scholarly journals Isolation and partial characterization of a lectin from Bauhinia pentandra (bong) vog. Ex. Steua.

2001 ◽  
Vol 13 (3) ◽  
pp. 262-269 ◽  
Author(s):  
ANDRÉ LUIS COELHO DA SILVA ◽  
ANA CECÍLIA GÓES HORTA ◽  
RENATO DE AZEVEDO MOREIRA
Keyword(s):  
Protein Band ◽  
Carbohydrate Specificity ◽  
Original Activity ◽  
Divalent Metal Cations ◽  
Ph Values ◽  
Seed Flour ◽  
Sds Page ◽  
Low Levels ◽  
Sepharose 4B

Bauhinia pentandra (Bong) Vog. ex. Steua seeds were investigated with respect to phenologic aspects (size, mass, hilum and length) and with respect to their chemical composition. The total nitrogen content of the seed flour was determined, and the flour was extracted in different pH values. A lectin was isolated from the seeds by Sepharose-4B affinity chromatography. The homogeneity of the lectin was demonstrated by SDS-PAGE in the presence of beta-mercaptoethanol. Only one protein band with an apparent molecular mass of 30 kDa was found. The B. pentandra lectin showed a carbohydrate specificity for D-galactose, a requirement for divalent metal cations (Ca2+ and Mn2+) for full activity and amino acid composition with a high content of aspartic acid, glutamic acid and alanine and low levels of methionine, cysteine and tryptophan. The lectin agglutinated rabbit and human A group erythrocytes and was relatively stable to heat treatment, retaining half of its original activity after 60 min at 70 ºC.

scholarly journals Characterization of the soluble, secreted form of urinary meprin

1996 ◽  
Vol 315 (2) ◽  
pp. 461-465 ◽  
Author(s):  
Robert J. BEYNON ◽  
Simon OLIVER ◽  
Duncan H. L. ROBERTSON
Keyword(s):  
Proteolytic Activity ◽  
Protein Band ◽  
Peptide Sequence ◽  
Soluble Form ◽  
Α Subunit ◽  
Alternative Processing ◽  
Sds Page ◽  
Processing Pathways ◽  
Membrane Bound

A soluble form of the kidney membrane metalloendopeptidase, meprin, is present in urine. Urinary meprin is expressed in BALB/C mice with the Mep-1a/a genotype (high meprin, expressing meprin-α and meprin-β) but not in BALB.K mice of the Mep-1b/b genotype (that only express meprin-β). Western blotting with antisera specific to the meprin-α and the meprin-β subunits established that the only form of meprin present in urine samples was derived from meprin-α. This form of meprin is partially active, and comprises at least three variants by non-reducing SDS/PAGE and by zymography and two protein bands on reducing SDS/PAGE. Sequencing of these two bands established that the N-terminus of the larger protein band begins with the pro-peptide sequence of the α-subunit (VSIKH..), whereas the smaller band possessed the mature meprin N-terminal sequence (NAMRDP..). Trypsin is able to remove the pro-peptide, with a concomitant activation in proteolytic activity. After deglycosylation, the size of the pro- and mature forms of urinary meprin are consistent with cleavage in the region of the X–I boundary. There is a pronounced sexual dimorphism in urinary meprin expression. Females secrete a slightly larger form, and its proteolytic activity is about 50% of that released by males. The urinary meprin is therefore a naturally occurring secreted form of this membrane-bound metalloendopeptidase and is more likely to be generated by alternative processing pathways than by specific release mechanisms.

scholarly journals Identification of rat liver phosphatidylinositol synthase as a 21 kDa protein

1994 ◽  
Vol 304 (1) ◽  
pp. 301-305 ◽  
Author(s):  
M E Monaco ◽  
M Feldman ◽  
D L Kleinberg
Keyword(s):  
Heat Treatment ◽  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Rat Liver ◽  
Protein Band ◽  
Absolute Requirement ◽  
Sds Page ◽  
Sepharose 4B ◽  
Post Mitochondrial Supernatant

