The Lysines in Liver Alcohol Dehydrogenase

McKinley-McKee, John S.; Morris, David L.

doi:10.1111/j.1432-1033.1972.tb01877.x

Cited by 45 publications

(17 citation statements)

References 31 publications

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“…7). This could be due to reverse reaction of the Schiff base by dilution of PLP in the assay of transport activity (32). The PLP concentration requiring for maximum inhibition of P i transport was consistent with the result in Stappen and Krä mer (31).…”

Section: Nucleotide Binding Site and Lys 185 In The Phosphate Carriersupporting

confidence: 88%

Specific Labeling of the Bovine Heart Mitochondrial Phosphate Carrier with Fluorescein 5-Isothiocyanate

Majima

Ishida

Miki

et al. 2001

Journal of Biological Chemistry

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The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys 185 in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0°C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424 -432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys 185 . In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.There are various solute carriers in the mitochondrial inner membrane to support ATP synthesis by oxidative phosphorylation. The 30-kDa solute carriers, consisting of a three-repeat structure containing a certain consensus sequence, are members of the mitochondrial solute carrier family (1). Of these, the ADP/ATP carrier mediating transport of ADP and ATP, the phosphate carrier mediating the symport of orthophosphate (P i ) and H ϩ , and the type 1 uncoupling protein forming the short circuit of the proton current (2-4) have received considerable attention. These carriers take similar topologies of six transmembrane helices with three large hydrophilic loops facing the matrix, and their homodimers are thought to be their functional units (2,3,5,6). However, their precise structural characteristics are not fully understood in relation to their transport functions.Because fluorescein derivatives have been thought to have similar structural features to those of adenine nucleotides (7, 8), they have been used as fluorescent probes in studies on the kinetics and conformational changes caused by their interactions with the adenine nucleotide binding sites of proteins such as ATPases (9 -11), NAD(P) ϩ -dependent dehydrogenases (12, 13), and kinases (7,8). In fact, we recently reported that the geometric and electronic structures of fluorescein analogs are very similar to those of ADP/ATP (14). In addition, we found that various fluorescein derivatives have high affinities to the ADP/ATP carrier in bovine heart mitochondria, and the binding leads to inhibition of the transport activity (4,14,15). Of the fluorescein analogs, the SH reagent eosin 5-maleimide (EMA) 1 most significantly interacts with the ADP/ATP carrier; it quickly and specifically labels Cys 159 in the second loop facing the matrix of the bovine ...

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Section: Nucleotide Binding Site and Lys 185 In The Phosphate Carriersupporting

confidence: 88%

Specific Labeling of the Bovine Heart Mitochondrial Phosphate Carrier with Fluorescein 5-Isothiocyanate

Majima

Ishida

Miki

et al. 2001

Journal of Biological Chemistry

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“…In this context it is of interest to note that modification of lysine residues by pyridoxal phosphate abolishes enzymatic activity [38]. The substitutions of valine residues 235 and 241 by leucine and isoleucine respectively, found in the human as well as in the rat enzymes, increase the hydrophobicity of the a-C and /3-C structures.…”

Section: Discussionmentioning

confidence: 99%

Structural Studies of Human‐Liver Alcohol‐Dehydrogenase Isoenzymes

Berger

Wartburg

1974

European Journal of Biochemistry

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1. Alcohol dehydrogenase isoenzymes BB were isolated from human livers of both "normal" and "atypical" phenotypes [l]. Their pH-rate profiles, their behaviour on CM-cellulose and the number of hybrids formed with horse subunit A show that the "atypical" isoenzymes contain subunits B1 and B,, the "normal" one only subunit B,. These results indicate that most "atypical" individuals genetically represent heterozygotes.2. A high degree of homology between horse subunit A and human subunits B is found. However, the human isoenzymes have a molecular weight of 87000 and contain about 410 amino acid residues, 6 tyrosines and 3 tryptophans per subunit.3. The sequence of one tryptic peptide from subunit B, is Phe-Ala-Lys and is altered to Phe-ProLys in the subunit B,. This substituted residue is located in a region which corresponds to the coenzyme-binding site of the horse enzyme.The models of the subunit composition of the multiple molecular forms of human liver alcohol dehydrogenase suggest that the dimeric isoenzymes are formed by combination of four different subunits [l--41. A polymorphism of the human enzyme has been observed in addition to this heterogeneity [5], and the term "atypical" variant was introduced. Screening tests discriminating between the "normal" and "atypical" phenotypes were devised on the basis of several differences in catalytic parameters of the enzymes. With these a bimodal distribution has been found in several populations [6-91.In liver homogenate a higher specific activity for ethanol oxidation and a shift of the pH optimum to a lower value represent the main characteristics of the "atypical" enzyme when compared with the "normal" one. Both differences were found to pertain to isoenzymes BB isolated from "normal" and "atypical" livers, suggesting that subunit B exists in a variant form [l]. According to the genetic model proposed by Smith et al.[8] two alleles occur at the gene locus coding for subunit B; they are responsible for the synthesis of either the "normal" (B,) or "atypical" (B2) subunit. Therefore three groups of individuals are expected with respect to their subunit composition : homozygotes with subunit B1 only, homozygotes with subunit B2 only and heterozygotes with both subunits B1 and B2. In "atypical" livers two isoenzymes AB were found [l], one with "atypical" (AB,) and one with "normal" (AB,) catalytic properties. This result indicated that "atypical" livers could stem from heterozygous individuals.Based on the genetic model for the polymorphism and the known primary structures of two subunits of the horse enzyme [lo], as well as the sequence of some peptides from human liver alcohol dehydrogenase [ll], it seemed of interest to search for the difference in primary structure between subunits

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“…This could mean either that modification of other residues interferes with modification of the lysine residue of primary interest (cf. McKinley-McKee & Morris, 1972), or that modification of this residue does not abolish activity entirely.…”

Section: Resultsmentioning

confidence: 97%

Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5′-phosphate

Chen¹,

Engel²

1975

Biochemical Journal

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1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.

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The Lysines in Liver Alcohol Dehydrogenase

Cited by 45 publications

References 31 publications

Specific Labeling of the Bovine Heart Mitochondrial Phosphate Carrier with Fluorescein 5-Isothiocyanate

Specific Labeling of the Bovine Heart Mitochondrial Phosphate Carrier with Fluorescein 5-Isothiocyanate

Structural Studies of Human‐Liver Alcohol‐Dehydrogenase Isoenzymes

Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5′-phosphate

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