This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h with an increase in DH. The major amino acids were glutamic acid (16.6%) in thermolysin- myofibrillar hydrolysate followed by aspartic acid (11.1%) in sarcoplasmic protein fraction with no enzyme treatment and lysine (10%) in thermolysin-myofibrillar hydrolysate. The apparent Michaelis constant of proteinase K was lower than thermolysin for both sarcoplasmic and myofibrillar proteins. However, rate of turnover and enzyme efficiency suggested that sarcoplasmic and myofibrillar proteins are suitable substrates for proteinase K and thermolysin hydrolytic reaction, respectively.
The objective of the present study was to develop a rapid, reliable and yet inexpensive protocol for genomic DNA extraction from frozen and ethanol-preserved Asian green-lipped mussels for random amplified microsatelite (RAM) analysis. The procedure comprised of three major steps: (1) Tissue degradation by boiling in 6% Chelex 100 resin in TE buffer; (2) Protein digestion by Proteinase K; and (3) DNA precipitation by adding 2 volumes of cold absolute ethanol. The entire procedure can be completed within two hours. The resulting RAM profiles were clear and reproducible. Our results demonstrate that the combined protocol of Chelex 100-Proteinase K-ethanol precipitation is a powerful yet economical DNA isolation method for population genetic studies involving a large sample size.
In this study, a Lactic acid bacteria (LAB) strain was isolated on MRS medium from gastro-intestinal tissues of Broadhead catfish (Clarias macrocephalus). Out of 50 isolates, 25 isolates were found to be positive on lactose utilisation test and were identified to be gram positive cocci. Using disc diffusion methods, one out of 22 isolates, i.e., a strain A5 demonstrated inhibitions against three indicator organisms; Bacillus cereus, Staphylococcus aureus and Salmonella thyphimurium. Partial 16S rDNA sequencing identified isolate A5 as a member of Lactococcus lactis, with 100% DNA homology. Cell free supernatant fluid from Lactococcus lactis A5 showed inhibitory activities against both gram positive pathogens (Bacillus cereus and Staphylococcus aureus) and gram negative pathogens (Salmonella thyphimurium). Chloroform precipitated bacteriocin retained antagonistic activities in the presence of catalase and lysozyme; and was completely inactivated by Proteinase K treatment. The bacteriocin has a molecular weight of 3.4 kDa, based on SDS-PAGE analysis and the extract was heat stable at 37°C and 65°C, for 15 minutes. The antibacterial activity was suppressed with the addition of EDTA but was significantly increased with the addition of SDS, Triton X-100, Tween 20 and Tween 80. This bacteriocin belongs to class 1 bacteriocin, which was shown to have a nisin-like properties. This strain can be used as potential probiotics in animal or aquaculture feeding; and the bacteriocin it produces will be useful in food preservative.
There is a need for identification of new infection markers against common Leptospira isolates in Malaysia. To achieve this goal, seven-day-old cultures of Leptospira interrogans serogroup Icterohemorrhagiae (L44) and Leptospira interrogans serogroup Javanica (L55) were used for antigen preparation by sequential extraction method using 40 mM Tris, 8M Urea and 2M thiourea. Immunoblot analysis of the antigens were performed using serum samples from 46 local patients with confirmed acute leptospirosis, 28 patients with other infections and 14 healthy controls. The patients serum samples used in this study contained heterologous antibody against a number of different leptospira serovars. A strong IgM reactivity to a broad diffuse band of 10-15 kDa was observed. Combining results using L44 and L55 antigens showed sensitivity of 80.4% and specificity of 95.2% for detection of leptospirosis. Proteinase K and periodate treatment indicated that the band is likely to be lipopolysaccharide (LPS) in nature. This study showed that the 10-15 kDa antigen could potentially be useful for serodiagnosis of acute leptospirosis in Malaysia.
The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8(*)4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.
Some novel hydrazone derivatives 6a-o were synthesized from the key intermediate 4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide 5 and characterized using IR, ¹H-NMR, 13C-NMR, mass spectroscopy and elemental analysis. The inhibitory potential against two secretory phospholipase A₂ (sPLA₂), three protease enzymes and eleven bacterial strains were evaluated. The results revealed that all compounds showed preferential inhibition towards hGIIA isoform of sPLA₂ rather than DrG-IB with compounds 6l and 6e being the most active. The tested compounds exhibited excellent antiprotease activity against proteinase K and protease from Bacillus sp. with compound 6l being the most active against both enzymes. Furthermore, the maximum zones of inhibition against bacterial growth were exhibited by compounds; 6a, 6m, and 6o against P. aeruginosa; 6a, 6b, 6d, 6f, 6l, 6m, 6n, and 6o against Serratia; 6k against S. mutans; and compounds 6a, 6d, 6e, 6m, and 6n against E. feacalis. The docking simulations of hydrazones 6a-o with GIIA sPLA₂, proteinase K and hydrazones 6a-e with glutamine-fructose-6-phosphate transaminase were performed to obtain information regarding the mechanism of action.
Bacterial adherence to connective tissue, especially to collagen has been vastly known for their invasive and infectious activities. However, the ability to exploit the unique and specific interactions between bacteria and collagen as a novel approach in detection of placental collagen has never been explored. This study aimed to determine bacteria with binding specificity to placental collagen (Type IV) derived from human and sheep. In order to do this, total bacteria from small intestines of pig and cow were isolated and their ability to bind to Type IV placental collagen (human and sheep) was determined. Interestingly, three bacterial samples; P5, P9 (pig small intestine origin) and B7 (cow small intestine origin) were found to be able to bind strongly to the placental collagen. The bacterial binding to human placental collagen was however, diminished after the bacteria were treated with trypsin, proteinase K (for removal of surface protein) and guanidine hydrochloride (for S-layer removal), suggesting that the interaction of these bacteria to placental collagen was promoted by protein(s) present at the bacterial surface. In addition, significant reduction of placental collagen-binding ability of the bacteria pre-incubated with soluble human placental collagen showed that there is a specific interaction between the bacteria and collagen. P5, P9 and B7 bacteria were found to share 95-97% 16S rRNA sequence similarity to Enterococcus faecalis ZL, Enterococcus hirae ss33b and Enterococcus faecium M3-1, respectively. The results presented here may facilitate future studies in identifying bacterial surface protein(s) responsible for the specific binding of bacteria to collagen and opens new opportunity to utilize the protein(s) for the detection of placental collagen in nutraceutical and food supplements.
Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients.