The purification of protease IV of E.coli and the demonstration that it is an endoproteolytic enzyme

A strong proteolytic activity is unmasked and solubilized when $\text{E. coli}$ outer membrane fragments are preincubated with 0.083% sodium dodecyl sulfate. This proteolytic activity cleaves αS1 casein into the same degradation products as protease IV, a recently described protease of $\text{E.coli}$ located in the outer membrane (Ph. Régnier, preceding paper), it is concluded that sodium dodecyl sulfate solubilizes the same protease. Protease IV has been purified 11,200 fold, probably to homogenetiy, by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by elution of the protein from gel slices. The purified enzyme is fully active, its molecular weight, determined from its migration in denaturating gels is 23,500. αS1 casein is cleaved by protease IV into two large polypeptides which are not further degraded and some small peptides of about 5,000 daltons. The production of discrete polypeptide species suggests that protease IV is an endoproteolytic enzyme.     

Influence of protease inhibitors and energy metabolism on intracellular protein breakdown in starving Escherichia coli

It has been reported that certain inhibitors of serine proteases block intracellular protein breakdown in $\text{E. coli}$ subjected to nutritional deprivation. We show here that the protease inhibitors p-toluene sulfonyl fluoride and pentamidine isethionate inhibit protein breakdown in $\text{E. coli}$ deprived of glucose, but not in bacteria starved for inorganic phosphate or ammonia. Furthermore, we find that the protease inhibitors cause a drastic decline in cellular ATP levels when glucose is omitted from the incubation medium. It is concluded that these protease inhibitors influence protein breakdown by interfering with cellular energy production, rather than by interacting with a specific serine protease.     

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of $\text{Escherichia}\text{coli}$ by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of $\text{E.}\text{coli}$, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD⁺ and NADP⁺, but NADPH served to accelerate the rate of inhibition.     

Absence of ferric enterobactin receptor modification activity in mutants of Escherichia coli K-12 lacking protein a.

The modification activity for the ferric enterobactin receptor in the Triton X-100 solubilized outer membrane of $\text{Escherichia}\text{coli}$ K-12 was adsorbed to a column of $\text{p}$-aminobenzamidine-//-sepharose and eluted with free benzamidine. Recombination of the dialyzed eluate with the filtrate from the column reinstituted conversion of the receptor from 81K to 81K¹, the latter exhibiting an apparent molecular weight of 74,000 daltons in sodium dodecyl sulfate polyacrylamide gel analysis. The eluate from the $\text{p}$-aminobenzamidine column was shown to contain a component, coincident on gels with both protein and modification activity, which by mutational and other analyses appears to be identical with protein $\text{a}$ of the outer membrane.     

Galactosyltransferase: confirmation of equilibrium-ordered mechanism.

A thermostable protein that strongly inhibits the soluble $\text{E. coli}$ D-alanine carboxypeptidase was isolated from a cell-free extract of $\text{E. coli B}$. The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg⁺⁺ or Na⁺ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of $\text{E. coli}$ or the growth of the bacteria.     

Diffusion of tetracycline across the outer membrane of Escherichia coli K-12: involvement of protein Ia.

The possibility that passage of tetracycline across the outer membrane of $\text{E.}\text{coli}$ K-12 is controlled by one or more of the proteins I_a, I_b and II¹ (Henning's nomenclature) was investigated. A mutant lacking protein I_a (obtained by selection for resistance to phage TuI_a) was more resistant to tetracycline than wild-type strains or those lacking only proteins I_b or II¹. The envelope protein composition of a tetracycline-resistant mutant ($\text{cmlB}$) was altered in several respects, but the major change involved loss of protein I_a. These data support our previous suggestion [12] that tetracycline diffuses across the outer membrane through hydrophilic regions. Furthermore, they imply that only protein I_a plays a significant role in the passage of this antibiotic across the outer membrane.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

Restoration of active calcium transport in vesicles of an Mg2+-ATPase mutant of Escherichia coli by wild-type Mg2+-ATPase.

