Displaying publications 41 - 60 of 226 in total

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  1. Rice tungro bacilliform virus open reading frame 3 encodes a single 37-kDa coat protein
    Marmey P, Bothner B, Jacquot E, de Kochko A, Ong CA, Yot P, et al.
    Virology, 1999 Jan 20;253(2):319-26.
    PMID: 9918890
    Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  2. Detection of immunoglobulin antibodies in the sera of patients using purified latex allergens
    Kurup VP, Yeang HY, Sussman GL, Bansal NK, Beezhold DH, Kelly KJ, et al.
    Clin Exp Allergy, 2000 Mar;30(3):359-69.
    PMID: 10691894
    BACKGROUND: Latex allergy is largely an occupational allergy due to sensitization to natural rubber latex allergens present in a number of health care and household products. Although several purified allergens are currently available for study, information on the usefulness of these purified, native or recombinant allergens in the demonstration of specific immunoglobulin (Ig) E in the sera of patients is lacking.

    OBJECTIVE: To evaluate the purified latex allergens and to demonstrate specific IgE antibody in the sera of health care workers and spina bifida patients with clinical latex allergy.

    METHODS: Two radioallergosorbent and an enzyme-linked immunosorbent assay (ELISA) using latex proteins Hev b 1, 2, 3, 4, 6 and 7 along with two glove extracts and Malaysian nonammoniated latex (MNA) were evaluated to demonstrate IgE in the sera of health care workers and spina bifida with latex allergy and controls with no history of latex allergy.

    RESULTS: ELISA using the purified latex allergens demonstrated specific IgE in 32-65% health care workers and 54-100% of spina bifida patients with latex allergy. The corresponding figures for RAST were 13-48 and 23-85 for RAST-1 and 19-61 and 36-57 for RAST-2. These results were comparable with the results obtained with glove extracts and crude rubber latex proteins.

    CONCLUSIONS: When used simultaneously, latex proteins Hev b 2 and Hev b 7 reacted significantly with specific serum IgE in 80% of health care workers and 92% of spina bifida patients with latex allergy by ELISA technique, while this combination gave lower positivity when the RASTs were used. By the addition of Hev b 3, specific IgE was detected in all spina bifida patients with latex allergy. Both RASTs failed to show specific IgE in the control subjects, while the ELISA showed significant latex-specific IgE in 22% of controls.

    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  3. Fulltext A one dimensional PAGE studies on Puntius javanicus liver proteome affected by copper toxicity
    Mohd Khalizan Sabullah, Azlan Jualang Gansau, Mohd Rosni Sulaiman, Fisal Ahmad
    Bioremediation Science and Technology Research, 2015;3(2):26-29.
    MyJurnal
    Observations on the effects of copper on the liver proteome of Puntius javanicus based on the
    one dimensional PAGE was carried out. The liver was dissected from each fish, which was
    separately treated with different concentrations of copper sulfate ranging from 0.1 to 5.0 mg/L.
    The livers were extracted and one dimensional PAGE was performed under nonreducing
    (native) and reducing (SDS)-PAGE. Several bands were resolved in the native PAGE with
    probable candidates for the effect of copper observed showing an increased in the expression
    and downregulation strongly associated with increasing copper concentrations. This study
    showed that high concentrations of copper significantly alters P. javanicus liver at the proteome
    level, and preliminary screening based on one dimensional PAGE is considered rapid and
    simple to assess the toxicity effect of copper before more advanced and extensive assesment
    with a second dimensional PAGE is carried out.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  4. Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris
    Lau YL, Fong MY
    Exp Parasitol, 2008 Jul;119(3):373-8.
    PMID: 18457835 DOI: 10.1016/j.exppara.2008.03.016
    The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n=20), acute (IgM positive, IgG positive; n=20) and chronic (IgM negative, IgG positive; n=20) toxoplasmosis patients, and toxoplasmosis negative control patients (n=20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients' serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P<0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  5. Fulltext Extraction, purification and characterisation of a milk-clotting protease from ‘kesinai’ (Streblus asper Lour.) leaves
    Pagthinathan, M., Ghazali, H.M., Yazid, A.M., Foo, H.L.
    International Food Research Journal, 2019;26(3):913-922.
