Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.     

Bimodal effects of cellular amino acids on Na+-dependent amino acid transport in ehrlich cells

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K⁺] ($\text{10 mM ± 5 mM}$).With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of infiux is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells.Knowing that increases and decreases in the membrane potential are achieved by using varying external [K⁺] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the $\text{V}$ for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased $\text{V}$. Changes in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ also occur when the membrane potential is varied.     

Effect of phenethylbiguanide on sugar and amino acid transport in rabbit ileum

Phenethylbiguanide has been shown to be an inhibitor of sugar and amino acid uptake in both $\text{in vivo}$ and $\text{in vitro}$ conditions. This action could be due to a competition for sodium sites on the sugar and amino acid carrier molecules. The effects of phenethylbiguanide on $\text{in vitro}$ intestinal preparations indicate that this compound has a time-dependent effect, it is most effective when placed on the mucosal surface but is also effective on the serosal surface. Furthermore, competition studies indicate that it is a competitive inhibitor of sugar uptake and a non-competitive inhibitor of amino acid uptake. These results are consistent with the differences in the mechanism of coupled transport between sugars and amino acids, but, do not substantiate the idea that phenethylbiguanide competes for the sodium site on the ternary carrier.     

Simultaneous determination of cysteine sulfinic acid and cysteic acid in rat brain by high-performance liquid chromatography

A sensitive and specific assay method for cysteine sulfinic acid (CSA) and cysteic acid (CA) using high-performance liquid chromatography has been developed. The method includes post-column derivatization of various amino acids with o-phthalaldehyde in the presence of 2-mercaptoethanol. The column packed with cation-exchange resin ($\text{ISC-07}\text{S1504}$, Shimadzu Sci entific instruments, Inc., Kyoto, Japan) was used for obtaining general separation of amino acids except CSA and CA, while the separation of CSA and CA was achieved using a strong-base anion exchange ($\text{ISA-07}\text{S2504}$, Shimadzu Scientific Instruments) column. The fluorescence peak area for CSA was linear between 20 pmol and 5 nmol, whereas that for CA was 10 pmol to 5 nmol. The regional distribution of CSA, CA, and other amino acids in the rat brain was studied using this new assay method.     

Inhibition of macromolecular synthesis in tumors by L-1-tosylamido-2-phenylethyl chloromethyl ketone.

L-1-tosylamido-2-phenylethyl chloromethyl ketone was observed to inhibit the incorporation of [³H] amino acids into protein and [³H] thymidine incorporation into DNA in Novikoff hepatoma ascites cells $\text{in vitro}$ Similar effects were seen with several Morris hepatomas and a transplanted colon tumor in rats, and were accompanied by decreased uptake of isotope into acid soluble tissue fractions. Under the same conditions, there was no significant inhibition in regenerating liver and there was an increased uptake of [³H] amino acids in the livers of normal and tumor bearing rats.     

Repair of membrane hyperpermeability in L. plantarum during partial reversal of lipid deficiency

Previous studies suggest that the reduced amino acid accumulation capacity of pantothenate-deficient $\text{L. plantarum}$ is caused by a lipid deficiency which results in membrane hyperpermeability. The accumulation defect can be reversed by supplying such cells with saturated or unsaturated fatty acids which are incorporated into the major lipid constituents. Simultaneous measurement of ³H-amino acid uptake and ¹⁴C-fatty acid incorporation revealed that some unsaturated fatty acids promote an 80% reversal of the amino acid accumulation deficit when the cells have taken up only enough fatty acid to replace 12 to 20% of the lipid deficit. Apparently, only a small fraction of the absent lipid plays a decisive role in membrane permeability.     

Identification and characterization of a new methylated amino acid in ribosomal protein L33 of Escherichiacoli

Methylated amino acids from ribosomal protein L33 of various $\text{Escherichia}\text{coli}$ strains (Q13, B and MRE600) were analyzed. It was found that while protein L33 from $\text{E.}\text{coli}$ Q13 contains two methylated neutral amino acids (peaks I and II), only one methylated neutral amino acid (peak I) was found in protein L33 derived from both $\text{E.}\text{coli}$ strains B and MRE600. The methylated amino acid present in peak I was identified as N-monomethylalanine by ion-exchange column chromatography, high-voltage paper electrophoresis and descending paper chromatography using different solvent systems. This marks the first time that N-monomethylalanine was found in any ribosomal protein.     

Dihydroxyacid dehydratase: the site of hyperbaric oxygen poisoning in branch-chain amino acid biosynthesis.

Exposure of $\text{Escherichia}$$\text{coli}$ to hyperbaric oxygen results in rapid inactivation of dihydroxyacid dehydratase but not of other enzymes required for branched-chain amino acid biosynthesis. Unless branched-chain amino acids are supplied, protein synthesis and growth stops abruptly. The sensitivity of dihydroxyacid dehydratase thus accounts for the observed protective role of branched-chain amino acids which cannot be adequately synthesized during exposure to hyperoxia.     