Strain NR70, a mutant of $\text{E. coli}$ lacking the Mg²⁺-adenosine triphosphatase (E.C. 3.6.1.3.) was previously shown to be defective in amino acid and sugar transport in whole cells and right-side-out membrane vesicles. It is shown here that the mutant is also deficient in the uptake of calcium into inverted membrane vesicles. Treatment of inverted vesicles from the wild-type strain with ethylenediamine tetraacetate removes the Mg²⁺-adenosine triphosphatase and results in an inability to transport calcium. Addition of a crude fraction containing the wild-type Mg²⁺-adenosine triphosphatase restores active uptake of calcium both to vesicles from the mutant and depleted vesicles from the wild-type.     

Localization of the lactose permease protein(s) in the E. coli envelope.

The amino acid double labeling technique was used to identify and localize membrane-bound lactose operon proteins in $\text{E.}\text{coli}$. Both the “M” protein, thought to be the $\text{y}$ gene product, and a polypeptide of MW ～15,000 appeared in the membrane following $\text{lac}$ operon induction. The amounts of these two proteins were approximately equal.The inner and outer membrane layers of the cell envelope were separated by sucrose density gradient centrifugation or by selective solubilization of inner membranes with the detergent Sarkosyl. When gentle lysis conditions were employed to prepare membrane vesicles, both $\text{lac}$ induced proteins fractionated with the inner membrane. However, the “M” protein was more easily randomized in the envelope structure by sonication than the 15,000 dalton component or an inner membrane marker enzyme.     

Increased production of the outer membrane receptors for colicins B, D and M by Escherichia coli under iron starvation.

Growth of $\text{E. coli}$ K-12 under severe iron stress results in increased production of the outer membrane receptors for colicins B, D, Ib and M. The increase in colicin receptor activity coincides with the appearance of large amounts of two high molecular weight proteins in the outer membrane of the cells. These proteins are identified as the outer membrane receptors for colicins B and D and for colicin M. Mutants lacking a functional outer membrane receptor for colicins B and D are defective in the uptake of iron complexed with the siderochrome enterochelin, and are thus comparable with $\text{tonA}$ mutants which lack a functional receptor for colicin M and are defective in the uptake of iron complexed with ferrichrome (6). The colicin B and D receptor may therefore function in the uptake of ferri-enterochelin.     

Interaction between the nitrate respiratory system of Escherichia coli K12 and the nitrogen fixation genes of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{chl}$^? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from $\text{K. pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$_Kp⁺ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by $\text{chl}$ genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity.     

Mutational Analysis of the Amino Acid Residues Essential for the cis and trans Cleavage Activity of the Potato Virus Y 50-kDa Protease

To examine, the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234→Tyr, Asp269→Asn, or Cys339→Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339→Ser replacement retained a reduced proteolytic activity.     

Peptide chain initiation with chemically formylated Met-tRNAs from E. coli and yeast

Chemically formylated Met-tRNA_m^Met and Met-tRNA_f^Met species from $\text{E.}$$\text{coli}$ and yeast were tested for their capacity to serve as chain-initiators in a cell-free system from $\text{E.}$$\text{coli}$. In the presence of R 17 mRNA, initiation factors and $\text{E.}$$\text{coli}$ ribosomes, all four Met-tRNAs could form functional initiation complexes as measured by ribosomal binding kinetics, fMet-puromycin formation and synthesis of a dipeptide fMet-Ala. Unformylated Met-tRNA_f^Met from $\text{E.}$$\text{coli}$ displayed significantly less activity as a peptide chain-initiator than the formylated Met-tRNA_m^Met species from $\text{E.}$$\text{coli}$ and yeast. Although the latter tRNAs were less effective initiators than the “physiological” initiator tRNAs, the data seem to indicate that a blocked α-amino group represents the major token of identification by which Met-tRNA is admitted to function in $\text{E.}$$\text{coli}$ peptide chain initiation.     

Occurrence in mammalian liver of a protein which replaces the B protein of E. coli quinolinate synthetase.

