scholarly journals PROTEASE INHIBITOR FROM THE CRUDE EXTRACT OF PLANT SEEDS AFFECTS THE DIGESTIVE PROTEASES IN HYPHANTRIA CUNEA (LEP.: ARCTIIDAE)

2013 ◽  
Vol 53 (4) ◽  
pp. 338-346 ◽  
Author(s):  
Negar Aghaali ◽  
Mohammad Ghadamyari ◽  
Vahid Hosseininaveh ◽  
Nasir Saberi Riseh
Keyword(s):  
Gel Filtration ◽  
Total Activity ◽  
Inhibitory Effect ◽  
Relative Activity ◽  
Trypsin Activity ◽  
Hyphantria Cunea ◽  
Plant Seeds ◽  
The Stability ◽  
Cellulose Membranes ◽  
Lathyrus Sativus L

Abstract Proteases are one of the most important digestive enzymes in the midgut of Hyphantria cunea Drury. Proteases are responsible for protein digestion. In the present study, we evaluated the efficiency of some plant inhibitors on proteases in the gut of the H. cunea. Last instar larvae were collected from mulberry trees. The digestive system of the larvae was used as an enzyme source. The total proteolytic and trypsin activity were assessed by the hemoglobin and BApNA, respectively, as the substrate. The evaluation of the total proteolytic and trypsin activities in various pHs showed the highest relative activity at a pH of 11. Also, the inhibitory effect of inhibitors extracted from Alhagi maurorum Medik., Lathyrus sativus L., Vicia faba L., Prosopis farcta (Banks & Sol.) Eig., and Panicum miliaceum L. on the digestive protease of the fall webworm was measured. Protease inhibitors extracted from A. maurorum, P. farcta and P. miliaceum showed negligible inhibition but L. sativus was able to inhibit 34.72% and 100% of the total activity of proteolytic and trypsin, respectively. Also, the total proteolytic and trypsin activities were inhibited by the inhibitor from V. faba, at 22.27% and 100%, respectively. The zymogram pattern of trypsin with nitro-cellulose membranes showed 2 isoforms in the gut of H. cunea. The inhibitor from L. sativus completely inhibited both isoforms. Gel electrophoresis of proteolitytic activity revealed at least 6 isoforms the inhibitor extracted from L. sativus; completely inhibiting some of them. The inhibitor from L. sativus was purified by ammonium sulfate precipitation and gel-filtration. The molecular mass of the inhibitor was determined as 45 kDa. The highest inhibition of trypsin activity by the inhibitor from L. sativus occurred at a pH of 10. The stability of the inhibitor from L. sativus was evaluated at different pHs and temperatures. The results showed that the inhibitor from L. sativus was stable at a pH of 11.0, and showed 45% inhibition on trypsin activity at a pH of 11. Also, this inhibitor revealed stability up to 50°C.

Purification of the NADP+: F420oxidoreductase ofMethanosphaera stadtmanae

2000 ◽  
Vol 46 (11) ◽  
pp. 998-1003 ◽  
Author(s):  
Dwayne A Elias ◽  
David F Juck ◽  
Karin A Berry ◽  
Richard Sparling
Keyword(s):  
Gel Filtration ◽  
Total Activity ◽  
Purification Process ◽  
Oxidoreductase Activity ◽  
Native Page ◽  
Sds Page ◽  
Methanosphaera Stadtmanae ◽  
Low Levels ◽  
The Stability ◽  
Optimal Ph

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420oxidoreductase activity. The NADP+:F420oxidoreductase, the enzyme which catalyses the electron transfer between NADP+and F420in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420reduction was 6.0, and 8.5 for NADP+reduction. During the purification process, it was noted that precipitation with (NH4)2SO4increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of α, β, and γ subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Kmvalues were 370 µM for NADP+, 142 µM for NADPH, 62.5 µM for F420, and 7.7 µM for F420H2. These values were different from the Kmvalues observed in the cell-free extract.Key words: methanogen, NADP:F420oxidoreductase, NADP reductase, F420, NADP+.