    MyJurnal
    Extracts from ‘kesinai’ (Streblus asper) leaves were investigated as a potential source of enzymes that can serve as an alternative to calf rennet in cheese making. Different types of extraction buffers were investigated namely sodium acetate buffer (pH 4.2-5.0), phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally, the milk-clotting enzyme was extracted using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone, 0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out using acetone precipitation, and ion-exchange and size-exclusion chromatographic techniques. Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer among the buffers used in the extraction study. After the final purification step of size-exclusion chromatography, the enzyme was purified 3.3-fold with 42.3% of recovery. The enzyme showed an optimum temperature and pH at 60°C and pH 7.4, respectively. The enzyme was stable up to 70°C for one hour and the partially purified enzyme retained 83% and 96% of its original activity at pH 6.0 and 8.0, respectively. The molecular weight of the partially enzyme was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of ‘kesinai’ enzyme was found to be lower than that of commercial Mucor rennet.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  6. Proteomics of Grade 3 infiltrating ductal carcinoma in Malaysian Chinese breast cancer patients
    Othman MI, Majid MI, Singh M, Subathra S, Seng L, Gam LH
    Biotechnol Appl Biochem, 2009 Mar;52(Pt 3):209-19.
    PMID: 18564057 DOI: 10.1042/BA20070271
    Breast cancer is the leading cause of cancer-related mortality and morbidity among women worldwide and IDC (infiltrating ductal carcinoma) is the most common type of invasive breast cancer. The changes in the biological behaviour of cancer tissue can be predicted by measuring the differential protein expression of normal and cancerous tissues. Using a combination of SDS/PAGE and LC (liquid chromatography)-MS/MS (tandem MS), we identified 82 common and differentially expressed proteins from normal and cancerous breast tissues in 20 Malaysian Chinese patients with IDC. These proteins are extracted from the normal and cancerous tissue of patients and therefore represent the actual proteins involved in cancer development. Proteins identified possibly have significant roles in the development of breast cancer in Malaysian Chinese patients in view of their consistent expression in most of the patients, although some of the proteins had not been detected in earlier studies that were mostly carried out in Western countries. This observation suggests that molecular mechanisms leading to breast cancer development in this region may not be identical with those leading to IDC in Western regions.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  7. Method development for analysis of proteins extracted from the leaves of Orthosiphon aristatus
    Koay SY, Gam LH
    J Chromatogr B Analyt Technol Biomed Life Sci, 2011 Jul 15;879(22):2179-83.
    PMID: 21689998 DOI: 10.1016/j.jchromb.2011.05.041
    Orthosiphon aristatus is a traditionally used medicinal plant. In order to study the proteome of the plant, we have developed a simple plant protein extraction method by direct extraction of protein using a modified 2D-gel compatible tris-sucrose buffer followed by a double TCA-acetone precipitation. This method omitted the use of toxic phenol which is widely used in the studies of plants proteins. Moreover, it shortens the lengthy extraction procedure of phenol extraction and back-extraction method and therefore reduced the extraction time (by 2h) while increased in protein yields (by 50%). Comparison of the 2D-gel images of the two extracts revealed that >60 extra protein spots were detected in the extract of our current method. The method was applied on the leaves of O. aristatus collected from six geographical areas in Malaysia. The correlation coefficient of each replicate gels from the six areas ranged from 0.70 to 0.90 indicating good reproducibility of the method.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  8. Identification of lactic acid bacteria from chili bo, a Malaysian food ingredient
    Leisner JJ, Pot B, Christensen H, Rusul G, Olsen JE, Wee BW, et al.
    Appl Environ Microbiol, 1999 Feb;65(2):599-605.
    PMID: 9925588
    Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28 degreesC with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg-1 (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  9. Proteomic investigation of Sri Lankan hump-nosed pit viper (Hypnale hypnale) venom
    Tan CH, Tan NH, Sim SM, Fung SY, Gnanathasan CA
    Toxicon, 2015 Jan;93:164-70.
    PMID: 25451538 DOI: 10.1016/j.toxicon.2014.11.231
    The hump-nosed pit viper, Hypanle hypnale, contributes to snakebite mortality and morbidity in Sri Lanka. Studies showed that the venom is hemotoxic and nephrotoxic, with some biochemical and antigenic properties similar to the venom of Calloselasma rhodostoma (Malayan pit viper). To further characterize the complexity composition of the venom, we investigated the proteome of a pooled venom sample from >10 Sri Lankan H. hypnale with reverse-phase high performance liquid chromatography (rp-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide sequencing (tandem mass-spectrometry and/or N-terminal sequencing). The findings ascertained that two phospholipase A2 subtypes (E6-PLA2, W6-PLA2) dominate the toxin composition by 40.1%, followed by snake venom metalloproteases (36.9%), l-amino acid oxidase (11.9%), C-type lectins (5.5%), serine proteases (3.3%) and others (2.3%). The presence of the major toxins correlates with the venom's major pathogenic effects, indicating these to be the principal target toxins for antivenom neutralization. This study supports the previous finding of PLA2 dominance in the venom but diverges from the view that H. hypnale venom has low expression of large enzymatic toxins. The knowledge of the composition and abundance of toxins is essential to elucidate the pathophysiology of H. hypnale envenomation and to optimize antivenom formulation in the future.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  10. Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein
    Levitskiy SA, Sycheva AM, Kharlampieva DD, Oberto J, Kamashev DE, Serebryakova MV, et al.
