Ligand-induced internalization and increased cell calcium are mediated via distinct structural elements in the carboxyl terminus of the epidermal growth factor receptor.

Signals that can mediate ligand-induced receptor internalization and calcium regulation are present in a 48-amino acid "calcium-internalization" domain in the C' terminus of the epidermal growth factor (EGF) receptor. The basis of calcium and internalization regulation signalled by this 48-amino acid sequence was analyzed using deletion and substitution mutant receptors. Cells expressing truncated receptors containing either the NH2- or COOH-terminal portion of the 48-residue domain displayed high affinity EGF-dependent endocytosis and receptor down-regulation. These endocytosis-competent EGF receptor mutants that lacked any autophosphorylation site were unable to increase the concentration of intracellular calcium. To investigate the role of self-phosphorylation in EGF-induced calcium mobilization, phenylalanine was substituted for the single autophosphorylated tyrosine residue in this region of an internalization-competent truncated receptor. The receptor-mediated calcium response was abolished, while ligand-dependent receptor internalization was unimpaired. These results demonstrate that EGF-dependent receptor endocytosis and calcium mobilization are separate events. Tyrosine self-phosphorylation is required for increased [Ca2+]i, while structural features distinct from autophosphorylation are required for receptor internalization.     

Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo.

The influence of topoisomerase I and gyrase mutations in Escherichia coli on the supercoiled density of recombinant plasmids and the stability of left-handed Z-DNA was investigated. The formation of Z-DNA in vivo by dC-dG sequences of different lengths was used to determine the effective plasmid supercoil densities in the mutant strains. The presence of Z-DNA in the cells was detected by linking number and EcoRI methylase inhibition assays. A change in the unrestrained superhelical tension in vivo directly effects the B- to Z-DNA transition. Alterations in the internal or external environment of the cells, such as the inactivation of gyrase or topoisomerase I, a gyrase temperature-sensitive mutant, or starvation of cells, have a dramatic influence on the topology of plasmids. Also, E. coli has significantly more superhelical strain than Klebsiella, Morganella, or Enterobacter. These studies indicate that linking deficiency and effective supercoil density are mutually independent variables of plasmid tertiary structure. A variety of factors, such as protein-DNA interactions, activity of topoisomerases, and the resulting supercoil density, contribute to the B to Z transition inside living cells.     

Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.     

ATP-citrate lyase from rat liver. Characterisation of the citryl-enzyme complexes.

The mechanism of ATP-citrate lyase has been proposed to involve a citryl-enzyme intermediate. When the enzyme is incubated with its substrates ATP and [14C]citrate, but in the absence of the final acceptor, two distinct types of citrate-containing complex can be isolated. At early time points, a highly unstable complex can be isolated by gel filtration which has a half-life of 36 s at 25 degrees C. This complex reacts rapidly with CoA, but cannot be acid-precipitated; behaviour consistent with its identification as enzyme-citryl phosphate. However, ATP-citrate lyase is also capable of undergoing a slow time-dependent covalent incorporation of radiolabel from [14C]citrate. This modification is acid-stable, non-specific, and cannot be reversed by the addition of CoA. When cytochrome is included in the reaction mixture as a heterologous acceptor, it is also citrylated. These reactions require that when ATP-citrate lyase is incubated with all its substrates except for CoA, a freely diffusible citrylating species is generated within the active site. This evidence suggests that there is no requirement for the mechanism of ATP-citrate lyase to proceed via a covalent citryl-enzyme intermediate. By analogy with succinyl-CoA synthetase, an enzyme which has a high degree of sequence similarity with ATP-citrate lyase, a simple mechanism is proposed for the enzyme in which citryl-CoA is produced by direct nucleophilic attack on citryl phosphate.     

Differentiation of free-living Anabaena and Nostoc cyanobacteria on the basis of fatty acid composition.

The cellular fatty acids of free-living, nitrogen-fixing cyanobacteria belonging to the genera Anabaena and Nostoc were analyzed to differentiate the genera. The fatty acid compositions of 10 Anabaena strains and 10 Nostoc strains that were grown for 12 days on BG-11o medium were determined by gas-liquid chromatography-mass spectroscopy. Of the 53 fatty acids detected, 17 were major components; the average level for each of these 17 fatty acids was at least 0.9% of the total fatty acids (in at least one of the genera). These fatty acids included (with mean percentages in the Anabaena and Nostoc strains, respectively) the saturated fatty acids 16:0 (30.55 and 23.23%) and 18:0 (0.77 and 1.27%); several unsaturated fatty acids, including 14:1 cis-7 (2.50 and 0.11%), 14:1 cis-9 (3.10 and 3.41%), a polyunsaturated 16-carbon (sites undetermined) fatty acid with an equivalent chain length of 15.30 (1.20 and 1.03%), 16:4 cis-4 (0.95 and 0.87%), 16:3 cis-6 (2.16 and 1.51%), 16:1 cis-7 (1.44 and 0.36%), 16:1 cis-9 (6.53 and 18.76%), 16:1 trans-9 (4.02 and 1.35%), 16:1 cis-11 (1.62 and 0.42%), 18:2 cis-9 (10.16 and 12.44%), 18:3 cis-9 (18.19 and 17.25%), 18:1 cis-9 (4.01 and 5.10%), and 18:1 trans-9 (0.92 and 1.94%); and the branched-chain fatty acids iso-16:0 (2.50 and 1.14%) and iso-15:1 (0.34 and 2.05%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E.T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants.(ABSTRACT TRUNCATED AT 250 WORDS)     

A hybrid cell mapping panel for regional localization of probes to human chromosome 8

We have characterized a panel of somatic cell hybrids that carry fragments of human chromosome 8 and used this panel for the regional localization of anonymous clones derived from a chromosome 8 library. The hybrid panel includes 11 cell lines, which were characterized by Southern blot hybridization with chromosome 8-specific probes of known map location and by fluorescent in situ hybridization with a probe derived from a chromosome 8 library. The chromosome fragments in the hybrid cell lines divide the chromosome into 10 intervals. Using this mapping panel, we have mapped 56 newly derived anonymous clones to regions of chromosome 8. We have also obtained physical map locations for 7 loci from the genetic map of chromosome 8, thus aligning the genetic and physical maps of the chromosome.     

Cytosine methylation enhances Z-DNA formation in vivo.

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.