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161.
As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochodrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the completing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channeling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction. 相似文献
162.
UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases. 总被引:7,自引:0,他引:7 下载免费PDF全文
J. Hempel J. Perozich H. Romovacek A. Hinich I. Kuo D. S. Feingold 《Protein science : a publication of the Protein Society》1994,3(7):1074-1080
The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD(+)-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microhetergeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of approximately 26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
163.
Neisseria gonorrhoeae possesses two nicotinamide adenine dinucleotide-independent lactate dehydrogenases 总被引:3,自引:0,他引:3
Randy S. Fischer Gaines C. Martin Premila Rao Roy A. Jensen 《FEMS microbiology letters》1994,115(1):39-44
Abstract An important metabolic capability of Neisseria gonorrhoeae is the utilization of host-derived lactate. Two isoenzymes of the membrane-associated, pyridine dinucleotide-independent type of lactate dehydrogenase (iLDH) participate in lactate assimilation, but exhibit distinctive properties. Isoenzyme iLDH-I utilized lactate exclusively as substrate, exhibiting a preference for the D-isomer. In contrast, isoenzyme iLDH-II exhibited broad substrate specificity (lactate, phenyllactate, and 4-hydroxyphenyllactate), but was stereospecific for the L-isomers. These results explain the difficulty in isolating mutants unable to utilize lactate. 相似文献
164.
Brenda S. Speer Ludmila Chistoserdova Mary E. Lidstrom 《FEMS microbiology letters》1994,121(3):349-355
Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well. 相似文献
165.
Calvin cycle genes in Nitrobacter vulgaris T3 总被引:1,自引:0,他引:1
Maren Strecker Eva Sickinger Robert S. English Jessup M. Shively 《FEMS microbiology letters》1994,120(1-2):45-50
Abstract The genes encoding the Calvin cycle enzymes of Nitrobacter vulgaris T3 are found as two separate clusters on the chromosome. One cluster contains the genes for the large and small subunits of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), glyceraldehyde-3-phosphate dehydrogenase, and one encoding a regulatory protein of the LysR family. The other cluster contains the genes for fructose-1,6-/sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, and fructose-1,6-/sedoheptulose-1,7-biphosphate aldolase. With the exception of the LysR-like gene, the genes in each cluster are apparently transcribed in the same direction. The deduced amino acid sequence of both the large and small subunits of RuBisCO are most similar (84–86%) to those of Thiobacillus ferrooxidans and Chromatium vinosum . The deduced sequences of phosphoribulokinase and fructose/sedoheptulose bisphosphatase are 67–73 aand 44–46% similar to those reported for other autotrophic bacteria, respectively. 相似文献
166.
In Drosophila melanogaster transformants, the alcohol dehydrogenase (Adh) genes from D. affinidisjuncta and D. grimshawi show similar levels of expression except in the adult midgut where the D. affinidisjuncta gene is expressed about 10- to 20-fold more strongly. To study the arrangement of cis-acting sequences responsible for this regulatory difference, homologous restriction sites were used to create a series of chimeric genes that switched fragments from the 5 and 3 flanking regions of these two genes. Chimeric genes were introduced into the germ-line of D. melanogaster, and Adh gene expression was analyzed by measuring RNA levels. Various gene fragments in the promoter region and elsewhere influence expression in the adult midgut and in whole larvae and adults. Comparison of these results with earlier studies involving chimeras between the D. affinidisjuncta and D. hawaiiensis genes indicates that expression in the adult midgut is influenced by multiple regulatory sequences and that distinct arrangements of regulatory sequences can result in similar levels of expression both in the adult midgut and in the whole organism. 相似文献
167.
In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO2 , the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle. 相似文献
168.
Ian M. Møller Allan G. Rasmusson Kenneth M. Fredlund 《Journal of bioenergetics and biomembranes》1993,25(4):377-384
Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca2+ with aK
0.5 of about 1 µM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca2+ with aK
0.5 of 3 µM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer. 相似文献
169.
Osmium tetroxide (OsO4) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO4 hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO4 will also identify other torsional perturbations of DNA. In this study, OsO4 was used to footprint an AT-rich region (between –780 and –500) of the maizeAdh1 promoter. Hyperreactive sites were identified bothin vitro andin vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO4 hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei. 相似文献
170.
Robert B. Kirkpatrick Zahida Parveen Presley F. Martin 《Genesis (New York, N.Y. : 2000)》1994,15(2):188-200
A transient expression assay has been used to investigate the cause of a tissuespecific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4-kb genomic clone containing the 3.2-kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue-specific pattern resembling the abnormal expression pattern of the position effect. The 3.2-kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2-kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2-kb fragment indicates that it is a noncoding region. A 17-bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2-kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an α1-tubulin promoter-LacZ fusion construct or an hsp70 promoter-Ach fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5′ silencer and the 3′ element act together to create the tissue specific pcsition effect characteristic of the GC-1 line. © 1994 Wiley-Liss, Inc. 相似文献