As in the case of the succinate and sarcosine dehydrogenases of liver mitochondria, the flavin prosthetic group of the bacterial sarcosine dehydrogenase can be released from the enzyme by proteolytic digestion with trypsin and chymotrypsin. The flavin, isolated in the dinucleotide form and covalently bound to a peptide fragment, is converted to the mononucleotide and purified by sequential chromatography on Sephadex G-25, DEAE-Sephadex A-25, followed by preparative paper chromatography and high voltage electrophoresis.The absorption maxima of the purified flavin at pH 7 are found at 268, 350, and 447 nm, with 268:447 nm and 350:447 nm ratios of 3.08 and 0.79, respectively. The fluorescence excitation and emission maxima, 450 and 530 nm, respectively, are similar to those of flavin mononucleotide. The fluorescence of the flavin-peptide is maximal at pH 3.0–3.1.Amino acid analysis of the flavin-peptide (riboflavin form) gave the following molar ratios of amino acids to flavin: Lys(1), Asp(2), Thr(1), Ser(1), Glu(1), Gly(1), and Ala(1). Aspartic acid was the N-terminal amino acid. Upon more extensive hydrolysis, histidine was obtained in 71–84% yields. Employing aminopeptidase M, the partial sequence of amino acids in the flavin-peptide was determined to be as follows: Flavin -Asp-Lys-Ser-Glu-Gly-His-(Asp,Ala,Thr)-Evidence is presented that the isoalloxazine ring is linked covalently via its 8 α-methyl group to N-3 of histidine. 相似文献
The activity of a sarcosine dehydrogenase isolated from a strain of Pseudomonas is enhanced by the addition of Triton X-100, Brij 35, and Tween 80, and is inhibited by deoxycholate and Sarkosyl NL-97. 2,6-Dichlorophenolindophenol, which is used as the oxidant in the dehydrogenase assay, has also been employed as an indicator in the spectrophotometric determination of the critical micelle concentrations (CMC) of both the nonionic and anionic detergents under conditions optimal for the enzyme analyses. A correlation between the activation or inhibitory activities of the surfactants and their CMC values has been established. 相似文献
The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation. 相似文献
Reviews in Environmental Science and Bio/Technology - The growing and pervasive presence of plastic pollution has attracted considerable interest in recent years, especially small... 相似文献
Apoptosis‐inducing protein of 56 kDa (AIP56) is a major virulence factor of Photobacterium damselae subsp. piscicida, a gram‐negative pathogen that infects warm water fish species worldwide and causes serious economic losses in aquacultures. AIP56 is a single‐chain AB toxin composed by two domains connected by an unstructured linker peptide flanked by two cysteine residues that form a disulphide bond. The A domain comprises a zinc‐metalloprotease moiety that cleaves the NF‐kB p65, and the B domain is involved in binding and internalisation of the toxin into susceptible cells. Previous experiments suggested that disruption of AIP56 disulphide bond partially compromised toxicity, but conclusive evidences supporting the importance of that bond in intoxication were lacking. Here, we show that although the disulphide bond of AIP56 is dispensable for receptor recognition, endocytosis, and membrane interaction, it needs to be intact for efficient translocation of the toxin into the cytosol. We also show that the host cell thioredoxin reductase‐thioredoxin system is involved in AIP56 intoxication by reducing the disulphide bond of the toxin at the cytosol. The present study contributes to a better understanding of the molecular mechanisms operating during AIP56 intoxication and reveals common features shared with other AB toxins. 相似文献