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Biomedical subjects

M Inouye

Publications and source records attributed to M Inouye.

At least 505 records · Page 28Linked to original sources
PubMed1977

A new form of structural lipoprotein of outer membrane of Escherichia coli.

Among the membrane proteins synthesized in toluene-treated cells of Escherichia coli were two distinct membrane proteins of different molecular weights, which were cross-reactive with antiserum against a structural lipoprotein of the outer membrane. One was thought to be the known membrane lipoprotein since it migrated to the same position as that of the lipoprotein (Mr = 7,200) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, the other protein migrated slower than the lipoprotein. No protein corresponding to the slower-migrating species was detected in the membrane proteins synthesized in vivo. The apparent molecular weight of the protein at the new peak was estimated to be between 10,000 and 15,000. Both the new protein and the lipoprotein were found to be synthesized from stable mRNA(s) in the toluene-treated cells. The synthesis of the new protein as well as the lipoprotein was sensitive to chloramphenicol, indicating that both proteins were synthesized on ribosomes. Peptides mapping of the new protein revealed the same COOH-terminal sequence as in the lipoprotein. This indicates that the new protein has an extra sequence at the NH2-terminal end. This hypothesis is supported by the finding that the NH2 terminus of the new lipoprotein is methionine, while that of the lipoprotein is a substituted cysteine. From double label experiments with each of 17 different amino acids and arginine, the amino acid composition of the extra region was deduced. The new protein was found to contain at least 18 to 19 extra amino acid residues over the lipoprotein, if it is assumed that the new protein has no extra arginine residues. It was found that 4 out of the 5 amino acids which were deficient in the lipoprotein (phenylalanine, tryptophan, proline, and histidine) were also deficient in the new protein, but the fifth one, glycine, was present in the new protein. From these results, it seems possible that this new form of the lipoprotine is a precursor of the lipoprotein (prolipoprotein) in the process of biosynthesis and assembly of the lipoprotein in the outer membrane.

Amino Acids↗
PubMed1977

Optical properties of an outer membrane lipoprotein from Escherichia coli.

The infrared spectrum of a structural lipoprotein from the Escherichia coli outer membrane indicated the lipoprotein had an alpha-helical conformation but no sign for the existence of beta-structures. From circular dichroism spectra of the lipoprotein, the alpha-helical content of the protein was found to be as high as 88% in 0.01-0.03% sodium dodecyl sulfate in the presence of 10(-5) M Mg2+ at pH 7.1 and 23 degrees C. When sodium dodecyl sulfate concentration increased higher than 0.1%, the alpha-helical content of the lipoprotein decreased to about 57%. Divalent cations, such as Mg2+ and Mn2+, were found to increase the helical content of the lipoprotein. The high alpha-helical content of the lipoprotein was observed in a wide range of temperatures (23 to 55 degrees C). The significance of the high alpha-helical content of the lipoprotein is discussed in light of the three-dimensional molecular models of the lipoprotein proposed previously.

Bacterial Proteins↗
PubMed1977

Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane.

The messenger RNA for the lipoprotein of the E. coli outer membrane was found to code for a putative precursor, prolipoprotein, which has 20 additional amino acid residues extending from the amino terminus of the lipoprotein. Using the prolipoprotein synthesized in an E. coli cell-free system directed by purified messenger RNA for the lipoprotein, the complete amino acid sequence of the amino-terminal precursor region was determined to be as follows: (formula: see text). It was also found that the prolipoprotein that accumulates in toluene-treated cells has the same sequence. The significance of the amino acid sequence is discussed in terms of the mechanism of biosynthesis and assembly of the lipoprotein in the E. coli outer membrane.

Amino Acid Sequence↗
PubMed1977

Amino acid replacement in a mutant lipoprotein of the Escherichia coli outer membrane.

The primary structure of a mutant lipoprotein of the outer membrane of Escherichia coli was investigated. This mutant was previously described as a mutant that forms a dimer of the lipoprotein by an S-S bridge (H. Suzuki et al., J. Bacteriol. 127:1494-1501, 1976). The amino acid analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of tryptic peptides, it was found that the arginine residue at position 57 was replaced with a cysteine residue. The amino terminal structure of the mutant lipoprotein was found to be glycerylcysteine, as in the case of the wild-type lipoprotein. The present results show that the mutation that was previously determined to map at 36.5 min on the E. coli chromosome occurred in the structure gene (lpp) for the lipoprotein. This was further confirmed by the fact that a merodiploid carrying both lpp+ and lpp produces not only the wild-type lipoprotein but also the mutant lipoprotein.

Amino Acids↗
PubMed1976

Isolation and identification of the messenger ribonucleic acid for a structural lipoprotein of the Escherichia coli outer membrane.