Substantial purification of rat liver phosphatidylinositol (PtdIns) synthase has been achieved by a combination of Hecameg extraction, heat treatment, affinity chromatography and chromatography on PBE-94. The activity chromatographs as a single peak which has an apparent molecular mass between 150 and 200 kDa on Sepharose 4B. When analysed by SDS/PAGE, two major bands are seen. The enzyme activity is correlated with a protein band of 21 kDa. A second band, at 51 kDa, is eluted from a PBE-94 column slightly ahead of the activity. Manganese is an absolute requirement for stabilization of activity in the presence of detergent. The effect of manganese is optimal at 0.5 mM; magnesium at a concentration of 10 mM is only minimally effective. Substrate Kms are 1.3 mM and 9.5 microM for inositol and CDP-diacylglycerol respectively. The activity eluting from the PBE-94 column is purified 5000-fold over the post-mitochondrial supernatant.

Cloning and Characterization of a Novel N-acetylglucosaminidase (AtlD) from Enterococcus faecalis

2018 ◽  
Vol 28 (1) ◽  
pp. 14-27 ◽  
Author(s):  
Carlos Eduardo Serrano-Maldonado ◽  
Israel García-Cano ◽  
Augusto González-Canto ◽  
Eliel Ruiz-May ◽  
Jose Miguel Elizalde-Contreras ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Heterologous Protein ◽  
Biochemical Characterization ◽  
Ph Values ◽  
Food Borne ◽  
Sds Page ◽  
Exclusion Chromatography

The <i>atlD</i> gene from<i></i> an <i>Enterococcus faecalis</i> strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in <i>Escherichia coli</i> in order to perform a biochemical characterization<i>.</i> A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62–75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6–7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn<sup>2+</sup>. It showed antibacterial activity against <i>Listeria monocytogenes</i>,<i> Staphylococcus aureus</i>, and enterococcal<i></i> strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.

scholarly journals Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease

1996 ◽  
Vol 313 (2) ◽  
pp. 423-429 ◽  
Author(s):  
Rajamma USHA ◽  
Manoranjan SINGH
Keyword(s):  
Physiological Role ◽  
Immunoblot Analysis ◽  
Protein Band ◽  
Winged Bean ◽  
Psophocarpus Tetragonolobus ◽  
Ph Optimum ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Storage Protein Mobilization

Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca2+-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

PURIFICATION AND CHARACTERIZATION OF THE PROCOAGULANT OF THE VENOM OF TROPIDECHIS CARINATUS

1987 ◽  
Author(s):  
P P Masci ◽  
A N Whitaker ◽  
J J Morrison ◽  
E A Bennett
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor V ◽  
Crude Venom ◽  
Adult Rats ◽  
Sds Page ◽  
Sepharose 4B ◽  
Blue Sepharose

Tropidechis carinatus is a venomous elapid snake distributed throughout Eastern Queensland. It has been considered as a tropical relative of Notechis scutatus and, similarly, the crude venom contains an indirect prothrombin activator, which will clot plasma provided that Factor V is present. Myotoxins and neurotoxins are also present. Envenomated patients regularly develop disseminated intravascular coagulation. The crude whole venom of T.carinatus was shown to have five major components by gel filtration, SDS PAGE and HPLC, and even more components by isoelectric focusing. The procoagulant eluted with a molecular weight of 55,000, being found in peak II on gel filtration on Sephadex-G150. The procoagulant was purified using a combination of Sephadex-G150 chromatography and ion-exchange on DEAE Sephadex-A50 and shown to migrate as a single band of molecular weight 55.000 by SDS PAGE. On reduction by β -mercaptoethanol this component was resolvec into u heavy chain of molecular weight 30.000 and a light chain of 25,000. The procoagulant was shown to bind to con A-Sepharose 4B and Blue Sepharose 4B. Coagulation studies using this purified procoagulant confirmed a factor Xa-like activity activating prothrombin in the presence of factor V. The purified fraction is unstable in buffer solutions at 4°C, probably because of trypsin - like autodigestion. Ouchterlony studies of the procoagulants of T.carinatus and N.scutatus show both lines of homogeneity and spurring, indicating similarities but also significant differences between the two proteins. The purified procoagulant was lethal to adult rats, an IV injection of 10 μg killing in 1 - 2 minutes. Death was prevented by prior heparinization, suggesting that the procoagulant is toxic in the absence of neurotoxin and other components.

