RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : III. Electron Microscopy and Analysis of the Cytochromes

The membranous nature of pellets obtained from broken Escherichia coli spheroplasts by successive centrifugation at 3500 g (P₁), 20,000 g (P₂), and 105,000 g (P₃), has been established by electron microscopy. Spectrophotometric analysis has shown that about 90% of the cytochromes are concentrated in the particulate fractions. The crude ribosomal pellet (P₃) contained as much of the total cytochromes as did the pellet obtained at 20,000 g (P₂). The high cytochrome content of P₃ is consistent with its high oxidative activity (1) and the presence of membrane vesicles in this fraction. Analysis at 77°K intensified the optical extinction of all the cytochrome absorption bands, but the degree of intensification was not uniform for each fraction nor for each band within a given fraction. Carbon monoxide had little or no inhibiting effect on NADH oxidation. Reduced plus carbon monoxide difference spectra yielded artifactual absorption bands in the wave length regions where reduced vs. oxidized absorption bands normally occur. Succinate and NADH, either together or separately, reduced nearly all of the cytochromes, indicating that the cytochrome portion of the electron-transport chain is shared by both substrates. A tentative formulation of the electron-transport chain is presented.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : II. Effects of Fatty Acids and Albumin on Respiration

Fatty acids inhibited the ability of Escherichia coli membrane-envelope fragments to catalyze the oxidation of succinate and nicotinamide adenine dinucleotide, reduced form (NADH) and also inhibited the response of the Clark oxygen electrode to nonenzymatic oxygen uptake. In all cases, unsaturated fatty acids were much more inhibitory than saturated fatty acids. Albumin afforded complete protection from inhibition in the nonenzymatic oxygen-uptake experiments but only partial protection for the respiratory activities of the membrane fragments. The succinoxidase activity was totally inhibited by bovine serum albumin at concentrations that inhibited succinate dehydrogenase only slightly and NADH oxidase not at all. The E. coli acellular preparation showed no dehydrogenase or oxidase activity for any of the fatty acids under a variety of conditions. These conditions included variations of pH, concentration of fatty acids, and the presence or absence of albumin, CoA, ATP, NAD, cysteine, succinate, and carnitine. It thus appears that E. coli grown in the absence of fatty acid can not use fatty acids as an energy source.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : V. On the Reduction of Nonheme Iron and the Cytochromes by Nicotinamide Adenine Dinucleotide and Succinate

The sensitivity of nicotinamide adenine dinucleotide (NADH) oxidase and succinoxidase to metal chelators, the generation of an electron paramagnetic resonance (EPR) signal upon addition of these substrates, and the rate of formation of the EPR signal relative to the rate of the cytochrome reduction suggest the participation of nonheme iron proteins in the respiratory process of Escherichia coli. The most inhibitory metal chelator, thenoyltrifluoro acetone, inhibited the reduction of nonheme iron and cytochromes but did not prevent the reoxidation of the reduced forms. The EPR signal, dehydrogenase, and oxidase activities evoked by NADH are considerably greater than the corresponding activities evoked by succinate. Because both substrates can reduce almost all of the cytochromes, a model in which fewer succinate dehydrogenase-nonheme iron protein complexes are linked to a common cytochrome chain than NADH dehydrogenase-nonheme iron protein complexes is considered likely.     

Studies in the Nitrogen Metabolism of Apple Fruits: THE CLIMACTERIC RISE IN RESPIRATION IN RELATION TO CHANGES IN THE EQUILIBRIUM BETWEEN PROTEIN SYNTHESIS AND BREAKDOWN

1. A close parallelism in the drift of the rate of respirationand the protein-N/ non-protein-N ratio is shown to occur bothin apple fruits attached to the tree and when detached fromthe tree at various stages of development and stored for severalmonths at 12 C.
2. In detached fruits the fall in respirationwhich occurs immediately(during the first 48 hours) after pickingis only accompaniedby a concomitant fall in net protein invery young fruits inwhich active cell division is taking place.Subsequently, infruit of all ages when a climacteric rise inrespiration occursit is accompanied by a net increase in protein.
3. It is argued that the climacteric rise in respiration is aresultof increase in the level of protein which will be expectedtoreduce the ATP/ADP ratio.
4. Over the climacteric period,although rate of respiration andnet protein content both rise,R rises more rapidly than proteinand, subsequently, falls ata faster rate than P. It is suggestedthat this may be due tothe ‘new’ protein containinga higher proportionof enzyme(s) directly involved in respirationand leading, forexample, to a reduction in the ATP/ADP ratio.

