Proteins Ia Research Articles

Membrane Proteins of Escherichia coli K-12: Two-Dimensional Polyacrylamide Gel Electrophoresis of Inner and Outer Membranes

Protein compositions of the inner and outer membranes of Escherichia coli K-12 have been analyzed by two-dimensional gel electrophoresis in which proteins are separated according to apparent isoelectric point (first dimension) and to apparent molecular weight (second dimension). Membrane proteins except for a pair of major outer membrane proteins (proteins Ia and Ib) were found to be solubilized effectively by lysis buffer containing urea, Triton X-100, ampholines and 2-mercaptoethanol. The latter two proteins could be solubilized after precipitation of membrane fraction with trichloroacetic acid; they formed a pair of spots at an acidic region on the electropherogram. Another major protein of the outer membrane, protein II, was also identified. Most of the inner and outer membrane proteins were shown to be focused at a pH range between 4 and 6.5. Specific protein patterns characteristic for both the inner and outer membranes could thous be visualized by the present system. At least 120 and 50 protein species were detected for the inner and outer membranes, respectively.

Adenosine 3':5'-monophosphate-regulated phosphoprotein system of neuronal membranes. I. Solubilization, purification, and some properties of an endogenous phosphoprotein.

An endogenous substrate for adenosine 3':5'-monophosphate-dependent protein kinase has been solubilized, and purified about 5,000-fold to apparent homogeneity, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes. This endogenous substrate, referred to as Protein I, is apparently specific to nervous tissue, and is composed of two types of polypeptides, present in a proportion of 1 (Protein Ia, 86,000 daltons) to 2 (Protein Ib, 80,000 daltons). In the presence of cAMP-dependent Protein I kinase purified from the same membrane fractions, Proteins Ia and Ib incorporated 0.83 and 0.81 mol of phosphate into serine/mol of peptide, respectively. Proteins Ia and Ib have similar amino acid compositions and have isoelectric points of 10.3 and 10.2, respectively. Both types of polypeptide have a relatively high content of glycine and proline, and both are degraded to a peptide of 48,000 daltons by highly purified collagenase, suggesting that Proteins Ia and Ib contain some sequences similar to those observed in collagen. The sedimentation coefficient of Protein Ia and Protein Ib was determined to be 2.9 S. The data suggest that both Protein Ia and Protein Ib have an elongated shape.

Escherichia coli K-12 tolF mutants: alterations in protein composition of the outer membrane

Outer membrane materials prepared from three independently isolated spontaneous Escherichia coli tolF mutants contained no detectable protein Ia. The loss of this protein was nearly completely compensated for by an increase in other major outer membrane proteins, Ib and II. Thus, the major outer membrane proteins accounted for 40% of the total cell envelope protein in both tol+ and tolF strains. No changes were found in the levels of inner membrane proteins prepared from tolF strains when compared with similar preparations from the tol+ strain. Phage-resistant mutants were selected starting with a tolF strain by using either phage TuIb or phage PA2. These phage-resistant tolF strains contained neither protein Ia nor protein Ib. The mutation leading to the loss of protein Ib in these strains is independent of the tolF mutation and is located near malP on the E. coli genetic map.

Major proteins of the outer cell envelope membrane ofEscherichia coli K-12: Multiple species of protein I

Protein I, one of the major outer membrane proteins ofE. coli, in a number of strains exists as two electrophoretically separable species Ia and Ib. Two phages, TuIa and TuIb, have been found which use, as receptors, proteins Ia and Ib, respectively. Selection for resistance to phage TuIb yielded mutants still possessing protein Ia and missing protein Ib (Ia+ Ib-). Selection in this background, for resistance to phage TuIa yielded one class of mutants missing both species of protein I and another synthesizing a new species of protein I, polypeptide Ic. Tryptic fingerprints of Ia and Ic are very similar and the sequence of 8 N-terminal amino acids is identical for Ia and Ic. Yet, Ic showed an entirely different pattern of cyanogen bromide fragments than that of protein Ia. With another example (cyanogen bromide fragments of protein II*, with and without performic acid oxidation) it is shown that protein modification can lead to gross changes of the electrophoretic mobility of cyanogen bromide fragments. It is not unlikely that all protein I species observed so far represent in vivo modifications of one and the same polypeptide chain. A genetic analysis together with data from other laboratories revealed that at least 4 widely separated chromosomal loci are involved in the expression of the protein I species known to date.

Outer cell envelope membrane and shape of Spirillum serpens

“Ghosts” have been isolated from Spirillum serpens that are free of murein, are surrounded by a unit membrane (derived from the outer membrane of the cell envelope), have lost all intracellular material (except for some poly-β-hydroxybutyrate), and still maintain Spirillum's shape. The ghost membrane contains about 50% protein which is resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into three bands corresponding to apparent molecular weights between 21,000 and 40,000, and the major protein band I (40,000) consists of at least two (Ia and Ib) but not many more polypeptide chains. HP-layer protein (hexagonally packed surface protein) is absent. At least one of the latter polypeptide chains is required for the establishment of the long-range order apparent in ghosts since proteases degrade band I proteins and concomitantly destroy the ghost. The other polypeptides (II and III) do not appear to be required for maintenance of shape of the Spirillum ghost since their amounts can vary widely from preparation to preparation. Ghosts as well as cells can be cross-linked with dimethyl diimidoesters. Such ghosts proved to be cross-linked over their entire surface, and a covalently closed macromolecule of the size of the cell had been created. Under certain conditions of cross-linking these ghosts upon extraction with hot sodium dodecyl sulphate were pure protein. Ammonolysis of this material liberated band I protein. These findings strongly suggest that there is a rather dense packing of the protein in the ghost membrane, and proteins Ia and Ib may be arranged as repeating subunits in the sense that protein-protein interaction exists along the whole membrane. Several observations also suggest that the ghost membrane concerning the arrangement of these proteins does not represent a gross artifact regarding the outer cell envelope membrane. The possibility exists that the assembly of polypeptides Ia and Ib participates in the determination of cellular shape.