 with (S)-lipoylated H-protein may be attributed to the contamination of (R)-lipoate.  Kinetic Properties of Lipoyltransferase-The high rate of lipoylation with GTP in the coupled method described above strongly indicates that lipoyltransferase is able to utilize lipoyl-GMP as a substrate. To confirm this, we examined the kinetic properties of lipoyltransferase using chemically synthesized lipoyl-GMP. As shown in Table III, lipoyltransferase utilized lipoyl-GMP as well as lipoyl-AMP, although the affinity for lipoyl-GMP was about three times lower than that for lipoyl-AMP.
Cloning and Sequencing of cDNAs for LAE-The N-terminal amino acid sequence analysis showed that the purified LAE had heterogeneous N termini. They start from leucine (58%, amino acid 32 (GenBank TM accession number AB048289)), serine (8%, amino acid 33), glycine (9%, amino acid 34), and alanine (25%, amino acid 35). We then obtained several lysylpeptides and analyzed the N-terminal amino acid sequence to synthesize oligonucleotide probes based on the sequences. The oligonucleotides were employed for the screening of a gt10 bovine liver cDNA library. Approximately 4 ϫ 10 5 independent clones were screened, and 18 positive clones were obtained. Five clones containing full-length cDNA for LAE were chosen randomly, and the nucleotide sequences were analyzed. The longest cDNA consists of 2114 base pairs and contains an open reading frame of 1731 base pairs. The cDNA encodes for 577 amino acids, including a mitochondrial presequence of 31 amino acid residues and a mature LAE of 546 amino acid residues. The predicted presequence shows a characteristic feature, being rich in basic amino acid residues, and an absence of acidic amino acid residues. The nucleotide sequence has been submitted to the DDBJ/EMBL/GenBank TM with accession number AB048289. The calculated molecular mass of the mature LAE of 61,146 Da is in good agreement with that determined by SDS-PAGE. The sequence CCACTATGC surrounding the first in-frame ATG codon matches the optimum translation initiation sequence, CC(A/G)CCATGG, described by Kozak (20). A putative polyadenylation signal, AATAAA, is located 18 base pairs upstream from the poly(A) tail. The amino acid sequence of LAE contains a putative AMP/ATP binding motif common to acyl-CoA synthetase (21). Amino acid sequence homology search by FASTA and BLAST showed that the sequence of LAE was identical to that of xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III from bovine liver, although the mitochondrial presequence has not been predicted (10). The cDNA obtained extends by 28 nucleotides upstream from the reported 5Ј-end. Furthermore, the following differences from the sequence reported were observed. First, a single nucleotide substitution of G for A at nucleotide position 1244 (GenBank TM accession number AB048289) was found in four of five clones analyzed, and consequently an amino acid substitution of alanine for threonine was predicted at position 372. Amino acid sequence analysis of a lysylpeptide obtained from purified LAE showed the amino acid residue at position 372 to be alanine. Second, an insertion of 39 nucleotides (nucleotides 80 -118 (GenBank TM accession number AB048289)) due to an alternative splicing was found in two of five clones.
Southern blot analysis was carried out to examine whether more than one gene encoding LAE was present in a genome, because two distinct cDNA containing a different nucleotide at the position 1244 were isolated from a cDNA library. As shown in Fig. 2, a single band was detected in each of the enzyme digests, indicating the presence of a single copy of the gene encoding LAE. The result suggests that the difference of the nucleotide at position 1244 should be due to a single nucleotide polymorphic mutation.
Relationship between Medium-chain Acyl-CoA Synthesis and Lipoate Activation-We next examined whether the purified LAE can catalyze acyl-CoA synthesis. Table IV shows that the purified enzyme exhibited activities of medium-chain acyl-CoA synthesis with the highest specific activity with hexanoic acid. The data are consistent with those of bovine mitochondrial xenobiotic-metabolizing/medium chain-fatty acid:CoA lygase III reported previously (15,22). Because LAE utilizes GTP, acyl-GMP formation was examined. A broad substrate specificity was observed with respect to the chain length of fatty acids (Table IV). The formation of lipoyl-GMP was comparable with that of decanoyl-GMP.