INHIBITION BY HUMAN SERUM OF THE ADIPOKINETIC EFFECT OF A HUMAN PITUITARY LIPID-MOBILIZING FACTOR

1972 ◽  
Vol 71 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Olav Trygstad ◽  
Irene Foss
Keyword(s):  
Human Serum ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Disc Electrophoresis ◽  
Inhibitory Protein ◽  
Human Pituitary ◽  
Albumin Fraction ◽  
Pituitary Glands ◽  
Normal Human

ABSTRACT A lipid-mobilizing factor (LMF) with an adipotrophic effect in human and animal fat tissue has been prepared from human pituitary glands. The addition of normal human serum to LMF reduced its lipolytic effect, and it was completely abolished by serum from a group of obese patients, whereas the lipolysis was not influenced by serum from patients with generalized lipodystrophy. By DEAE-cellulose chromatography of human serum the inhibitory effect on LMF was found to be present in a protein fraction less acidic than the main serum albumin fraction. The inhibitory fraction was deprived of some contaminants by Sephadex gel filtration. Disc electrophoresis demonstrated the presence of three components in the inhibitory protein (IP), and they were identified as albumin, transferin, and haemopexin by immuno-electrophoresis. Precipitation of these proteins by their rabbit antisera demonstrated that the inhibitory effect was present in the albumin fraction. Insulin like activity was not observed in IP. A protein binding of LMF by IP could not be demonstrated. Incubation at 37°C for one hour of a mixture of LMF and IP eliminated the electrophoretic picture of LMF. It is concluded that the inhibitory effect of human serum may be due to proteolysis of LMF.

scholarly journals Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease

Catalysts ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 55 ◽  
Author(s):  
Muhammad Syafiq Mohd Razib ◽  
Raja Noor Zaliha Raja Abd Rahman ◽  
Fairolniza Mohd Shariff ◽  
Mohd Shukuri Mohamad Ali
Keyword(s):  
Structural Analysis ◽  
Organic Solvent ◽  
Average Diameter ◽  
Relative Activity ◽  
Biochemical Properties ◽  
Biochemical Interaction ◽  
Organic Solvent Tolerant ◽  
Random Aggregate ◽  
Solvent Tolerant ◽  
The Stability

Cross-linked enzyme aggregates (CLEAs) is an immobilization technique that can be used to customize enzymes under an optimized condition. Structural analysis on any enzyme treated with a CLEA remains elusive and has been less explored. In the present work, a method for preparing an organic solvent tolerant protease using a CLEA is disclosed and optimized for better biochemical properties, followed by an analysis of the structure of this CLEA-treated protease. The said organic solvent tolerant protease is a metalloprotease known as elastase strain K in which activity of the metalloprotease is measured by a biochemical interaction with azocasein. Results showed that when a glutaraldehyde of 0.02% (v/v) was used under a 2 h treatment, the amount of recovered activity in CLEA-elastase was highest. The recovered activity of CLEA-elastase and CLEA-elastase-SB (which was a CLEA co-aggregated with starch and bovine serum albumin (BSA)) were at an approximate 60% and 80%, respectively. The CLEA immobilization of elastase strain K allowed the stability of the enzyme to be enhanced at high temperature and at a broader pH. Both CLEA-elastase and CLEA-elastase-SB end-products were able to maintain up to 67% enzyme activity at 60 °C and exhibiting an enhanced stability within pH 5–9 with up to 90% recovering activity. By implementing a CLEA on the organic solvent tolerant protease, the characteristics of the organic solvent tolerant were preserved and enhanced with the presence of 25% (v/v) acetonitrile, ethanol, and benzene at 165%, 173%, and 153% relative activity. Structural analysis through SEM and dynamic light scattering (DLS) showed that CLEA-elastase had a random aggregate morphology with an average diameter of 1497 nm.

scholarly journals Analysis of the forms of acetylcholinesterase from adult mouse brain

1975 ◽  
Vol 147 (2) ◽  
pp. 205-214 ◽  
Author(s):  
E D Adamson ◽  
S E Ayers ◽  
Z A Deussen ◽  
C F Graham
Keyword(s):  
Enzyme Activity ◽  
Mouse Brain ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Acetylcholinesterase Activity ◽  
Total Activity ◽  
Polyacrylamide Gel ◽  
Soluble Extract ◽  
Molecular Sizes

