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Biomedical subjects

I Rasched

Publications and source records attributed to I Rasched.

At least 19 recordsLinked to original sources
PubMed1992

Subtilisin removes the surface layer of the phage fd coat.

The major coat protein of native filamentous phage fd is vulnerable to digestion by subtilisin, but not by any of a number of other proteolytic enzymes. Degradation by the non-specific protease subtilisin occurs at specific sites in the N-terminal portion of g8p. The N-terminal part of the protein is considered to be the outer layer of a two-layered coat. Thus, subtilisin treatment results in a monolayered phage particle. These particles possess the morphology and stability of native phage fd. Furthermore, subtilisin proteolysis proved to be an efficient instrument in detecting variations in the topology of the g8p of related filamentous phages.

Amino Acid Sequence↗
PubMed1992

The adsorption protein of phage IKe. Localization by deletion mutagenesis of domains involved in infectivity.

We constructed a set of plasmid-encoded internal deletion mutants within the gene for the adsorption protein (g3p) of phage IKe. All mutant proteins still contain the signal and membrane anchor sequence, as those are known to be indispensable for proper localization and hence assembly of the g3p into phage. These various deletions comprise all internal parts of the protein and are properly incorporated into phage, which remarkably shows that signal and anchor sequence are sufficient for incorporation of g3p. The data furthermore reveal that two separate sections within the IKe g3p are essential for infection: one amino-terminal, preceding the glycine-rich stretch, and the other carboxy-terminal. We conclude that this latter domain is involved in penetration because mutants lacking it are not infectious, but still bind to the receptor. The amino-terminal region, essential for infection, bears the receptor-recognizing domain and a sequence homologous to the penetration domain of the evolutionary related Ff phages, which is probably also involved in penetration of phage IKe. The prominent glycine-rich stretch of the IKe g3p is not essential for infection but significantly promotes it.

Adsorption↗
PubMed1991

Release of periplasmic proteins induced in E. coli by expression of an N-terminal proximal segment of the phage fd gene 3 protein.

We used the enzymes beta-lactamase and alkaline phosphatase to quantitatively evaluate the release of periplasmic proteins from E. coli cells transformed by plasmids harboring gene 3 of phage fd. Different deletion mutants of gene 3 released varying fractions of the enzymes. From these results we conclude that essentially the amino-terminal proximal part, upstream of the first glycine-rich region but not this region itself, is responsible for the excretion of periplasmic proteins in E. coli cells expressing the gene 3 protein of phage fd.

Alkaline Phosphatase↗
PubMed1990

Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli.

The cDNA of human medium chain acyl-CoA dehydrogenase (MCADH) was modified by in vitro mutagenesis, and the sequence encoding the mature form of MCADH was introduced into an inducible expression plasmid. We observed synthesis of the protein in Escherichia coli cells transformed with this plasmid with measurable MCADH enzyme activity in cell extracts. Glutamic acid 376, which has been proposed by Powell and Thorpe (Powell, P. J., and Thorpe, J. (1988) Biochemistry 27, 8022-8028) as an essential residue and the proton-abstracting base at the active site of the enzyme, was mutated to glutamine. After expression in bacteria of this plasmid, the corresponding extracts show no detectable MCADH activity, although mutant MCADH-protein production was detected by protein immunoblots. The mature enzyme and the Gln376 mutant were purified to apparent homogeneity. The wild-type enzyme is a yellow protein due to the content of stoichiometric FAD and had a specific activity which is 50% of MCADH purified from pig kidney. The Gln376 mutant is devoid of activity (less than 0.02% that of wild type, expressed enzyme) and is green because of bound CoA persulfide. Properties of the mutant enzyme suggest that the Glu376----Gln change specifically affects substrate binding. These results prove that Glu376 plays an important role in the initial step of dehydrogenation catalysis.

Acyl-CoA Dehydrogenase↗
PubMed1990

Dissection of functional domains in phage fd adsorption protein. Discrimination between attachment and penetration sites.

We constructed a set of deletion mutants in the attachment protein of phage fd. These mutants lack sequences coding for sections in the amino-terminal half. All the mutants that comprise a leader sequence are incorporated into phage particles. Our data strongly suggest a bipartite organization of the amino-terminal domain with (1) a region for receptor recognition and (2) a region that is necessary for penetration of the DNA into the host cell. These regions were mapped. Some evidence suggesting different roles for gene 3 protein in penetration of the outer and inner membrane are discussed. We demonstrate that the phenotypes caused by gene 3 protein in host cells can be subdivided into two groups with different sequence requirements: (1) phenotypes related to outer membrane disturbance; and (2) phenotypes related to the tolQRA transport system.

Amino Acid Sequence↗
PubMed1989

Pore-forming properties of the adsorption protein of filamentous phage fd.

The gene 3-encoded adsorption protein (g3p) of filamentous phage fd has been purified to homogeneity by using high-performance liquid chromatography. Removal of SDS from the SDS-solubilized g3p results in spontaneous oligomerization of the g3p. Reconstitution into artificial lipid bilayer membranes shows that the oligomer forms large aqueous pores that remain open for seconds and are insensitive to changes in membrane potential. The estimated diameter of the pores suggest that they are large enough to allow passage of phage single-stranded DNA. The implications of these findings for phage infection are discussed.

Adsorption↗
PubMed1988

Functional analysis of the adsorption protein of two filamentous phages with different host specificities.