     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : IV. HeLa Tumor Strain Cells

Interferometric and photometric measurements have been made on HeLa cells, a strain of cells originally derived from a human carcinoma. From a study of the relations between successive physical measurements on individual cells, it was confirmed that, whereas the net syntheses of nuclear RNA and nuclear protein are closely associated during interphase, they are dissociated from DNA replication to a significant extent. These results on nuclear metabolism agree with others previously reported in cell strains derived from tumors; they contrast with results from freshly prepared normal cells, where the net syntheses of DNA, nuclear RNA, and protein are closely associated during interphase. Cytoplasmic measurements on HeLa cells showed that much of the net synthesis of cytoplasmic RNA is associated with DNA replication as in normal cells, and they failed to detect transfer from the nucleus of a stable RNA component synthesized independently from DNA replication. In auxiliary experiments, an inhibition of the onset of DNA synthesis was produced by a dose of X-rays; under these conditions it was shown that the major part of the accumulation of nuclear protein was independent of DNA replication and that the accumulation of nuclear RNA was equivalent to or slightly less than that of nuclear protein. About half the accumulation of cytoplasmic RNA was inhibited when DNA synthesis was blocked.     

LOCALIZATION OF MACROMOLECULES IN ESCHERICHIA COLI : II. RNA and its Site of Synthesis

The distribution of RNA in cells of E. coli 15 T^-U^- labeled with uridine-H³ was studied by methods involving the analysis of radioautographic grain counts over random thin cross-sections and serial sections of the cells. The results were correlated with electron microscope morphological data. Fractionation and enzyme digestion studies showed that a large proportion of the label was found in RNA uracil and cytosine, the rest being incorporated as DNA cytosine. In fully labeled cells the distribution of label was found to be uniform throughout the cell. The situation remained unchanged when labeled cells were subsequently treated with chloramphenicol. When short pulses of label were employed a localization of a large proportion of the radioactivity became apparent. The nuclear region was identified as the site of concentration. Similar results were obtained when cells were exposed to much longer pulses of uridine-H³ in the presence of chloramphenicol. If cells were subjected to a short pulse of cytidine-H³, then allowed to grow for a while in unlabeled medium, the label, originally concentrated to some extent in the nuclear region, was found dispersed throughout the cell. The simplest hypothesis which accounts for these results is that a large fraction of the cell RNA is synthesized in a region in or near the nucleus and subsequently transferred to the cytoplasm.     

Defoliation-Induced Stress in Nodules of White Clover: I. CHANGES IN PHYSIOLOGICAL PARAMETERS AND PROTEIN SYNTHESIS

Nodule function and protein synthesis were studied in defoliationstressed white clover plants. Uncut control plants (C) werecompared with plants from two defoliation treatments: (1) continuousdefoliation (CD) where all leaves and petioles were removedeach day; and (2) defoliated/recovered (DR) where, after removalof all leaves and petioles, new leaves were then allowed toregrow. After a single defoliation N₂ fixation (acetylene reductionactivity) and nitrogenase-linked respiration declined by morethan 80% within 3 h and by nearly 100% by 24 h. DR plants beganto fix nitrogen again at a very low level 3 d later and thereafterrose to control levels by 15 d. Continuously defoliated plantsnever recovered N₂ fixation capacity. Nodule protein complementwas assessed by polyacrylamide gel electrophoresis. Major changesoccurred in buffer soluble protein band patterns by 6 d in CDplants, but few changes were evident in SDS soluble proteins.By 9 and 14 d significant disruption of all proteins was evident.The prominent host plant protein, leghaemoglobin (Lb) had disappearedby 14 d. In DR plants the intensity of staining was reducedbut no major changes in band patterns were evident and by 21d nodules were rejuvenated. [³⁵S]-labelled methionine was incorporated into nodule proteinsfrom all treatments throughout the experiment. However, continuousdefoliation caused increasing variability between replicatesin the labelled band patterns. By 21 d CD, much of the labelledprotein was present as amorphous low M_r material which suggestseither disruption of the protein synthesizing machinery or rapidhydrolysis by proteolytic enzymes. Surprisingly [³⁵S]-methionine was never found in Lb from nodulesof any treatment. It is possible that white clover Lb does notcontain any methionine residues or that no synthesis of Lb occurred. Key words: Trifolium repens, white clover, defoliation, protein synthesis, nodules     