The solubilization of 80% of the acetylcholinesterase activity of mouse brain was performed by repeated 2h incubations of homogenates at 37 degrees C in an aqueous medium. Analysis of the soluble extract by gel filtration on Sephadex G-200 showed that up to 80% of the enzyme activity was eluted in a peak which was estimated to consist of molecules of about 74000mol.wt. This peak was called the monomer form of the enzyme. After 3 days at 4 degrees C, the soluble extract was re-analysed and was eluted from the column in four peaks of about 74000, 155000, 360000 and 720000 mol.wt. Since the total activity of the enzyme in these peaks was the same as that in the predominantly monomer elution profile of fresh enzyme, we concluded that the monomer had aggregated, possibly into dimers, tetramers and octomers. Extracts of the enzyme were analysed by polyacrylamide-gel electrophoresis and the resulting multiple bands of enzyme activity on gels were shown to separate according to their molecular sizes, that is by molecular sieving. All these forms had similar susceptibilities to the inhibitors eserine, tetra-isopropyl pyrophosphoramide and compound BW 284c51 [1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide]. Thus the forms of the enzyme in mouse brain which can be detected by gel filtration and polyacrylamide-gel electrophoresis may all be related to a single low-molecular-weight form which aggregates during storage. This supports similar suggestions made for the enzyme in other locations.

scholarly journals RGS4 and RGS2 Bind Coatomer and Inhibit COPI Association with Golgi Membranes and Intracellular Transport

2000 ◽  
Vol 11 (9) ◽  
pp. 3155-3168 ◽  
Author(s):  
Brandon M. Sullivan ◽  
Kimberly J. Harrison-Lavoie ◽  
Vladimir Marshansky ◽  
Herbert Y. Lin ◽  
John H. Kehrl ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Rgs Proteins ◽  
Placental Alkaline Phosphatase ◽  
Protein Signaling ◽  
Golgi Membranes

COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit β′-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on Giα. In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi–plasma membrane or intra-Golgi transport.

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.

Isomerization of Peroxidizing Thiadiazolidine Herbicides Is Catalyzed by Glutathione S-Transferase

1996 ◽  
Vol 51 (5-6) ◽  
pp. 342-354 ◽  
Author(s):  
Beate Nicolaus ◽  
Yukiharu Sato ◽  
Ko Wakabayashi ◽  
Peter Böger
Keyword(s):  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Total Activity ◽  
Naphthalic Anhydride ◽  
Molecular Weights ◽  
Isomerase Activity ◽  
Ammonium Sulfate Precipitate ◽  
Catalyzed Reactions

Abstract Thiadiazolidine-converting activity (isomerase), detected in a 45-75% ammonium sulfate precipitate from corn seedlings extracts, was purified by chromatography on hydroxyapatite and by anion exchange on Mono Q Sepharose. Two fractions 1 and 2 with isomerase activity were separated on Mono Q by combination of a stepwise elution and continuous salt gradient; fraction 2 eluting at higher salt concentrations was found the most active. Total activity could be enhanced by treatment of seedlings with naphthalic anhydride. Both fractions containing isomerase activity were further purified by glutathione-(GSH) agarose affinity chromatography and characterized by their specificity for different thiadiazolidines. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration revealed that the isomerase of fraction 2 consists either of a homodimer or a heterodimer of two proteins with apparent molecular weights of 28 and 31 kDa, respectively. The protein pattern as well as the strict dependence of activity on thiol groups (GSH or dithiothreitol) suggested a glutathione Stransferase (GST) catalyzing the thiadiazolidine conversion. Further evidence was obtained by measuring reactions specific for GSTs in both purified fractions, namely the conjugating activity for l-chloro-2,4-dinitrobenzene (CDNB ). atrazine and metazachlor. While no atrazine turnover was found, metazachlor and CDNB conjugation occurred rapidly. Both fractions differed in their activities to several GST substrates with fraction 2 being more effective in metazachlor but less active in C DN B conjugation. Inhibitors specific for GST-catalyzed reactions also inhibited thiadiazolidine conversion confirming that isomerizing activity is attributed to a GST form. We conclude that GST isoforms with different affinities towards thiadiazolidines have been isolated. CDNB activity, molecular weight, the protein pattern on SDS-PAGE as well as the amino acid sequence of one of its polypeptides suggest that fraction 1, less active in thiadiazolidine isomerization, is identical to GST I. The second peptide of this fraction was resistant to Edman degradation probably due to N-terminal blockage. The properties of the high isomerase activity found in fraction 2 are in agreement with characteristics of a GST previously termed as isoform II.