    Biochimie, 2011 Jul;93(7):1102-9.
    PMID: 21443922 DOI: 10.1016/j.biochi.2011.03.005
    HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  11. Galactose-binding lectin from the seeds of champedak (Artocarpus integer): sequences of its subunits and interactions with human serum O-glycosylated glycoproteins
    Abdul Rahman M, Anuar Karsani S, Othman I, Shafinaz Abdul Rahman P, Haji Hashim O
    Biochem Biophys Res Commun, 2002 Jul 26;295(4):1007-13.
    PMID: 12127996
    Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  12. Temporal variation of Malaysian rotavirus electropherotypes
    Rasool N, Othman RY, Adenan MI, Hamzah M
    J Clin Microbiol, 1989 Apr;27(4):785-7.
    PMID: 2470775
    An analysis of rotavirus electropherotypes circulating in Kuala Lumpur, Malaysia, over 7 years showed that all except 1 of the 360 electropherotypes encountered were characteristic of group A rotaviruses. The long electropherotype predominated annually, and there was a rarity of short electropherotypes. Extensive genome variability and cocirculation of different electropherotypes were observed annually. A sequential appearance of the predominant electropherotype was observed in all years of the study, except for 1985 and 1988, when one electropherotype predominated throughout the study periods. There was no shift in the predominant electropherotype over a 6-year period.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  13. Fulltext A High-Yield Two-Hour Protocol for Extraction of Human Hair Shaft Proteins
    Wong SY, Lee CC, Ashrafzadeh A, Junit SM, Abrahim N, Hashim OH
    PLoS One, 2016;11(10):e0164993.
    PMID: 27741315 DOI: 10.1371/journal.pone.0164993
    Proteome analysis of the human hair remains challenging due to the poor solubility of hair proteins and the difficulty in their extraction. In the present study, we have developed a rapid extraction protocol for hair shaft protein using alkaline-based buffer. The new protocol accelerated the procedure by reducing the extraction time from at least a day to less than two hours and showed a protein recovery of 47.3 ± 3.72%. Further analyses of the extracted protein sample through sodium dodecyl sulfate polyacrylamide gel electrophoresis and Quadrupole-time-of-flight mass spectrometry analysis unveiled a total of 60 proteins, including 25 that were not previously reported. Identification of these proteins is anticipated to be crucial in helping to understand the molecular basis of hair for potential applications in the future.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  14. Infeksi Plasmodium berghei dan kesannya ke atas pengisyaratan MAP kinase eritrosit perumah
    Mohd Fakharul Zaman Raja Yahya, Hasidah Mohd Sidek
    Sains Malaysiana, 2009;38.
    Kajian ini melibatkan pemantauan perkembangan parasitemia dan taburan morfologi Plasmodium berghei sewaktu infeksi parasit dalam mencit, serta penentuan kesan infeksi P. berghei ke atas pengisyaratan MAP kinase eritrosit perumah. Analisis mikroskop ke atas slaid calitan darah terwarna-Giemsa yang disediakan daripada mencit terinfeksi-P. berghei (strain PZZ1/00) menunjukkan darjah parasitemia mencapai sehingga 70% dalam masa dua minggu selepas penyuntikan parasit. Morfologi cecincin dan trofozoit parasit dicerap dengan jelas sepanjang tempoh infeksi manakala morfologi skizon parasit hanya dicerap dengan ketara selepas hari ketiga selepas penyuntikan parasit. Pemblotan Western [antibodi primer: anti-MAP kinase (ERK-1/2 tak terfosfat) monoklon; antibodi sekunder: anti-IgG, poliklon terkonjugat-HRP] ke atas protein sitosol eritrosit terinfeksi-P. berghei (70% parasitemia) susulan pemisahan SDS-PAGE menunjukkan bahawa keamatan protein imunoreaktif-MAP kinase eritrosit berberat molekul 42 dan 44 kDa didapati meningkat secara signifikan (p<0.05) pada 70% iaitu peningkatan sebanyak 21.5% dan 22.3% masing-masing berbanding sampel kawalan tanpa infeksi. Samada kesan infeksi P. berghei (70% parasitemia) ke atas pengisyaratan MAP kinase perumah ini berkaitan dengan pengaktifan enzim ini perlu dikaji dengan lebih lanjut.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  15. Actions of three clostridial IgA proteases on distinct forms of immunoglobulin A molecules
    Hashim OH, Hassan H
    Immunology, 1991 Jun;73(2):235-8.