Effects of glucagon and insulin on liver lysosomes

Recent experimental evidence has been obtained, principally in the laboratory of Glenn Mortimore, that hepatic lysosomes can act as a pool of amino acids during fasting. This pool is generated through $\text{autophagy}$, whereby intracellular proteins are somehow captured by the lysosomes and then rapidly hydrolyzed to free amino acids by the lysosomal proteinases. Two important metabolic fates of these lysosomal digestive products can be: 1) conversion of the glucogenic amino acids into glucose, and 2) conversion of trimethyl-lysine into carnitine. The latter metabolite is required to transfer fatty acids to the mitochondrial site of β-oxidation. Most interesting is the observation that glucagon appears to induce lysosomal autophagy and the resulting degradation of intracellular proteins by decreasing the size of amino acid pools in the perfused liver. This effect of the hormone may be directed at the single amino acid glutamine, since adding it alone to the perfusate can prevent the increase in autophagy caused by glucagon. Insulin also rapidly inactivates hepatic autophagy and its ensuing proteolysis. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the rate of los of autophagic vocuoles from the insulin-treated liver (or animal) is approximately 8 min. Thus, glucagon and insulin actively control intracellular protein catabolism that takes place within hepatic lysosomes, and this regulation by the two hormones may be one of their major molecular effects on gluconegenesis in the liver.     

Presence of glycerol and fatty acids in the C-terminal end of a variant surface glycoprotein from Trypanosoma equiperdum

The composition of the C-terminal end of a variant surface glycoprotein from $\text{Trypanosoma equiperdum}$ (BoTat-1 VSG) has been examined. It has been reported for two $\text{Trypanosoma brucei}$ VSGs (Holder, A.A., Biochem. J. (1983), $\text{209}$, 261–262) that ethanolamine was involved in binding the C-terminal amino acid to an oligosaccharide side chain. Tryptic glycopeptides were prepared from BoTat-1 VSG and analyzed. One of them was found to contain ethanolamine and consequently was assumed to be C-terminal. It was shown that the glycopeptide also included phosphate, glycerol and fatty acids. The fatty acid composition was representative of that of glycerolipids. All the results suggest that the end of the molecule is a core of phosphatidylethanolamine.     

The amino acid sequence of the Azotobacter vinelandii flavodoxin.

The amino acid sequence of a group II flavodoxin, the $\text{Azotobacter vinelandii}$ flavodoxin has been determined. The FMN-redox protein was shown to exist as a single polypeptide chain and to contain 179 amino acids. Despite the rather low amino acid sequence homology with the other flavodoxins sequenced, it is concluded that sequences of the group I and group II flavodoxins are homologous. The major differences between the group I and group II flavodoxins appears to be a lengthening in the C-terminal region in the group II flavodoxins.     

Relationship of metabolite inhibition of growth to flow-of-carbon patterns in nature

Various genetic diseases arise from biochemical imbalances that are relatively subtle in the sense that the original mutations are not lethal, that the organism is most vulnerable to damage during certain phases of rapid development, and that in well-managed cases it may be possible to avoid damaging effects through the use of appropriate nutritional manipulations. Analogous imbalances occur in lower organisms. Data obtained with $\text{Pseudomonas}$$\text{putida}$ illustrate that susceptibility to metabolic imbalance is conditionally dependent upon the nutritional regimen.Stereoisomers of leucine, isoleucine and valine, except for L-allo-isoleucine, are metabolized as sole sources of carbon and energy by $\text{P.}$$\text{putida}$. Although the cell yields calculated following utilization of $\text{D}$-leucine and $\text{L}$-leucine were similar, the rate of growth on D-leucine was seven-fold faster than on $\text{L}$-leucine. Slower growth on the $\text{L}$-isomer is not explained as 2-ketoisocaproate limitation since 2-ketoisocaproate production from $\text{L}$-leucine appears to occur more readily than from $\text{D}$-leucine. Spontaneous mutants were obtained which grew 2–10 times more rapidly than wild type on $\text{L}$-leucine, $\text{L}$-isoleucine, or $\text{L}$-valine. It is concluded that the true growth potential (rate) of wild type on any of the branched-chain amino acids is masked by a partial, sustained inhibitory effect produced by the corresponding keto acids or their derivative metabolites. Inhibition of growth rate was only found during utilization of branched-chain amino acids as the sole source of carbon and energy, indicating that the metabolite vulnerability is unique to particular flow-of-carbon patterns during growth. The partial and sustained depression of growth rate by branched-chain amino acids in the absence of other carbon sources cannot be attributed to mis-regulation events localized within the biosynthetic pathway. It is concluded that the catabolism of branched-chain amino acids produces a generalized state of metabolic imbalance owing to the existence of abnormally high levels of degradative metabolites such as keto acids of Coenzyme-A derivatives. Such compounds could (1) interfere with keto acid (e.g. pyruvate) metabolism, (ii) cause feed-forward inhibition of rate-limiting steps in the pathways of branched-chain amino acid catabolism, (iii) perturb fatty acid composition or disrupt the biochemical integrity of membrane material, or (iv) react with substrate-ambiguous enzymes, either slowing essential biochemical reactions to rates that are growth-limiting or producing erroneous products having antimetabolite properties.These effects of branched-chain amino acids in $\text{P.}$$\text{putida}$ may be quite relevant to the molecular events that characterize maple syrup urine disease in man. Metabolite inhibition is probably more common in nature than is generally appreciated, and an appreciation of the molecular basis for anomalous inhibitions of growth in prokaryotic systems should help supply insight into various molecular diseases in man, many of them yet to be described.     

