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Urease from seeds of water melon was purified to apparent homogeniety upto a sp act of 3750 units/mg protein with 31% recovery. Enzyme showed single protein band on native PAGE by urease specific staining. The mol wt of the enzyme was 4,70,000 and the preparation was free from bound nucleotides (A280/A260=1.14). The enzyme exhibited maximum activity in 50 mM Tris-acetate buffer (pH 8.5). The Km for urease was 8 mM. The enzyme was not inhibited by 25 mM of EDTA in 50 mM Tris-acetate buffer (pH 8.0 and 8.5). 相似文献
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Anurima Majumder Kota N. Gopalakrishna Pallavi Cheguru Lokesh Gakhar Nikolai O. Artemyev 《The Journal of biological chemistry》2013,288(29):21320-21328
Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA. 相似文献
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Jayant Pralhad Rathod Gunjan Prakash Reena Pandit Arvind M. Lali 《Photosynthesis research》2013,118(1-2):141-146
Parachlorella kessleri is a unicellular alga which grows in fresh as well as marine water and is commercially important as biomass/lipid feedstock and in bioremediation. The present study describes the successful transformation of marine P. kessleri with the help of Agrobacterium tumefaciens. Transformed marine P. kessleri was able to tolerate more than 10 mg l?1 hygromycin concentration. Co-cultivation conditions were modulated to allow the simultaneous growth of both marine P. kessleri and A. tumefaciens. For co-cultivation, P. kessleri was shifted from Walne’s to tris acetate phosphate medium to reduce the antibiotic requirement during selection. In the present study, the transfer of T-DNA was successful without using acetosyringone. Biochemical and genetic analyses were performed for expression of transgenes by GUS assay and PCR in transformants. Establishment of this protocol would be useful in further genetic modification of oil-bearing Parachlorella species. 相似文献
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Gunjan Arora Andaleeb Sajid Mary Diana Arulanandh Richa Misra Anshika Singhal Santosh Kumar Lalit K. Singh Abid R. Mattoo Rishi Raj Souvik Maiti Sharmila Basu-Modak Yogendra Singh 《Biometals》2013,26(5):715-730
Bacillus anthracis Ser/Thr protein kinase PrkC (BasPrkC) is important for virulence of the bacterium within the host. Homologs of PrkC and its cognate phosphatase PrpC (BasPrpC) are the most conserved mediators of signaling events in diverse bacteria. BasPrkC homolog in Bacillus subtilis regulates critical processes like spore germination and BasPrpC modulates the activity of BasPrkC by dephosphorylation. So far, biochemical and genetic studies have provided important insights into the roles of BasPrkC and BasPrpC; however, regulation of their activities is not known. We studied the regulation of BasPrkC/BasPrpC pair and observed that Zn2+ metal ions can alter their activities. Zn2+ promotes BasPrkC kinase activity while inhibits the BasPrpC phosphatase activity. Concentration of Zn2+ in growing B. anthracis cells was found to vary with growth phase. Zn2+ was found to be lowest in log phase cells while it was highest in spores. This variation in Zn2+ concentration is significant for understanding the antagonistic activities of BasPrkC/BasPrpC pair. Our results also show that BasPrkC activity is modulated by temperature changes and kinase inhibitors. Additionally, we identified Elongation Factor Tu (BasEf-Tu) as a substrate of BasPrkC/BasPrpC pair and assessed the impact of their regulation on BasEf-Tu phosphorylation. Based on these results, we propose Zn2+ as an important regulator of BasPrkC/BasPrpC mediated phosphorylation cascades. Thus, this study reveals additional means by which BasPrkC can be activated leading to autophosphorylation and substrate phosphorylation. 相似文献
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Singh Manisha Sharma Deepak Chauhan Rajni Goel Gunjan 《Probiotics and antimicrobial proteins》2019,11(3):850-856
Probiotics and Antimicrobial Proteins - The present study investigated skimmed milk and alginate-based encapsulation for protection of a probiotic strain, Lactobacillus gastricus BTM7 during... 相似文献
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We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo. 相似文献
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Saxowsky TT Choudhary G Klingbeil MM Englund PT 《The Journal of biological chemistry》2003,278(49):49095-49101
In higher eukaryotes, DNA polymerase (pol) beta resides in the nucleus and participates primarily in DNA repair. The DNA polymerase beta from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol beta-like proteins. One is approximately 70% identical to the C. fasciculata pol beta and is likely the homolog of this enzyme. The other, although approximately 30% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different. 相似文献