    PMID: 2071167
    Three bacterial species of Clostridium (septicum, tertium and sporogenes) were identified to produce extracellular proteases cleaving IgA to Fab and Fc fragments, as demonstrated by SDS-PAGE and immunoelectrophoretic procedures. These enzymes acted on monometric IgA1 paraproteins and normal serum IgA1 but had no activity on IgA2 paraproteins and intact secretory IgA1 from human colostrum. Their action on polyclonal serum IgA1 suggested the absence of neutralizing anti-clostridial IgA protease activity. Although the enzymes were shown not to act on secretory IgA1, they were, however, able to digest free alpha-heavy chains of the dimeric IgA molecules. Susceptibility of the alpha-heavy chain to the proteases was more likely due to the change to a more accessible conformation than because of the absence of neutralizing anti-enzymic activity.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  16. First Report of Hibiscus Chlorotic Ringspot Carmovirus in Tuvalu
    Jones P, Devonshire J, Dabek A, Howells C
    Plant Dis, 1998 May;82(5):591.
    PMID: 30857000 DOI: 10.1094/PDIS.1998.82.5.591C
    In September 1997, plants of Hibiscus manihot (locally called nambele) were observed on Vaitupu Island, Tuvalu, exhibiting an angular leaf mosaic and chlorosis that was not always clearly discernible. Electron microscopy of negatively stained sap from affected leaves revealed the presence of numerous isometric virus particles 28 nm in diameter. Poly-acrylamide gel electrophoresis of purified virus gave a single protein band of Mr 38,000 similar to that of the carmoviruses. Immunosorbent electron microscopy tests with antisera kindly provided by N. Spence showed the virus to be hibiscus chlorotic ringspot carmovirus (HCRSV) (1). This virus is also reported from El Salvador, the U.S., Australia, Thailand, Malaysia, Fiji, the Solomon Islands, and Vanuatu. It is not known how the virus reached Tuvalu but we suspect it was via infected cuttings, which were imported for the production of food supplements to combat acute deficiencies of vitamins A and C in the population. The virus is most likely to have been disseminated throughout the islands and atolls of Tuvalu through infected cuttings. Local spread within fields could occur through contaminated hands and cutting implements because of the ease with which the virus is mechanically transmitted. Reference: (1) H. E.Waterworth et al. Phytopathology 66:570, 1976.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  17. Isolation and characterization of the major phospholipase A2 from the venom of Trimeresurus purpureomaculatus (shore pit viper)
    Nget Hong Tan, Chon Seng Tan, Hun Teck Khor
    Int. J. Biochem., 1989;21(12):1421-6.
    PMID: 2612728
    1. The major phospholipase A2 (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity. 2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC greater than PE greater than PS =0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme. 4. The phospholipase A2 exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 micrograms/g by i.v. route.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  18. Fulltext Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence
    Karim KMR, Husaini A, Sing NN, Sinang FM, Roslan HA, Hussain H
    3 Biotech, 2018 Apr;8(4):204.
    PMID: 29607285 DOI: 10.1007/s13205-018-1225-z
    In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca2+-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  19. Fulltext Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata
    Liew YJ, Soh WT, Jiemy WF, Hwang JS
    Toxins (Basel), 2015 Feb;7(2):407-22.
    PMID: 25654788 DOI: 10.3390/toxins7020407
    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
  20. Fulltext Partial Purification and Characterisation of Pectinase Produced by Aspergillus niger LFP-1 Grown on Pomelo Peels as a Substrate
    Jalil MTM, Ibrahim D
    Trop Life Sci Res, 2021 Mar;32(1):1-22.
    PMID: 33936548 DOI: 10.21315/tlsr2021.32.1.1
    In the present study, pectinase was produced by local fungal isolate, Aspergillus niger LFP-1 grown on pomelo peels as a sole carbon source under solid-state fermentation (SSF). The purification process begins with the concentration of crude enzyme using ammonium sulfate precipitation and followed by purification using anion-exchange column chromatography (DEAE-Sephadex) and subsequently using gel filtration column chromatography (Sephadex G-100). On the other hand, the molecular weight of the purified enzyme was determined through SDS-PAGE. The findings revealed the crude enzyme was purified up to 75.89 folds with a specific activity of 61.54 U/mg and the final yield obtained was 0.01%. The molecular mass of the purified pectinase was 48 kDa. The optimum pH and temperature were 3.5 and 50°C, respectively. This enzyme was stable at a range of pH 3.5 to 4.5 and a relatively high temperature (40°C-50°C) for 100 min. The Km and Vmax were found to be 3.89 mg/mL and 1701 U/mg, respectively. Meanwhile, pectin from citrus fruit and the metal ion (Co2+) were the best substrate and inducer to enhance pectinase yield, respectively.
    Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
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