$\text{Escherichia coli}$ contains two proteins (A and B) which together convert dihydroxyacetone phosphate and aspartate to quinolinic acid, a precursor of NAD. Although mammalian liver homogenate does not catalyze this reaction it contains a protein which will replace the B protein of the $\text{E. coli}$ system. The behavior of the liver protein on Sephadex G-75 suggests it is much smaller than the $\text{E. coli}$ B protein. Liver B protein also appears to contain tightly bound FAD while FAD is easily removed from the $\text{E. coli}$ B protein. The pH optimum for the hybrid system $\text{E. coli}$ A protein-liver B protein is 9.0 while in the pure $\text{E. coli}$ system the optimum is pH 8.0. The hybrid system is inhibited by NAD to the same extent as the pure $\text{E. coli}$ system.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

Protein synthesis in rabbit reticulocytes: control of protein synthesis initiation by a met-tRNA Met f deacylase and peptide chain initiation factors

The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA_f^Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$). Other aminoacyl tRNAs tested including fMet-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$), Phe-tRNA ($\text{E.}$$\text{coli}$), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA_f^Met forms the initiation complex Met-tRNA_f^Met:IF1:GTP (2), and in this ternary complex Met-tRNA_f^Met is not degraded by the deacylase. $\text{E.}$$\text{coli}$ Met-tRNA_f^Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA_f^Met is degraded by the deacylase.Prior incubation of f1 with Met-tRNA_f^Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA_f^Met (retic.) was pre-incubated with peptide chain initiation factors.     

Proteolysis of chloroplast thylakoid membranes. I. Selective degradation of thylakoid pigment-protein complexes at the outer membrane surface

Isolated pea thylakoids were experimentally unstacked in low-salt buffer and incubated with Pronase or trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that brief treatment with a very low concentration (1 μg/ml) of either enzyme had an effect primarily on the light-harvesting chlorophyll $\text{a}\text{b}$-protein complexes, which are more sensitive to proteolytic attack than the other proteins of the thylakoid membranes. This mild proteolysis cleaves a ~1000-dalton portion from the predominant 28,000-dalton polypeptide of these complexes. Extensive proteolysis (100 μg Pronase/ml for 15 min) degraded almost all membrane polypeptides not associated with the pigment-protein complexes and degraded the chlorophyll $\text{a}\text{b}$-protein complexes further than milder proteolysis. Pronase treatment of thylakoids in the presence of horseradish peroxidase was used to monitor membrane breakage during proteolysis. Treatment with 100 μg Pronase/ ml enabled considerable amounts of peroxidase activity, and presumably, proteolytic enzymes to enter into the intrathylakoid space. This trapping of peroxidase activity was seen only minimally with milder proteolysis (1 μg Pronase/ml). These results suggest that brief exposure to low concentrations of proteolytic enzymes affects only the outer, stromal thylakoid surface, while at higher concentrations, significant proteolysis takes place at both sides of the membrane.     

Outer membrane of Escherichia coli K-12: Tsx mutants (resistant to bacteriophage T6 and colicin K) lack an outer membrane protein

$\text{Tsx}$ mutants of $\text{Escherichia coli}$ are fully resistant to a set of T6-like bacteriophage and are resistant to colicin K. We demonstrate that these mutants are missing an outer membrane protein (the tsx-protein) of molecular weight 32,000 as measured by SDS-polyacrylamide gel electrophoresis. $\text{Tsx}$ mutants are receptor mutants which are unable to absorb either the bacteriophages or the colicin and the loss of receptor function can be demonstrated using outer membrane preparations.We suggest that the tsx-protein is the receptor for both the bacteriophage and colicin.     

Cell surface proteins of E. coli

Cell surface proteins of $\text{E. coli}$ K12 have been labelled with ¹²⁵I using a lactoperoxidase method. Results suggest that most outer membrane proteins so far characterised appear to have at least part of their polypeptide chain on the cell surface. These include major outer membrane protains I and II¹, the maltose and vitamin B₁₂ binding proteins and proteins involved in iron transport. The labelling of an antibiotic sensitive mutant and its parent were compared but their labelling patterns did not appear to differ in any way which would suggest the cause of the permeability difference between these two strains.     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic $\text{dna}\text{A}$ mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in $\text{dna}$A mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or $\text{dna}\text{A}$ mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of $\text{dna}\text{A}$ mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or $\text{dna}\text{A}$ membrane preparations. Thus, the $\text{dna}\text{A}$ mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.