedure ◽  
Partial Purification ◽  
Step Procedure ◽  
Entamoeba Invadens

The behavior of an RNAase, present in centrifugally clarified homogenates of axenic trophozoites of Entamoeba invadens, on isoelectric focusing (IEF) and agarose–poly(G) chromatography is described. The results led to a simple two-step procedure for partial purification of the RNAase in which fractionation of the homogenate by IEF is followed by agarose–poly(G) chromatography. Recovery of enzyme activity has ranged from 50 to 80% of that present in homogenates, and increases of 80- to 160-fold in the specific activity have been obtained using the procedure. A single zone of activity was observed on analysis of the partially purified RNAase by sucrose gradient IEF and velocity zonal ultracentrifugation.

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 Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

scholarly journals Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T1 seedlings of tomato exposed to metal ions

2013 ◽  
Vol 170 (3) ◽  
pp. 346-354 ◽  
Author(s):  
Hossein Kamaladini ◽  
Siti Nor Akmar Abdullah ◽  
Maheran Abdul Aziz ◽  
Ismanizan Bin Ismail ◽  
Fatemeh Haddadi
Keyword(s):  
Metal Ions ◽  
Oil Palm ◽  
Specific Activity ◽  
Gene Promoter ◽  
Tissue Specific

scholarly journals Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 325 ◽  
Author(s):  
He Chen ◽  
Jie Huang ◽  
Binyun Cao ◽  
Li Chen ◽  
Na Song ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Plantarum ◽  
Industrial Development ◽  
Extraction Efficiency ◽  
Specific Activity ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Kinetic Studies ◽  
Predicted Values ◽  
Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

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