scholarly journals Purification and biochemical characterization of an extracellular lipase produced by a new strain of Rhizopus sp.

2006 ◽  
Vol 30 (3) ◽  
pp. 494-502 ◽  
Author(s):  
Maria Gabriela Bello Koblitz ◽  
Gláucia Maria Pastore
Keyword(s):  
Molecular Mass ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Extracellular Lipase ◽  
Optimum Activity ◽  
Ph Values ◽  
Sds Page ◽  
Rhizopus Sp

The present study had as a goal to purify and characterize the lipolytic fraction secreted by a strain of Rhizopus sp. Only 3 steps of purification were necessary to achieve SDS-PAGE homogeneity. One band with 37.5 KDa molecular mass and with 1446 U/mg specific activity was obtained. The purified fraction presented 2 lipase isoforms; both showed optimum activity at 50ºC, and were stable between 6.5 and 7.5 pH values and at temperatures below 50ºC and also kept their activity in hexane. The lipase was inactivated by Hg+2 and by n-bromosuccinimide and activated by Na+.

scholarly journals The murine lymphocyte receptor for IgE. II. Characterization of the multivalent nature of the B lymphocyte receptor for IgE.

1984 ◽  
Vol 159 (6) ◽  
pp. 1790-1795 ◽  
Author(s):  
W T Lee ◽  
D H Conrad
Keyword(s):  
B Cell ◽  
B Lymphocyte ◽  
Affinity Column ◽  
Column Eluate ◽  
Sds Page ◽  
Murine Lymphocyte ◽  
Low Levels

The murine B lymphocyte Fc epsilon R is functionally multivalent. Radiolabeled rat IgE, when bound to the B cell Fc epsilon R will co-isolate with the Fc epsilon R on a rat IgE affinity column; examination of the affinity column eluate by SDS-PAGE reveals the component previously identified as the Fc epsilon R as well as E and L chains from IgE. At low levels of Fc epsilon R saturation, up to 30% of the Fc epsilon R bound IgE becomes bound to IgE-Affi-Gel. By using a biotin-avidin system, the coprecipitation of non-haptenated IgE with haptenated IgE was examined and the results suggest (but do not prove) a divalent receptor.

scholarly journals Partial purification and characterization of a superoxide distimutase (SOD1) from black tiger shrimps Penaeus monodon

2020 ◽  
Vol 42 (1) ◽  
Author(s):  
Tran Minh Hien ◽  
Nguyen Thi Hong Loan ◽  
Trinh Dinh Quynh ◽  
Ngo Thi Trang ◽  
Dang Thi Lua ◽  
...  
Keyword(s):  
Specific Activity ◽  
Penaeus Monodon ◽  
Protein Band ◽  
Partial Purification ◽  
Sod Activity ◽  
Black Tiger Shrimp ◽  
Tiger Shrimp ◽  
Sds Page ◽  
Muscular Tissues

Superroxide dismutase (SOD, EC.1.15.1.1) is the enzyme which dismutates superoxide radicals and plays an important role in protection of living cells against oxidative stress. SOD is also involved in immune response in shrimps. In this study, it was found that the total SOD activity of black tiger shrimp muscular tissues is 10 fold higher than that of the haemolymph, however, the specific activity of SOD in the shrimp haemolymph is 9.2 fold higher than that of muscular tissues. By using active gel electrophoresis, 2 different SOD forms were found in black tiger shrimps (one in muscular tissues and two in haemolymph).Using DE-52 cellulose and Q-Sepharose ion exchange column chromatography, one SOD (SOD1) from black tiger shrimp haemolymph was partially purified, and its purity was 31.2 times higher than that of the starting haemolymph. The SOD1 was shown to have mainly one protein band of approximately 24 kDa on SDS-PAGE. SOD1 was most active at 45oC and pH of 5.5. At a concentration of 5 mM, Mn2+ strongly activated SOD1 (up 200% activity), Ca2+ và Zn2+ could increase approximately 20% activity while Cu2+ inhibited more than 60% ativity of the enzyme. 