Transport of arginine by an in vitro system

Cell surface glycoproteins of $\text{Neurospora crassa}$ conidia have been shown to bind amino acids and to be genetically associated with the previously defined amino acid transport systems of that organism. L-arginine does not readily permeate a film of $\text{Neurospora}$ conidial lipids. Addition of glycoprotein extracts from $\text{Neurospora}$ to the lipid film enhances permeation of arginine at an initial rate 1000 times the rate of permeation through lipid alone. The initial rate of passage exceeds the rate of unhindered passage (no lipid film) through the same cross sectional area by 10 fold.     

Evidence for a change in catalytic properties of glyceraldehyde 3-phosphate dehydrogenase monomers upon their association in a tetramer

The complete amino acid sequence of human spleen apoferritin has been determined. It consists of 174 amino acids, corresponding to M_r20017. The sequence is very similar to that of horse spleen apoferritin (14% difference between the two sequences). Some peptides were isolated and sequenced which could not be placed in the sequence but which are homologous with part of the principal sequence. Automatic sequence determination of a large peptide resulting from acid cleavage allows us to establish the presence of two homologous sequences (in the ratio $\text{80}\text{20}$).     

Carboxylation, the completion step in prothrombin biosynthesis

It has been found that [¹⁴C]CO₂ is incorporated into prothrombin $\text{in vivo}$ in two hours. The amount of incorporation is increased 3 to 4 fold by the administration of vitamin K₁ to the warfarin-treated vitamin K-deficient rat, over incorporation in the “normal” rat. The radioactivity is found in one acidic peptide following trypsin digestion and following pronase and aminopeptidase digestion is found in one acidic amino acid. The [¹⁴C] is lost on heating of this amino acid at pH 2, leaving unlabeled glutamic acid. It appears that the vitamin K-dependent step in the “completion” of prothrombin is carboxylation of a glutamyl residue of the preformed protein molecule.     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Mechanism of phospholipid biosynthesis in Escherichia coli: Acyl-CoA synthetase is not required for the incorporation of intracellular free fatty acids into phospholipid

Previous results have shown that acyl-CoA synthetase is required both for the incorporation of exogenous fatty acids into the phospholipids of $\text{E. coli}$ and for the transport of fatty acids into the cell. We have demonstrated that acyl-CoA synthetase is not required for the incorporation of intracellular free fatty acids into phospholipid. This finding indicates that the role of this enzyme in the incorporation of exogenously supplied fatty acids is primarily at the level of fatty acid transport.     

The influence of mitochondria on ribosomes. Cycloheximide resistance of ribosomes from petite mutants of saccharomyces cerevisiae

The influence of cycloheximide on amino acid incorporation into protein of standard strain ? + and cytoplasmic mutant ? ? of $\text{S.}\text{cerevisiae}$ was determined $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$. $\text{In}\text{vivo}$ cycloheximide at the concentration which inhibits protein synthesis in ? + strain by over 60% has litle or no effect in mutant ? ? strain. $\text{In}\text{vitro}$ cycloheximide in the range of 0.05 to 0.3 ug/ml of incubation medium inhibits polyphenylalanine synthesis in ? + strain by 50% and in ? ? strain by less than 10%. Similar resistance to this antibiotic are shown in standard strain grown in anaerobic conditions. It has been found that the resistance to cycloheximide is associated with changes in cytoplasmic ribosomes and may depend on the integrity of mitochondrial system.     

Biochimica et Biophysica Acta: Vol. 307 (1973)

The present study evaluates the unsaturated fatty acid requirement in Escherichia coli. A derivative of a double mutant defective both in unsaturated fatty acid biosynthesis and in fatty acid degradation has been selected which grows equally well on anteisopentadecanoate ($\text{12-Me-14:0}$) or $\text{cis-Δ}{}^9\text{-}\text{octadecenoate}$ ($\text{cis-δ}{}^9\text{-18:1}$). When this strain is grown for many generations on $\text{12-Me-14:0}$, there is extensive incorporation of this analogue into the membrane phospholipid and essentially no detectable unsaturated fatty acids residues in any lipid-containing structures of the cell envelope. Secondly, as the maximal growth temperature of E. coli is approached, the minimum content of unsaturated fatty acid required by this strain for growth decreases to a few percent and is associated with the appearance of substantial amounts of $\text{12:0}$ (8%) and $\text{14:0}$ (50%) in the phospholipid. These experiments demonstrate that the cis unsaturated fatty acids of E. coli phospholipids can be replaced by residues which possess no special electronic configuration. Hence, the unsaturated fatty acids do not participate in specific interactions with other membrane components but serve a general role of controlling the packing of paraffin chains in the membrane bilayer.