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S Kocabiyik

Publications and source records attributed to S Kocabiyik.

13 recordsLinked to original sources
PubMed2001

Induced CYP1A mRNA, protein and catalytic activity in the liver of feral fish, leaping mullet, Liza saliens.

In this study, we examined whether levels of P4501A mRNA expression were naturally induced in feral fish, Liza saliens, and whether CYP1A protein levels and associated enzyme activity, EROD, were also increased. Induction of mRNA was measured using a nucleic acid hybridization technique. For the hybridization studies, a new 33-mer oligonucleotide probe 5'-dCTC ATC CAG CTT CCT GTC CTC GCA GTG ATC AAT-3' was designed, which corresponded to the totally conserved amino acid motif of CYP1A protein from positions 291 to 301 among the various fish species. Results of Northern blot analysis revealed that RNA isolated from the liver of mullet collected from the highly contaminated region of Izmir Bay with a dissolved and dispersed petroleum hydrocarbon content of 12.45 microg l(-1) gave a strong hybridization signal, whereas only a weak hybridization signal was detected in the liver RNA of fish caught from the reference site containing less than 1 microg l(-1) of petroleum hydrocarbons. Similarly, fish from the contaminated site had approximately 80 times more EROD activity than the feral fish captured from the reference site. Studies using polyclonal antibodies produced against purified mullet CYP1A also showed the similar trend. In conclusion, feral leaping mullet caught from contaminated water displayed induction of CYP1A at three levels of expression, namely, mRNA, apoprotein and catalytic activity.

Amino Acid Sequence↗
PubMed2000

The effect of valine substitution for glycine in the dimer interface of citrate synthase from Thermoplasma acidophilum on stability and activity.

To determine the role of hydrophobic interactions in the dimer interface of citrate synthase (CS) from Thermoplasma (Tp) acidophilum in thermostabilization, we have used site-directed mutagenesis to replace Gly 196 by Val on the helix L of the subunit interface. Recombinant wild-type and Gly 196 mutant TpCS enzymes were largely identical in terms of substrate specificities (K(m) for oxaloacetate and acetyl CoA). However, the mutation not only reduced catalytic activity (about 10-fold) (i.e., V(max), k(cat) and specific activity) of the TpCS, but also decreased its thermal and chemical stability. Archaeal citrate synthase is active as a dimer, since residues from both monomers participate in the active site. Our results suggest that Gly196 --> Val mutation interferes with dimerization, so that improper dimerization or dissociation of the dimer would have a profound affect on the activity as well as the conformational stability of TpCS.

Base Sequence↗
PubMed1998

Amino acid substitutions in the subunit interface enhancing thermostability of Thermoplasma acidophilum citrate synthase.

We have used citrate synthase from Thermoplasma (Tp.) acidophilum as a thermostable model system to investigate the role of hydrophobic interactions in dimer interface for maintaining high temperature stability. Three mutant enzymes were constructed by single amino acid substitutions in the interface helices: Ala97-->Ser, Ala104-->Thr, and Gly209-->Ala. All of the mutations enhanced the thermostability of Tp. citrate synthase, while improving its catalytic properties (Km, Vmax, and specific activity). The highest thermostability was achieved by the Gly209-->Ala substitution. The half-life of irreversible inactivation of the G209A mutant enzyme at 85 degreesC was about 57 min, and the midpoint of guanidinium chloride (GdmCl) induced irreversible denaturation was at 2.0 M GdmCl. Our results showed that amino acid substitutions increasing or decreasing interface hydrophobicity could further increase the thermostability of the Tp. citrate synthase. Thus, interface substitutions affecting the entropy of the unfolded state did not prove to be so critical in protein thermostabilization at higher temperatures.

Chemical Phenomena↗
PubMed1996

The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum.

In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.

Citrate (si)-Synthase↗
PubMed1995

Cloning and expression of a haloacid dehalogenase from Pseudomonas sp. strain 19 S.

A dehalogenase gene specifying the utilization of a variety of haloacids by Pseudomonas sp. Strain 19S has been cloned and expressed in E. coli. Our cloning strategy employed specific amplification of a fragment homologous to Pseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digested Pseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified in E. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.

Cloning, Molecular↗
PubMed1995

Effects of metals on elastase from Pseudomonas aeruginosa SES-938-1.

Among the numerous virulance factors produced by Pseudomonas aeruginosa, elastase is the one most often associated with pathogenesis. In this study, effects of various metal ions on elastase from a new isolate of P. aeruginosa (Strain SES-938-1) was investigated. Crude elastase was prepared from culture supernatant via salting out by ammonium sulfate, and then desalting and concentrating the sample using a centricon microconcentrator. Activities were measured at 450 nm using N-succinyl-L-(ala)3-p-nitroanilide as the substrate. The metal chelating agents EDTA and EGTA inhibited the Pseudomonas elastase, which shows that the enzyme is a typical metalloproteinase. At a 10-mM concentration, Mn2+, Ni2+, and Zn2+ strongly inhibited the elastase, whereas Mg2+ effect was negligable. There was a gradual decrease in the enzyme activity in accordance with an increase in the concentration of metal ions.

