BBA - General Subjects (v.1800, #1)

Complete analysis of single substrate enzyme-catalyzed reactions has required a separate use of two distinct approaches. Steady state approximations are employed to obtain substrate affinity and initial velocity information. Alternatively, first order exponential decay models permit simulation of the time course data for the reactions. Attempts to use integrals of steady state equations to describe reaction time courses have so far met with little success.Here we use equations based on steady state approximations to directly model time course plots.Testing these expressions with the enzyme β-galactosidase, which adheres to classical Michaelis–Menten kinetics, produced a good fit between observed and calculated values.This study indicates that, in addition to providing information on initial kinetic parameters, steady state approximations can be employed to directly model time course kinetics.Integrated forms of the Michaelis–Menten equation have previously been reported in the literature. Here we describe a method to directly apply steady state approximations to time course analysis for predicting product formation and simultaneously obtain multiple kinetic parameters.
Keywords: β-galactosidase; Enzyme kinetics; Equation modeling; Global data fitting;

Niacin improves renal lipid metabolism and slows progression in chronic kidney disease by Kyu-hyang Cho; Hyun-ju Kim; Vaijinath S. Kamanna; Nosratola D. Vaziri (6-15).
Mounting evidence points to lipid accumulation in the diseased kidney and its contribution to progression of nephropathy. We recently found heavy lipid accumulation and marked dysregulation of lipid metabolism in the remnant kidneys of rats with chronic renal failure (CRF). Present study sought to determine efficacy of niacin supplementation on renal tissue lipid metabolism in CRF.Kidney function, lipid content, and expression of molecules involved in cholesterol and fatty acid metabolism were determined in untreated CRF (5/6 nephrectomized), niacin-treated CRF (50 mg/kg/day in drinking water for 12 weeks) and control rats.CRF resulted in hypertension, proteinuria, renal tissue lipid accumulation, up-regulation of scavenger receptor A1 (SR-A1), acyl-CoA cholesterol acyltransferase-1 (ACAT1), carbohydrate-responsive element binding protein (ChREBP), fatty acid synthase (FAS), acyl-CoA carboxylase (ACC), liver X receptor (LXR), ATP binding cassette (ABC) A-1, ABCG-1, and SR-B1 and down-regulation of sterol responsive element binding protein-1 (SREBP-1), SREBP-2, HMG-CoA reductase, PPAR-α, fatty acid binding protein (L-FABP), and CPT1A. Niacin therapy attenuated hypertension, proteinuria, and tubulo-interstitial injury, reduced renal tissue lipids, CD36, ChREBP, LXR, ABCA-1, ABCG-1, and SR-B1 abundance and raised PPAR-α and L-FABP.Niacin administration improves renal tissue lipid metabolism and renal function and structure in experimental CRF.
Keywords: Inflammation; Proteinuria; Hypertension; Atherosclerosis; Reverse cholesterol transport; Oxidative stress; Anti-inflammatory-antioxidant agent;

The human hGSTA5 gene encodes an enzymatically active protein by Sharda P. Singh; Ludwika Zimniak; Piotr Zimniak (16-22).
Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional.Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity.Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity. hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme.Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions.
Keywords: Glutathione transferase; Alpha-class glutathione transferase; 4-hydroxynonenal; 4-HNE; Lipid peroxidation; Pseudogene;

Sinapinic acid can replace ascorbate in the biotin switch assay by Vasantha Madhuri Kallakunta; Andrea Staruch; Bulent Mutus (23-30).
Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO2 - released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ±  S-nitrosocysteine (CysNO) exposure.We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.
Keywords: Modification of the biotin switch assay; Sinapinic acid-mediated denitrosation; Ascorbic acid-mediated denitrosation; S-nitrosoprotein determination;

A novel protein found in the I bands of myofibrils is produced by alternative splicing of the DLST gene by Sadayuki Matuda; Takuro Arimura; Akinori Kimura; Hiroaki Takekura; Shigeo Ohta; Kyoko Nakano (31-39).
It is not known if the dihydrolipoamide succinyltransferase (DLST) gene, a mitochondrial protein, undergoes alternative splicing. We identified an uncharacterized protein reacting with an anti-DLST antibody in the I bands of myofibrils in rat skeletal muscle.Immunocytochemical staining with an anti-DLST antibody, the purification and amino acid sequence analysis of the protein, and the isolation and sequencing of the protein's cDNA were carried out to clarify the properties of the protein and its relationship to the DLST gene.A pyrophosphate concentration >10 mM was necessary to extract the protein from myofibrils in the presence of salt with a higher concentration than 0.6 M, at an alkaline pH of 7.5–8.0. The protein corresponded to the amino acid sequence of the C-terminal side of DLST. The cDNAs for this protein were splicing variants of the DLST gene, with deletions of both exons 2 and 3, or only exon 2 or 3. These variants possessed an open reading frame from an initiation codon in exon 8 of the DLST gene to a termination codon in exon 15, generating a protein with a molecular weight of 30 kDa.The DLST gene undergoes alternative splicing, generating the protein isolated from the I bands of myofibrils.The DLST gene produces two different proteins with quite different functions via alternative splicing.
Keywords: Dihydrolipoamide succinyltransferase (DLST) gene; α-Ketoglutarate dehydrogenase complex; Alternative splicing; Myofibril-protein; Skeletal muscle; I band;

γ-Irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells by Yasuhiro Ohshima; Mitsutoshi Tsukimoto; Takato Takenouchi; Hitoshi Harada; Akina Suzuki; Mitsuru Sato; Hiroshi Kitani; Shuji Kojima (40-46).
Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined γ-irradiation-induced ATP release and its mechanism in B16 melanoma.Extracellular ATP was measured by luciferin–luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X7 receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X7 receptor.Cells were exposed to 0.5–8 Gy of γ-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X7 receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X7 receptor-knockdown cells. Our results indicate that γ-irradiation evokes ATP release from melanoma cells, and P2X7 receptor channel plays a significant role in mediating the ATP release.We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.
Keywords: ionizing irradiation; ATP release; P2X7 receptor;