Characterization of Carrot Cell Wall Protein : I. EFFECT OF alpha, alpha'-DIPYRIDYL ON CELL WALL PROTEIN SYNTHESIS AND SECRETION IN INCUBATED CARROT DISCS

A single glycoprotein accounts for the majority of radioactivity secreted to the cell wall when incubated carrot (Daucus carota) discs are labeled with radioactive proline or arabinose. The ferrous chelator α,α′-dipyridyl prevents the synthesis of this protein. A new proline-labeled protein is made in the presence of α,α′-dipyridyl and is secreted to the cell wall. The protein has little, if any, carbohydrate attached to it and has a molecular weight of 55,000 daltons. This protein appears to be the nonhydroxylated, nonglycosylated form of the major cell wall glycoprotein. α,α′-Dipyridyl does not prevent proline label from becoming tightly (presumably covalently) bound to the cell wall, providing further evidence that hydroxylation and arabinosylation are not required for the covalent attachment of proteins to the cell wall. Messenger RNA extracted from incubated carrot discs produces a product which electrophoreses similarly to the protein made in the presence of α,α′-dipyridyl. The possible use of the carrot disc system to study gene structure and regulation is discussed.     

Characterization of the Operator Sites of the exu Regulon in ESCHERICHIA COLI K-12 by Operator-Constitutive Mutations and Repressor Titration

In Escherichia coli, the exu regulon of the hexuronate system involves the three exuT, uxaCA and uxaB operons and is under the negative control of the exuR regulatory gene product. The technique developed by Casadaban, Chou and Cohen was employed to construct two plasmids containing operon fusions in which the lactose genes were fused to the uxaCA and exuT operons. These fusions were transferred into the chromosome by a reciprocal recombination event, and the resulting strains were used for isolation of mutants defective in repression. Two types of operator-constitutive mutants were obtained: one specific for the uxaCA operon expression and the other affecting the exuT gene expression. This genetic evidence confirms that these two operons which are divergently transcribed each possess their own operator site.--The derepressed expression of the two exuT-lac and uxaCA-lac operons and the uxaB gene was also examined upon introduction of plasmids bearing various operators of the exu regulon. The results of testing exuR repressor titration by multiple copies of the exu operators allowed us to show a gradation in the affinity degrees for the three exu operators: uxaBo has the strongest affinity for the exuR repressor and uxaCo the weakest, although that of exuTo seems to be just slightly greater. This gradation may play a role in the control of the exu regulon expression.     

SPECIFIC PROTEIN SYNTHESIS IN CELLULAR DIFFERENTIATION : Production of Eggshell Proteins by Silkmoth Follicular Cells

Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3–1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30–37 codons per ribosome) is similar to that encountered in other eukaryotic systems.     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : I. Fresh Embryo Human Cells

Interferometric and photometric measurements have been made on replicating embryo human cell cultures. From a study of the relations between successive physical measurements on individual cells, it was found that the net syntheses of DNA, nuclear RNA, nuclear protein, and cytoplasmic RNA are closely associated during interphase. Additional measurements of DNA and cytoplasmic RNA on freshly prepared replicating monkey kidney cells gave similar results. In auxiliary experiments with embryo human cells, an inhibition of the onset of DNA synthesis (produced by a dose of X-rays) was found to block the majority of the accumulation of nuclear protein and RNA and about half the accumulation of cytoplasmic RNA. These results are consistent with others previously reported in dividing cell cultures freshly prepared from normal tissues.     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : III. Mouse Ascites Tumor Cells

Interferometric and photometric measurements have been made on replicating mouse ascites tumor cell cultures. From a study of the relations between successive physical measurements on individual cells, it was found that whereas the net syntheses of nuclear RNA and nuclear protein are closely associated during interphase, they are dissociated from DNA replication to a significant extent. These results agree with others reported in replicating cell strains derived from tumors. In auxiliary experiments an attempt was made to block the initiation of DNA synthesis by X-irradiation: although large amounts of nuclear protein accumulated in some cells in the absence of DNA synthesis, the inability to hold the DNA block for an interphase time prevented a quantitative analysis of the results.     