The cells of Escherichia coli strain CP 78 were labeled with [32P]orthophosphate and the total radioactive RNA was prepared from the cells. The mRNA that codes for a structural lipoprotein in the outer membrane was purified from the total RNA by three successive electrophoreses on polyacrylamide slab gels, twice at pH 8.3 and once at pH 3.5 in 7 M urea. Approximately 0.002% of the total radioactive phosphate used was incorporated into the fraction containing the most purified mRNA. The two-dimensional fingerprint of the T1 ribonuclease digest of the 32P-labeled mRNA showed that the purity of the mRNA was as high as 90%. A preliminary sequence analysis was carried out on the T1 ribonuclease oligonucleotides which had been separated by the fingerprinting procedure. By using the established amino acid sequence of the lipoprotein and the genetic code, three relatively long oligonucleotides were assigned to code for three different parts of the lipoprotein. From these data, the present RNA fraction was identified as the lipoprotein mRNA. From the analysis of the T1 ribonuclease oligonucleotides, the mRNA was estimated to be 360 +/- 10 nucleotides in length. Although the length of the mRNA was enough to code for 2 lipoprotein molecules, T1 ribonuclease digestion of the mRNA yielded only 1 mol/mol of mRNA of the individual oligonucleotides assigned to parts of the amino acid sequence of the lipoprotein. This suggests that the mRNA codes for only 1 molecule of the lipoprotein. It was also found that the mRNA has no polyadenylate sequence at the 3' end.

Base Sequence↗
PubMed1976

Puromycin-resistant biosynthesis of a specific outer-membrane lipoprotein of Escherichia coli.

The reported puromycin resistance of the in vivo biosynthesis of a specific outer-membrane lipoprotein of Escherichia coli was further investigated. The biosynthetic machinery making the lipoprotein was made more accessible to puromycin by disruption of the cell structure using ethylenediaminetetracetate or toluene, and finally in an in vitro protein biosynthesis system using polyribosomes. Puromycin sensitivity of overall protein synthesis increased by about 10-fold for each method of disruption of the cell structure; 50% inhibitions were obtained at 330, 35, 2.7, and 0.22 mug of puromycin per ml for intact cells, ethylenediaminetetraacetate-treated cells, toluene-treated cells, and the polyribosome system, respectively. However, the lipoprotein biosynthesis remained more resistant to puromycin than the biosynthesis of other proteins in all systems tested. These results strongly suggest that puromycin resistance of the lipoprotein biosynthesis is due to an intrinsic property of the lipoprotein biosynthetic machinery.

Bacterial Proteins↗
PubMed1976

Lipoprotein from the outer membrane of Escherichia coli: purification, paracrystallization, and some properties of its free form.

In the envelope of Escherichia coli, is a lipoprotein of molecular weight 7,200 as a major envelope protein. This lipoprotein was previously shown to exist in two different forms in the outer membrane of E. coli: the free form and the boundform, which is covalently linked to the peptidoglycau. The free form of the lipoprotein has been purified and paracrystallized by adding acetone to a sodium dodecyl sulfate solution in the presence of magnesium ion. The paracrystals were needle shaped. An electron micrograph of the negatively stained paracrystals showed a highly ordered ultrastructure. The chemical structure of the free form was compared with that of the bound form by (i) the amino acid composition, (ii) the fatty acid composition, and (iii) the peptide analysis after cyanogen bromide cleavage. The alpha-helical content of the free form of the lipoprotein was measured from the circular dichroism spectrum of the lipoprotein in 0.01% sodium dodecyl sulfate and found to be 87%. Using the purified lipoprotein as antigen, antiserum against the free form of the lipoprotein was obtained. Immunoprecipitation of the lipoprotein with the antiserum was found to be very specific, since only the free form of the lipoprotein was found as a major peak when the antiserum was reacted with the whole envelope proteins solubilized in 0.2% sodium dodecyl sulfate, and the immunoprecipitate thus formed was analyzed by polyacrylamide gel electrophoresis.

Amino Acids↗
PubMed1976

Ultrastructure of paracrystals of a lipoprotein from the outer membrane of Escherichia coli.

The highly purified lipoprotein of the outer membrane of Escherichia coli forms paracrystals. The ultrastructures of these paracrystals were examined by electron microscopy. The needle-shaped paracrystals show several different band patterns, depending on conditions of paracrystallization. Models are presented to explain possible arrangements of the lipoprotein molecules within the paracrystals.

Bacterial Proteins↗
PubMed1975

Biosynthesis of a specific lipoprotein of the Escherichia coli outer membrane on polyribosomes.

With use of polyribosomes isolated from Escherichia coli, biosynthesis of a specific lipoprotein of the E. coli outer membrane was investigated. The products of the cell-free system were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoprecipitation with anti-lipoportein serum, followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It was found that one of the major products was the lipoprotein of the E. coli outer membrane. The lipoprotein was also found to be preferentially synthsized on smaller polyribosomes.

Cell Fractionation↗
PubMed1975

Lysozymes from bacteriophages T3 and T5.

Lysozymes produced in host cells infected with bacteriophages T3 and T5 were found to have the same enzymatic specificity toward the peptidoglycan from Escherichia coli as T7 phage lysozyme, which has been shown to be an N-acetylmuramyl-L-alanine amidase.

Acetylglucosamine↗