Inhibition of Human Platelet Aggregation by the Proteolytic Effect of Streptokinase (SK). Role of the Factor VIII Related Antigen

1975 ◽  
Author(s):  
R. Castillo ◽  
S. Maragall ◽  
J. A. Guisasola ◽  
F. Casals ◽  
C. Ruiz ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Factor Viii ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Factor Viii Related Antigen ◽  
Human Factor Viii ◽  
Induced Aggregation

Defective ADP-induced platelet aggregation has been observed in patients treated with streptokinase. This same effect appears “in vitro” when adding SK to platelet rich plasma (PRP). Classic hemophilia and normal platelet poor plasmas (PPP) treated with SK inhibit the aggregation of washed platelets; plasmin-treated normal human serum also shows an inhibitory effect on platelet aggregation. However, von Willebrand SK-treated plasmas do not inhibit the aggregation of washed platelets. The same results appear when plasmas are previously treated with a rabbit antibody to human factor VIII.This confirms that the antiaggregating effect is mainly linked to the digested factor VIII related antigen.The inhibition of ADP-induced platelet aggregation has been proved in gel filtration-isolated and washed platelets from SK-treated PRP.Defective ristocetin-induced platelet aggregation has also been observed- This action does not appear in washed platelets from SK-treated PRP in presence of normal PPP, but it does in presence of SK-treated PPP, which suggests that the inhibition of the ristocetin-induced aggregation is due to the lack of factor VIII and not to the factor VIII-related products.Heparin, either “in vivo” or “in vitro”, has corrected the antiaggregating effect of SK.

Inhibitory effect of ethacrynic acid on chloride permeability

1976 ◽  
Vol 231 (5) ◽  
pp. 1485-1489 ◽  
Author(s):  
R Motais ◽  
JL Cousin
Keyword(s):  
Hydrophobic Interaction ◽  
Ethacrynic Acid ◽  
Chloride Transport ◽  
Direct Proof ◽  
Inhibitory Effect ◽  
Ringer Solution ◽  
Relative Activity ◽  
Chloride Permeability ◽  
Sh Group ◽  
Highly Correlated

Ethacrynic acid inhibits anion movements in ox red blood cells. The I50 for chloride is 7 X 10(-6) M. The inhibitory effect is instantaneous and completely reversed by washing the cells with a Ringer solution, suggesting that reaction with a membrane SH group is not involved in this process. Direct proof that ethacrynic acid does not act by its reactivity with thiol groups is given by experiments with dihydroethacrynic acid, a derivative that lacks the ability to combine with SH groups: the characteristics of inhibition are strictly identical (instantaneous and reversible; I50 equals 9 X 10(-6) M). All the phenoxyacetic derivatives tested were also more or less inhibitory. The relative activity of all the derivatives was highly correlated with their liposolubility, indicating that hydrophobic interaction is important in determining drug effect and influence of steric factors is minimal. The data suggest that inhibition essentially results from a hydrophobic interaction between ethacrynic acid and apolar regions of the membrane protein allowing chloride transport.

scholarly journals Conditions Contributing to the Stability of Factor VIII Coagulant Activity

1977 ◽  
Author(s):  
T. Exner ◽  
K.A. Rickard ◽  
H. Kronenberg
Keyword(s):  
Factor Viii ◽  
High Purity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Buffer System ◽  
Factor Viii Activity ◽  
Coagulant Activity ◽  
The Stability ◽  
Degree Of Purification

Factor VTII tends to become less stable the greater its degree of purification. The loss of factor VIII during preparation of high activity concentrates makes such processes uneconomical. Conditions contributing to the stability of factor VIII were investigated.High purity factor VIII was incubated with plasma components fractionated by gel filtration and by anion exchange chromatography. Factor VIII activity was assessed initially and after several hours incubation. Several fractions destroying factor VIII activity were clearly resolved. Fractions stabilizing factor VIII were associated only with albumin.Various buffer systems were investigated similarly. A non-chelating buffer system containing albumin was found to give optimal factor VIII stability.

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