Bovine pancreatic phospholipase A2 (PLA2), a small (13.8 kDa) Ca2+-dependent lipolytic enzyme, is rich in functional and structural character. In an effort to examine its detailed structure-function relationship, we determined its solution structure by multidimensional nuclear magnetic resonance (NMR) spectroscopy at a functionally relevant pH. An ensemble of 20 structures generated has an average root-mean-square deviation (RMSD) of 0.62 +/- 0.08 A for backbone (N, Calpha, C) atoms and 0.98 +/- 0.09 A for all heavy atoms. The overall structure shows several notable differences from the crystal structure: the first three residues at the N-terminus, the calcium-binding loop (Y25-T36), and the surface loop (V63-N72) appear to be flexible; the alpha-helical conformation of helix B (E17-F22) is absent; helix D appears to be shorter (D59-V63 instead of D59-D66); and the hydrogen-bonding network is less defined. These differences were analyzed in relation to the function of PLA2. We then further examined the H-bonding network, because its functional role or even its existence in solution has been in dispute recently. Our results show that part of the H-bonding network (the portion away from N-terminus) clearly exists in solution, as evidenced by direct observation (at 11.1 ppm) of a strong H-bond between Y73 and D99 and an implicated interaction between D99 and H48. Analyses of a series of mutants indicated that the existence of the Y73.D99 H-bond correlates directly with the conformational stability of the mutant. Loss of this H-bond results in a loss of 2-3 kcal/mol in the conformational stability of PLA2. The unequivocal identification and demonstration of the structural importance of a specific hydrogen bond, and the magnitude of its contribution to conformational stability, are uncommon to the best of our knowledge. Our results also suggest that, while the D99.H48 catalytic diad is the key catalytic machinery of PLA2, it also helps to maintain conformational integrity.
The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse Thymidylate synthase (TS) is a housekeeping enzyme that is responsible for the formation of thymidylic acid in the de novo biosynthetic pathway. The enzyme is present at a much higher level in proliferating cells that are engaged in DNA replication than in quiescent cells (4,26). We have been studying the mechanisms that are responsible for regulating expression of the mouse TS gene. We have cloned and analyzed the sequences of the cDNA (29) and gene (5) for this enzyme. The gene is 12 kb in length and has a 1-kb coding region interrupted by six introns. The G+C-rich promoter region lacks a TATAA box and initiates transcription at multiple sites over a 60-nucleotide region (5, 8). Mouse TS mRNA is highly unusual in that the predominant species lacks a 3' untranslated region (18). The upstream polyadenylation signal is AUUAAA and is located within the coding region (15).The amount of TS mRNA increases at least 20-fold during a serum-induced transition from the quiescent (GO) phase to the S phase of the cell cycle (8,19 (12,13,21,33,37).Transient transfection assays with TS minigenes have shown that normal levels of gene transcription and the normal pattern of transcriptional start sites are observed with TS 5'-flanking regions that retain as few as 15 nucleotides upstream of the first transcriptional initiation site (or 105 nucleotides upstream of the ATG start codon) (7). Introns are also important for efficient expression of the gene; addition of introns 5 and 6 to an intronless minigene increases the level of expression about eightfold (6). Similar stimulatory effects have been observed previously in other genes (2,3,14). However, addition of intron 4 did not lead to an increase in expression, indicating that the stimulatory effect is not universal for all TS introns (6).In this study, we analyzed the role of the 5'-flanking region in regulating mouse TS gene expression. We constructed a series of chimeric minigenes consisting of different promoters linked to the normal TS coding region and polyadenylation signal. The minigenes were transfected into cultured cells, and the level of expression was measured. Our results indicate that sequences upstream of the essential promoter element(s) are required for normal regulation of TS gene expression in growth-stimulated cells. However, the 5'-flanking sequences are not sufficient for normal regulation. MATERIALS AND METHODSConstruction of mmingenes. The structures of the minigenes used in these analyses are summarized in Fig. 1. The construction of pTTT (also named pTSMG2 [9]) and pTI56T (also named p156 [6]) has been described. Both of these minigenes have 1 kb of the normal 5'-flanking region and 0.25 kb of the normal 3'-flanking region of the mouse TS gene 1023
Degradation of perfluoropolyether lubricants (Fomblin Z-Dol and Fomblin AM 2001) on the magnetic recording thin-films disks are studied during sliding with an Al 2 O 3 ±TiC slider under high vacuum. Gaseous products generated from the head=disk interfaces are detected and monitored as a function of sliding distance using a quadrupole mass spectrometer. Effects of overcoats and surface roughnesses of disks on degradation of lubricants and coefficient of friction were also studied. Experimental results show that the gaseous products generated from Z-Dol and AM 2001 lubricants during sliding are quite similar. Degradation of both lubricants is found to take place from the beginning of sliding, and the evolution rate of decomposition fragments decreases with the sliding distance. Removal of the lubricants is accompanied by a rapid rise in the coefficient of friction of the interfaces, followed by gaseous products from the tribochemical reaction of the overcoat material. Surface roughness of the disk has an effect on the sliding distance to failure, but little effect on the decomposition of lubricant.
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