To explore the relationship between the lipoate activation and the acyl-CoA synthesis, the effects of lipoate and GTP on the acyl-CoA synthesis (with ATP) were investigated. When hexanoate was the fatty acid substrate, synthesis of hexanoyl-CoA was reduced by 65% (with 0.1 mM lipoate) and 52% (with 2 mM GTP). The inhibitions by lipoate and GTP were competitive with respect to hexanoate and ATP, respectively (K i for lipoate, 3.9 Ϯ 0.3 M; K m(app) for hexanoate, 5.9 Ϯ 1.3 M; K i for GTP, 0.58 Ϯ 0.13 mM; K m(app) for ATP, 0.32 Ϯ 0.10 mM; n ϭ 3). Similarly, lipoyl-GMP formation was inhibited by substrates   for acyl-CoA synthesis. The addition of 0.25 mM CoA and 0.1 mM hexanoate inhibited the reaction by 52 and 15%, respectively. The addition of 2 mM ATP inhibited the reaction nearly completely (residual activity was 1.4% of that of control) because the lipoyl-AMP formed could hardly dissociate from LAE. The inhibition by ATP was slightly relieved in the presence of 0.25 mM CoA to 5.6% residual activity (586 nmol/h/mg of protein) with concomitant synthesis of lipoyl-CoA (13269 nmol/ h/mg of protein). Although the rate of lipoyl-GMP formation in this situation is very low, it is about 40-fold greater than that of lipoyl-AMP formation (Table II). These results suggest that GTP and lipoate share the binding site with ATP and fatty acid, respectively, on LAE and that LAE can catalyze the lipoyl-GMP formation in mitochondria where ATP, CoA, and GTP coexist. It is unclear at present whether there is some biochemical mechanism responsible for the preferential production of lipoyl-GMP in mitochondria.
Conclusions-This work characterizes the LAE reaction and demonstrates a novel function of the medium-chain acyl-CoA synthetase in mitochondria. Although ATP has been recognized as a substrate for lipoate activation, the present study demonstrates that LAE catalyzes the activation of lipoate utilizing GTP and easily releases the product, lipoyl-GMP, from the active site to provide a substrate for lipoyltransferase. In contrast, ATP is an essential substrate for the acyl-CoA synthesis because the reaction intermediate, acyl-AMP, has to be retained on the active site of the enzyme to react with CoA. We could not detect a significant amount of lipoate-activating activity other than the purified LAE in mitochondria, and we therefore conclude that the enzyme reported here is primarily responsible for the activation of lipoate in mitochondria.Title: R.E.M.?? More like C.O.M.A. Product review: R.E.M. is dead. Kaput. Out of ideas. The critics say that R.E.M. is taking chances on this album. What chances?? Hey critics, can you say "FORMULA"? R.E.M. plods along and puts out another album of pure, utter, unadulterated CRAP.I used to be a huge R.E.M. fan. I saw them every time they came here. I bought every album, every single. They were on continuous play in my car's cassette player. That was a long time ago, though. Since the Green album, the quality R.E.M.'s output has been steadily declining. (And yes, this includes Automatic For The People. "The Sidewinder Sleeps Tonight" indeed. What a joke!)This CD is garbage. I hate it. Hate it. Hate it hate it hate it hate it hate it! I never thought I would say that about an R.E.M. album. These are mere shells of the guys that gave us Murmur, Reckoning, Document, and songs like "Begin The Begin." It's over, guys. Give it "UP." Would you say this review depicts the product in a flattering or unflattering light?
unflattering

Title: Excellent Documentary Product review: Very informative. I was never aware of the speeds that the electric car could attain and that on a single charge it could go at least 70 miles. It is a very practical unit for city driving but you would need something that would last a bit longer for long trips. The people that owned these cars seemed to have a lot of fun driving them.The premise of the film is that the oil companies put pressure on government officials to doom the original electric car. It seems like nobody in the government cares about global warming, pollution and protecting the environment. It was interesting because when the electric car was introduced they talk about gas prices of sixty cents a gallon! Would you say this review depicts the product in a flattering or unflattering light?
flattering

Title: just not into it. Product review: Now im not gunna bash this game because i bought not knowing what it was. However