Chelating Agents↗
PubMed1992

Site-specific mutations of conserved C-terminal residues in aminoglycoside 3'-phosphotransferase II: phenotypic and structural analysis of mutant enzymes.

In order to test the biological importance of amino acids in the C-terminal quarter of aminoglycoside 3'-phosphotransferase II enzyme, seven of the highly conserved residues in this region, His-188, Asp-190, Asp-208, Gly-210, Arg-211, Asp-216 and Asp-220, were changed via site-directed mutagenesis. The phenotype of each mutant was compared to wildtype in terms of antibiotic susceptibilities and enzymatic activities. All of the substitutions either abolished or significantly reduced the resistance of the resulting strains to kanamycin, neomycin, butirosin, ribostamycin, paromomycin, gentamicin A, and G-418. Similarly, enzyme activities in crude extracts were substantially reduced for the mutant strains. Affinity of the enzyme for Mg+2-ATP decreased with His-188, Asp-190, Asp-216 and Asp-220 substitutions as revealed by Km measurements. Secondary structure analysis predicted that substitutions at the conserved residues caused severe conformational distortions at the corresponding regions of the protein.

Amino Acid Sequence↗
PubMed1992

Altered substrate specificity by substitutions at Tyr218 in bacterial aminoglycoside 3'-phosphotransferase-II.

Mutant aminoglycoside 3'-phosphotransferase II enzymes were produced in which Tyr218 was changed to serine, aspartic acid, or phenylalanine. In each case the mutation resulted in increased bacterial susceptibility to neomycin and kanamycin, while simultaneously increasing the Km values for these substrates. For the Ser and Asp mutants, bacterial resistance to amikacin increased, with a concomitant increase in affinity for this drug. Initial velocity studies indicated that the wild-type and mutant enzymes all followed Michaelis-Menten kinetics. Although these mutagenic substitutions changed the substrate specificity of these enzymes they did not alter the enzyme affinity for Mg(2+)-ATP.

Amino Acid Sequence↗
PubMed1992

Structure-function analyses for aminoglycoside 3'-phosphotransferase II (APH(3')-II).

Mutant strains containing APH(3')-II were constructed via site-directed mutagenesis of the cloned gene and by random mutagenesis of a strain containing the APH(3')-II gene on a conjugative plasmid. Substitutions at highly conserved amino acid residues produced APH(3') enzymes which in general showed reduced activity and conferred reduced levels of resistance to their substrates. Substitutions at Tyr 218 altered substrate specificity for the enzymes. Random mutagenesis produced plasmid-borne mutations conferring amikacin resistance. Two of these mutations appeared to be localized to the APH(3')-II structural gene.

Amino Acid Sequence↗
PubMed1989

Analysis of plasmids mediating ANT (2") and AAC(3)-II based gentamicin resistance in clinical isolates of Enterobacteriaceae.

The genetics of ANT(2") and AAC(3)-II mediated gentamicin resistance in Gram-negative bacteria obtained from Hacettepe University Hospital in Turkey, was studied. The plasmid DNA analyzed by agarose gel electrophoresis revealed a heterogeneous plasmid population. Conjugal transfer appeared to be an efficient mechanism for the dissemination of resistance to gentamicin/tobramycin/kanamycin/streptomycin/chloramphenicol/cephalothin , which was the common resistance pattern in the population. Plasmids of 46, 60, 70 and 80 Mdal in size were found to be conjugative. Among these a 70 Mdal plasmid was isolated more frequently; it was harboured by the strains exhibiting either of the enzyme profiles, ANT(2") or AAC(3)-II. Like the substrate profiles of the donors, their enzymatic activities were also maintained in the respective transconjugants. Based on the donor and transconjugant resistance markers, agarose gel electrophoresis and transfer properties, ANT(2") and AAC(3)-II dependent gentamicin resistance was found to be plasmid-linked. However, the evidence of a possible chromosomal location for the gentamicin resistance suggested that the resistance might well be of a transposable nature.

Anti-Bacterial Agents↗
PubMedDate not supplied

Prevalence of monochloroacetate degrading genotypes among soil isolates of Pseudomonas sp.

This study reports the isolation of Pseudomonas sp. strains with monochloroacetate (MCA) degradation function, from uncontaminated soil, and the use of Southern blot hybridization technique to detect MCA degrading catabolic genes and their divergence. Based on their capacity to remove Cl- from MCA in a minimal medium containing 185 ppm Cl-, the strains were classified into three groups: poor degraders (Cl- release between 0-15 ppm), medium degraders (Cl- release between 16-30 ppm), and high degraders (Cl- release between 31-45 ppm). We have applied a gene probe assay for determining the diversity of MCA degradative genotypes of 61 strains. Two different gene probes, dehCI and dehCII were used in Southern blot hybridization assays. Majority of the DNA samples that produced signals on the membrane blots (18 out of 24) hybridized with only dehCI DNA probe, while 6 strains hybridized with only dehCII probe. On the other hand, 37 isolates did not hybridize to either of the gene probes used. The results indicated the high specificity of the DNA hybridization method and the divergence of metabolic functions and/or genotypes among the native MCA-degrading Pseudomonas sp. populations in the soil.

Acetates↗