PubMed Health⌕ Search

Biomedical subjects

L H Guo

Publications and source records attributed to L H Guo.

46 records · Page 3Linked to original sources
PubMed1990

Direct electrochemistry of protein-protein complexes involving cytochrome c, cytochrome b5, and plastocyanin.

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.

Cytochrome c Group↗
PubMed1990

Direct voltammetry of the Chromatium vinosum enzyme, sulfide:cytochrome c oxidoreductase (flavocytochrome c552).

The electrochemistry of the enzyme, sulfide:cytochrome c oxidoreductase, also known as flavocytochrome c552 from the purple sulfur bacterium, Chromatium vinosum, has been studied using several modified electrodes. Direct electron transfer between the heme of the flavocytochrome and an electrode is observed in the presence of a redox-inactive cationic species which promotes the voltammetry of the enzyme. Quasi-reversible electron transfer was achieved using the aminoglycoside, neomycin, as a promoter at either a modified gold or polished edge-plane graphite electrode. Further evidence for direct electron transfer is provided by the catalytic response of the enzyme at the electrode in the presence of substrate. Also reported is the direct spectroelectrochemistry of flavocytochrome c552 at an optically transparent thin layer gold electrode modified with Cys-Glu-Cys in the presence of neomycin.

Chromatium↗
PubMed1989

[Whole nucleotide sequence of penicillin G acylase gene and its flanking region from E. coli].

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)

Amidohydrolases↗
PubMed1983

An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid.

A strategy for kilo-base sequencing of a target DNA cloned in plasmid pWR34 is described. A long target DNA is progressively shortened from one end, by digestion with BAL31 nuclease or exonuclease III and nuclease S1, followed by cleaving off the shortened vector DNA. The family of the shortened target DNA molecule is next cloned in between the StuI site on one end, and a cohesive-ended restriction site on the other end, within the polylinker region of pWR34. DNA fragments cloned into this plasmid are sequenced directly by using a synthetic oligonucleotide primer, which binds to one side of the polylinker region using the dideoxynucleotide chain-termination method. The plasmid DNA, easily obtained by adoption of a rapid mini-preparation, is usually pure enough for direct DNA sequencing. Thus, both strands of any DNA several thousand base pairs in length can be completely sequenced (using two different primers) with ease within a short time, without the need for constructing a physical map.

Base Sequence↗
PubMed1983

Synthesis of the 3'-half molecule of yeast alanine tRNA.

This paper deals with the synthesis of the 3'-half molecule of yeast alanine transfer RNA (tRNAAlay) by ligation with T4 RNA ligase of three component oligonucleotide fragments corresponding to nucleotides 36-45(I), 46-57(II) and 58-76(III) in succession extending from the 3'-end to the 5'-end. First, in a ratio of acceptor to donor at 1.5 to 1, we adopted a method of three successive reactions, namely, the 5'-phosphorylation of the nonadecamer (III), ligation with the dodecamer (II) and the 5'-phosphorylation of the ligation product formed; with one isolation step and obtained the 5'-phosphorylated 31mer(46-76) (IV) in an overall yield of 70%. Then the 31mer(IV) as a donor was ligated with 3 times of decamer (I) to form the 41mer(36-76) (V), the 3'-half molecule of tRNAAlay. The yield was 67%. After 5'-phosphorylation, (V) was ligated with the natural 5'-half molecule to form the semi-synthetic tRNAAlay, which was biologically active, i.e. accepting and transferring (3H)-alanine into proteins.

Base Sequence↗
PubMed1983

Assay of biological activity of synthetic yeast alanine transfer RNA (tRNAAlay).

The biological activity of the synthetic tRNAAlay was studied with an extremely sensitive method. tRNAAlay accepted alanine in the presence of rat liver aminoacyl-tRNAAlay-synthetase (this was called the accepting activity). The aminoacylated tRNAAlay was conveniently precipitated by ethanol with good recovery. The efficiency of transferring alanine from the aminoacylated tRNAAlay into the protein was determined in in vitro rabbit reticulocyte lysate cell-free protein-synthesizing system (this was called the incorporation activity). Both accepting and incorporation activities could be determined in one assay with only 5-7 pmoles of tRNAAlay either in ligation mixture or in purified form. Our results show that the accepting activities of the synthetic products were 51.6-65.6% and 91.3-106.0% of that of natural and reconstituted natural tRNAAlay respectively. The efficiency of the incorporation of alanine in the aminoacylated tRNAAlay into the protein was 61.6-63.1%, corresponding to 90.6-91.7% and 97.2-115.8% of that of the natural and the reconstituted natural tRNAAlay respectively.

Animals↗
PubMed1982

New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis.

We describe improve enzymatic methods for sequencing method for sequencing DNA. They are based on partial digestion of duplex DNA with exonuclease III to produce DNA molecules with 3' ends shortened to varying lengths, followed by repair synthesis to extend and label the 3' ends. After asymmetrical cleavage of the DNA with a restriction enzyme, the labeled products are separated by gel electrophoresis and the sequence read from the autoradiogram. The entire procedures, beginning with unrestricted DNA and followed through gel electrophoresis, takes only one day for sequencing both strands of the DNA molecule. These methods are especially suitable for sequencing DNA cloned in plasmid vectors, and they greatly extend the usefulness of the dideoxynucleotide chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463, 1977). Using these methods we have determined the sequence of a 410 base pair fragment which includes the yeast SUP3 tyrosine tRNA gene.

Base Composition↗
PubMedDate not supplied

Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli.

We have constructed two families of plasmids suitable for the cloning of genes and for directing the synthesis of large amounts of fused proteins in Escherichia coli. The plasmids include the E. coli lac promoter and a portion of the coding sequence for beta-galactosidase, which can code for approx. 590 or 450 amino acids. The truncated beta-galactosidase gene ends with a poly-linker region at the 3' end, which can be cleaved by any one of the eight common restriction enzymes and joined to the gene coding for any desired protein. Each family includes three plasmids that enable fusion to be made in all three of the translational reading frames. We have cloned a synthetic human proinsulin gene into these plasmids, and 30% of the total E. coli protein was represented by the 590 amino acid-long truncated beta-galactosidase fused to proinsulin. The yield of proinsulin in this system is more than twice the amount produced by using a 1007 amino acid-long beta-galactosidase gene for fusion.

Base Sequence↗