Influence of pH on endo- and exo-pectinase production by Aspergillus sp. CH-Y-1043

1991 ◽  
Vol 37 (12) ◽  
pp. 912-917 ◽  
Author(s):  
Guillermo Aguilar ◽  
Blanca A. Trejo ◽  
Juan M. García ◽  
Carlos Huitrón
Keyword(s):  
Fungal Growth ◽  
Protein Band ◽  
Ph Values ◽  
Sds Page ◽  
Pectin Esterase ◽  
Aspergillus Sp ◽  
Pectinase Production ◽  
Key Words Aspergillus ◽  
Maximal Production

Aspergillus sp. CH-Y-1043 grown on pectins with various degrees of esterification produces endo- and exo-pectinases at pH values as low as 2.5. Maximal production was attained at this pH, although fungal growth only approximated 50% of that obtained at higher pH values. Endopectinase was produced at pH 2.5–3.5 when the fungus was grown on low degree esterified pectin. With higher degree esterified pectin this enzyme was produced at all pH values analyzed. Exopectinase production was less affected by pH values. Still, maximal production was also reached at pH 2.5–3.5. Exopectinase was found to be associated to the cell and could be released after incubation at different pH values, whereas endo pectinase was not detected in the cellular fraction. Results confirmed by SDS–PAGE coupled with in situ activity assays in pectin–agarose gels allowed the identification of a protein band corresponding to endopectinase and a band with pectin esterase activity. Stability of Aspergillus sp. CH-Y-1043 pectinases at various pH values was also evaluated. Key words: Aspergillus sp. CH-Y-1043, extreme acidic pH pectinase production, in situ pectinase detection, cell-associated exopectinase.

scholarly journals Purification and partial characterization of a novel lectin from elder (Sambucus nigra L.) fruit

1991 ◽  
Vol 278 (3) ◽  
pp. 667-671 ◽  
Author(s):  
L Mach ◽  
W Scherf ◽  
M Ammann ◽  
J Poetsch ◽  
W Bertsch ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Amino Acid ◽  
Carbohydrate Specificity ◽  
Sambucus Nigra ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Sambucus Nigra Agglutinin ◽  
Sds Page ◽  
Molecular Masses

A previously unknown haemagglutinin, named Sambucus nigra agglutinin-III (SNA-III), has been purified from the fruit of the elder (Sambucus nigra). Whereas elder bark agglutinin I (SNA-I) is highly specific for terminal alpha 2,6-linked sialic acid residues, SNA-III displays a high affinity for oligosaccharides containing exposed N-acetylgalactosamine and galactose residues. Different N-terminal sequences and the amino acid composition distinguish the fruit lectin from elder bark agglutinin II (SNA-II), which shows a similar carbohydrate specificity. The 40-fold higher affinity of SNA-III for asialofetuin than for human asialo-alpha 1-acid glycoprotein and human asialotransferrin respectively suggests a preference for O-linked glycans. SNA-III occurs mainly as a monomeric glycoprotein, but tends to form di- and oligo-meric aggregates. This aggregation seems to mediate the multivalent interaction, leading to agglutination. SDS/PAGE revealed two major polypeptides with apparent molecular masses of 32 and 33 kDa respectively. This heterogeneity is probably a result of proteolysis in the C-terminal region. Binding to concanavalin A and susceptibility to peptide: N-glycosidase F indicated the presence of N-glycosidically linked oligosaccharides.

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