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J Cha

Publications and source records attributed to J Cha.

25 records · Page 2Linked to original sources
PubMed1997

Clustering of GABAA receptors by rapsyn/43kD protein in vitro.

Rapsyn, a 43-kDa protein on the cytoplasmic face of the postsynaptic membrane, is essential for clustering acetylcholine receptors (AChR) at the neuromuscular junction. When transfected into nonmuscle cells (QT-6), rapsyn forms discrete membrane domains and can cluster AChR into these same domains. Here we examined whether rapsyn can cluster other ion channels as well. When expressed in QT-6 cells, the GABAA receptor (human alpha 1, beta 1, and gamma 2 subunits) and the skeletal muscle sodium channel were each diffusely scattered across the cell surface. Rapsyn, when co-expressed, clustered the GABAA receptor as effectively as it clustered AChR in previous studies. Rapsyn did not cluster co-transfected sodium channel, confirming that it does not cluster ion channels indiscriminately. Rapsyn mRNA was detected at low levels in the brain by polymerase chain reaction amplification of reverse-transcribed RNA, raising the possibility of a broader role for rapsyn.

Animals↗
PubMed1996

Metal and pH dependence of heptapeptide catalysis by human matrilysin.

Human matrilysin devoid of its propeptide is expressed in Escherichia coli and purified to homogeneity by heparin chromatography after refolding of the guanidine hydrochloride solubilized protein. Matrilysin autolytically removes its N-terminal tripeptide Met-Tyr-Ser during the refolding process. The enzyme contains 1.91 +/- 0.08 zinc atoms/mol of protein and retains full activity when stored several months at 4 degrees C. It hydrolyzes the fluorescent substrate Dns-PLALWAR at the Ala-Leu bond with a kcat of 3.1 s-1 and K(m) of 1.8 x 10(-5) M at pH 7.5, 37 degrees C, values closely similar to those for the matrilysin produced by activation of the Chinese hamster ovary and E. coli-expressed promatrilysin. The properties of this form of matrilysin demonstrate that the propeptide is not essential for proper folding or stability of the enzyme but likely determines the N-terminal amino acid of the mature enzyme. The pH dependence of kcat/K(m) for Dns-PLALWAR shows that matrilysin has a broad pH optimum (5.0-9.0) and the pKa values obtained are 4.3 and 9.6 at 25 degrees C. The activity is inhibited by several metal binding agents including 1, 10-phenanthroline, OP, but not by the nonchelating isomer, 1,7-phenanthroline. OP inhibits instantaneously by likely forming a transient ternary enzyme.metal.chelator complex. The zinc atom is then removed from the protein in a time-dependent manner. In agreement with the kinetic studies, dialysis in the presence of OP and CaCl2 removes only the catalytic zinc atom. The monozinc enzyme can be reactivated to 90%, 56%, 27%, and 17% of the native activity by addition of zinc, manganese, nickel, and cobalt, respectively. Cadmium, on the other hand, forms an inactive Cd/Zn hybrid. The differences in the chelator accessibility properties of the two zinc sites can thus be exploited to yield metallohybrids of matrilysin.

Amino Acid Sequence↗
PubMed1996

Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.

Bacteriophage lambda↗
PubMed1995

Probing the roles of active site residues in D-xylose isomerase.

The roles of active site residues His54, Phe94, Lys183, and His220 in the Streptomyces rubiginosus D-xylose isomerase were probed by site-directed mutagenesis. The kinetic properties and crystal structures of the mutant enzymes were characterized. The pH dependence of diethylpyrocarbonate modification of His54 suggests that His54 does not catalyze ring-opening as a general acid. His54 appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding. Phe94 stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp137 and Phe26. Lys183 and His220 mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic alpha-D-xylopyranose. Lys183 functions structurally by maintaining the position of Pro187 and Glu186 and catalytically by interacting with acyclic-extended sugars. His220 provides structure for the M2-metal binding site with properties which are necessary for extension and isomerization of the substrate. A second M2 metal binding site (M2') is observed at a relatively lower occupancy when substrate is added consistent with the hypothesis that the metal moves as the hydride is shifted on the extended substrate.

Aldose-Ketose Isomerases↗
PubMed1994

Perturbing the metal site in D-xylose isomerase. Effect of mutations of His-220 on enzyme stability.

The histidine residue at position 220 in the Streptomyces rubiginosus D-xylose isomerase is conserved in all D-xylose isomerases. The three-dimensional structure of D-xylose isomerase reveals that His-220 is part of the octahedral coordination sphere of M2, one of two metal ions (Mn2+) in the active site. This work describes the effects of replacing His-220 with Ser, Glu, Asn, and Lys. The consequences of these amino acid substitutions on enzyme activity, thermostability, and structure were analyzed by kinetic, denaturation, and crystallographic methods. The kcat values H220S, H220N, and H220E are only 0.3-0.5% of the wild-type values, and the Km for each of these mutant enzymes increased by 30-40-fold over the wild-type value. The mutant enzyme H220K did not exhibit any measurable activity. Thermal denaturation studies (Tm values) indicate that the H220S and H220N mutant enzymes are approximately 5-8 degrees C less stable than the wild-type enzyme, whereas H220E and H220K are 13-24 degrees C less stable than the wild-type enzyme. To analyze the molecular basis for this decreased thermostability, the crystal structures of the H220S, H220N, and H220E mutant enzymes complexed with Mn2+ have been determined at 1.95, 1.90, and 1.75 A, respectively. In the H220S structure, a water molecule effectively replaces the N epsilon-2 atom of the imidazole ring of His-220 and mediates the interaction between Mn2+ at the M2 site and Ser-220. A similar water-mediated interaction between the metal ion and Asn-220 is observed in H220N. No direct or water-mediated interactions between the carboxyl group of Glu-220 and the metal are observed in H220E. Whereas octahedral coordination is maintained for the metal at the M2 site in H220S and H220N, a pentahedral coordination with the metal at the M2 site is observed in H220E. Metal activation measurements support the observation that metal binding is perturbed and is responsible for thermal lability of His-220 mutants.

Aldose-Ketose Isomerases↗
PubMed1993

New vectors for direct cloning of PCR products.

We describe the construction of two new vectors for direct cloning of polymerase chain reaction (PCR) products. This was done by inserting a synthetic DNA fragment containing two adjacent XcmI sites between the Asp718 and BamHI sites of the M13mp18 and M13mp19 phages. Cleavage of these M13 derivatives with XcmI will result in a linearized vector with a single thymidine nucleotide at the 3' ends. Thus, these vectors would be very useful for direct cloning of PCR-generated products with high efficiency.

Bacteriophage M13↗