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES : IV. Biochemical Pathway of Catalase Synthesis

Early events in the biosynthesis of liver catalase were studied on female rats receiving [³H]leucine or [³H]δ-aminolevulinic acid or a mixture of [³H]leucine with [¹⁴C]δ-aminolevulinic acid by intraportal injection. Catalase antigen was selectively separated from homogenates by immunoprecipitation, both without and after partial purification of the enzyme. Label from both precursors appeared first in immunoprecipitable material which was lost upon purification of catalase; the label subsequently became associated with material indistinguishable from catalase. Kinetic analysis of the results indicates that the nonpurifiable material identified by early labeling consists of two distinct biosynthetic intermediates, the first lacking heme and representing about 1.6% of the total catalase content or 13 µg/g liver, the second containing heme and representing about 0.5% of the total catalase content or 4 µg/g liver. The first intermediate migrates at the same rate as catalase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and therefore has a monomeric molecular weight of about 60,000.     

The Role of Maturation Drying in the Transition from Seed Development to Germination: IV. PROTEIN SYNTHESIS AND ENZYME ACTIVITY CHANGES WITHIN THE COTYLEDONS OF RICINUS COMMUNIS L. SEEDS

Drying of immature seeds of Ricinus communis L. cv. Hale (castorbean) during the desiccation-tolerant phase of development causesthem to germinate upon subsequent rehydration. This desiccation-inducedswitch from development to germination is also mirrored by achange in the pattern of soluble and insoluble protein synthesiswithin the cotyledons of the castor bean. Following rehydrationof seeds prematurely dried at 40 d after pollination (DAP),cotyledonary proteins characteristic of development (e.g. storageproteins) are no longer synthesized; hydrolytic processes resultingin their degradation commence (after 12 h) in a manner similarto that observed following imbibition of the mature seed. Apattern of protein synthesis recognizable as germination/growth-associatedoccurs; premature drying has elicited a redirection in metabolismfrom a developmental to a germinative mode. Desiccation is alsorequired for the induction (within cotyledons of 35 DAP seeds)of enzymes involved in protein reserve breakdown (leucyl ß-naphthylamidase;LeuNAase) and lipid utilization (isocitrate lyase; ICL), anevent intimately associated with the post-germinative (growth)phase of seedling development. Thus, at a desiccation-tolerantstage of development, premature drying results in the suppressionof the developmental metabolic programme and a permanent switching-onof the germination/growth metabolic programme. Key words: Desiccation, metabolism, seed development, seed germination, castor bean, cotyledons     

LECITHIN SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION IN RAT LIVER : IV. A Radioautographic and Biochemical Study of Choline-Deficient Rats Injected with Choline-3H

Injection of choline-³H into choline-deficient rats resulted in an enhanced incorporation of the label into liver lecithin, as compared to the incorporation of label into liver lecithin of normal rats. The results obtained with the use of different lecithin precursors indicate that in the intact liver cell, both in vivo and in vitro, exchange of choline with phosphatidyl-choline is not significant. The synthesis and secretion of lecithins by the choline-deficient liver compare favorably with the liver of choline-supplemented rats, when both are presented with labeled choline or lysolecithin as lecithin precursors. Radioautography of the choline-deficient liver shows that 5 min after injection of choline-³H the newly synthesized lecithin is found in the endoplasmic reticulum (62%), mitochondria (13%), and at the "cell boundary" (20%). The ratio of the specific activity of microsomal and mitochondrial lecithin, labeled with choline, glycerol, or linoleate, was 1.53 at 5 min after injection, but the ratio of the specific activity of phosphatidyl ethanolamine (PE), labeled with ethanolamine, was 5.3. These results indicate that lecithin and PE are synthesized mainly in the endoplasmic reticulum, and are transferred into mitochondria at different rates. The site of a precursor pool of bile lecithin was studied in the intact rat and in the perfused liver. Following labeling with choline-³H, microsomal lecithin isolated from perfused liver had a specific activity lower than that of bile lecithin, but the specific activity of microsomal linoleyl lecithin was comparable to that of bile lecithin between 30 and 90 min of perfusion. It is proposed that the site of the bile lecithin pool is located in the endoplasmic reticulum and that the pool consists mostly of linoleyl lecithin.