The gene 3 coding for one minor coat protein (adsorption protein) of phage IKe was cloned into an expression plasmid and overproduced. The presence of a promoter for this gene could be demonstrated as well as the incorporation of the IKe gene 3 protein (g3p) into the cytoplasmic membrane of host cells. When 110 carboxy-terminal amino acids were deleted, the truncated protein was translocated across the cytoplasmic membrane into the periplasm. Thus the deleted amino acids bear a membrane anchor domain. In contrast to the partly homologous g3p of the Ff phages, IKe g3p did not alter the membrane properties of its host. IKe g3p was not incorporated into Ff phage particles in amounts detectable by our assays although the presence of IKe g3p may affect the efficiency of Ff phage production. The existence of a structural feature necessary for the specific recognition of the respective g3p during phage assembly is deduced.

Amino Acid Sequence↗
PubMed1987

Interaction between the gene 5 protein, gene 5 protein/single stranded fd DNA complex and gene 8 protein of the filamentous phage fd.

An affinity column consisting of gene 8 protein, the major coat protein of fd phage, bound to Sepharose was prepared. Isolated gene 5 protein/single stranded fd DNA complex was found to bind to this column and was eluted with fd phage single stranded fd DNA. pH changes, and 1 M CaCl2 were not effective in eluting the protein from the affinity column. Gene 5 protein/single stranded fd DNA complex from the crude extracts of fd-infected E. coli also bound to the column, as did isolated gene 5 protein; whereas fd single stranded DNA alone did not. These results may be relevant for the illucidation of the molecular events occurring in the early stages of fd phage assembly.

Arginine↗
PubMed1987

The inhibitory effect of dithiothreitol on the assembly of the filamentous phage fd.

Assembly of the filamentous phage fd is preceded by the formation of a complex between the viral single-stranded (ss) DNA and the virally coded gene 5 protein (gene 5 protein-ssDNA complex). The presence of 5 mM dithiothreitol in the growth medium prevents phage production; however, phage infection, phage DNA replication and phage genome expression are still observed. In contrast, the gene 5 protein-ssDNA complex is not formed in the presence of dithiothreitol in vivo, although the complex is not affected by the disulfide reducing agent in vitro. Furthermore, host lipid composition is altered by growth in the presence of dithiothreitol. The zwitterionic lipid, phosphatidylethanolamine, increases while the cationic phospholipid content, cardiolipin and phosphatidylglycerol, decreases. This suggests a role for lipids or membranous structures in the process of gene 5 protein-ssDNA complex formation.

Cardiolipins↗
PubMed1987

Molecular cloning of cDNA for rat liver general acyl CoA dehydrogenase and homology between the rat liver and pig kidney enzymes.

cDNA clone for general acyl CoA dehydrogenase (GAD) was isolated from a rat liver cDNA expression library in lambda gt11 using anti-pig kidney GAD antibody. Size of the isolated cDNA was estimated to be 1.5-1.6 kb. By immunological analysis of fusion protein and epitope selection, the cDNA clone was identified as that containing the GAD gene. Partial amino acid sequence deduced from nucleotide sequence of the cDNA coincided with that of the pig kidney enzyme. The antibody cross-reacted with rat liver enzyme and molecular weights of these enzyme proteins were shown to be almost the same. All these results indicate that rat liver GAD shares a common structure with pig kidney enzyme.

Acyl-CoA Dehydrogenases↗
PubMed1986

Demonstration of glutamate dehydrogenase isozymes in beef heart mitochondria.

Glutamate dehydrogenase (GDH) has been purified from beef heart mitochondria and compared with crystalline beef liver GDH. The specific activity of heart GDH was 127 units and of liver GDH 80 units. Heart GDH subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis had a protein corresponding to liver GDH and a smaller molecular weight protein. On agarose gel electrophoresis heart GDH activity was resolved into two fractions (with or without protease inhibitors) while liver had only one fraction. One of the heart fractions moved with liver GDH on electrophoresis. Thermal stability studies showed heart and liver GDH activity differed. Mouse antibodies to liver GDH precipitated both liver and heart GDH on double immunodiffusion. Mouse antibodies to liver GDH identified on nitrocellulose paper the polypeptide band of liver and heart GDH that were the same molecular weight but did not cross-react with the smaller molecular weight polypeptide present in heart GDH. Trypsin digestion of the two major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified GDH from beef heart mitochondria did not show any overlapping peptides. We conclude beef heart GDH activity is composed of two isozymes. One is the same as beef liver GDH, and the other is a smaller molecular weight protein. We propose the terms GDH-LM for the liver GDH isozyme and GDH-HM for the smaller molecular weight isozyme present in heart mitochondria but not liver.

Animals↗
PubMed1986

Versatile kanamycin-resistance cartridges for vector construction in Escherichia coli.

To generate polylinker sequences which can be transferred together with an adjacent selectable marker, two plasmids (pWW-84 and pWW-97) were constructed which contain a kanamycin-resistance gene (KmR) flanked by various restriction sites. From these plasmids KmR-cartridges can be obtained as EcoRI, BamHI, SalI, AccI or HincII fragments for insertion into the appropriate restriction site of any plasmid. The following restriction sites can be introduced with these cartridges: BamHI, SalI (AccI, HincII), EcoRI, SacI, SphI and KpnI (Asp718) all adjacent to KmR, XhoI and HindIII, both within KmR. If desired, KmR can be removed by PstI digestion and religation, creating a single PstI site and leaving all adjacent sites intact.

Base Sequence↗
PubMed1983

The lysine residues implicated in the gene 5 protein association sites.

Gene 5 protein, a DNA unwinding protein encoded by the bacteriophage fd, is self-associative in presence of DNA or oligonucleotides. The lysine residues implicated in the protein-protein binding domains have been identified after modification with acetimidate by means of peptide and amino acid analyses. These residues are Lys-7 and Lys-69.

Amino Acid Sequence↗