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.     

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.     

Analysis of the primary structure of collagen for the origins of molecular packing

The amino acid sequence in the triplet region of the α1 chain of collagen was analyzed for complementary relationships that would explain the stagger of multiples of 670 Å between the rod-like molecules in the fibril. The analysis was done by moving the sequence of 1011 amino acids past itself and scoring for complementarity between opposing amino acids allowing a range of ±2 to 3 residues. It was found that interactions between amino acids of opposite charge and between large hydrophobic amino acids in the overlapping region between two chains are maximal when the chains are staggered by 0D, 1D, 2D, 3D and 4D, where D = 234 ± 1 residues. The residue repeat derived from this value is 2.86 ± 0.02 Å. The existence of a D separation between interacting residues was shown to be reflected in the actual distribution of large hydrophobic amino acids. Surprisingly, the distribution approximates the pattern ($\text{2D}\text{11}$)₅($\text{D}\text{11}$) repeated over 4.4D intervals. The regularity may arise from structural constraints imposed by super-coiling. The distribution of charged residues is less regular and does not show a well-defined periodicity. However, positively-charged residues tend to be near negatively-charged residues, allowing intramolecular charge neutralization as well as strong intermolecular charge interactions at 0D.     

Transfer of methionyl residues by leucyl,phenylalanyl-tRNA-protein transferase

Leucyl, phenylalanyl-tRNA-protein transferase also catalyzes transfer of methionyl residues as indicated by (i) copurification over a 1000-fold range of transfer activities for all three amino acids and (ii) loss of methionyl transfer activity in a mutant of $\text{E.}\text{coli}$ lacking the transferase and reappearance of this activity in a transferase revertant. The purified enzyme was found to use Met-tRNA_m^Met in preference to Met-tRNA_f^Met as donor substrate. Peptides containing a basic amino acid at the NH₂-terminus functioned as acceptors for the transfer of methionyl residues.     

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.     

Studies on the purification and properties of teleost prolactin

A protein fraction containing prolactin activity from the pituitary tissue of a teleost fish, $\text{Tilapia mossambica}$, has been purified by a combination of ion-exchange and exclusion chromatographic procedures. The purified $\text{Tilapia}$ prolactin was characterized by disc gel electrophoresis, amino-terminal group identification, and amino acid analysis. Its amino acid composition was found to be similar to ovine prolactin. The purified fish prolactin was found to be 40–50 times more potent than ovine prolactin in the $\text{Tilapia}$ sodium-retaining bioassay. However, it was found to be devoid of $\text{Gillichthys}$ yellow pigment-dispersing activity which was previously thought to be a property of teleost prolactin.     

Structure of the "keratosulfate-like" material in liver from a patient with G M1 -gangliosidosis ( -D-galactosidase deficiency)

Large amounts of a glycopeptide containing galactose, $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and threonine in the ratio 4:3:1:1, together with smaller amounts of mannose, fucose, sialic acid, sulfate, serine, and other amino acids were isolated from the liver of a patient with G_M1-gangliosidosis. Treatment with mild alkali and sodium borohydride indicated an $\text{O}$-glycosidic linkage between $\text{N}$-acetylgalactosamine and threonine. All the hexosamine residues were resistant to sodium metaperiodate whereas 2 out of 4 D-galactose residues were destroyed. Further studies indicated that one of the galactose residues was 1→3 linked to $\text{N}$-acetylgalactosamine (as in G_M1) and the other 1→4 linked to $\text{N}$-acetylglucosamine as found in skeletal keratosulfate.     

EPR study of the effect of formate on cytochrome C oxidase

The addition of formate to oxidized cytochrome $\text{c}$ oxidase (ferrocytochrome $\text{c}$: oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high spin heme signal at g = 6 and a splitting of g = 3 signal to g = 2.98 and 3.07. When formate-cytochrome $\text{c}$ oxidase is reduced, the g = 2.98 signal decreases significantly. The spectrophotometric studies showed that formate is a specific ligand to cytochrome $\text{a}$₃. Data suggest that binding of formate to oxidized cytochrome $\text{c}$ oxidase produces a ligand-$\text{a}$₃ interaction leading to the splitting of g = 3 signal hitherto considered as due to cytochrome $\text{a}$. Thus both cytochrome $\text{a}$ and $\text{a}$₃ contribute to the resonance of g = 3 signal of cytochrome $\text{c}$ oxidase.     

Energization of sulfate transport in yeast

Sulfate uptake by Saccharomyces cerevisiae is stimulated about 12-fold by preincubation of cells with 1% d-glucose or 1% ethanol. The $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ remains unchanged (0.34–0.38 mM), the $\text{J}{}_{\mathrm{<mtext>mar</mtext>}}$ increase from 18–20 to 195–230 and 170–185 nmol/min per g dry wt., respectively, after glucose and ethanol preincubation. The stimulation involves protein synthesis (it is suppressed by cycloheximide), has a half-time of 18 min and requires mitochondrial respiration (no or low effect in respiration-deficient mutants and those lacking ADP-ATP transport in mitochondria, as well as after anaerobic preincubation of the wild-type strain, and in low-phosphate cells). The presence of NH₄⁺ and some amino acids (e.g., leucine, aspartate, cysteine and methionine) depressed the stimulation while that of cationic amino acids (typically arginine and lysine) and of K⁺ increased it by 50–80%. The stimulated (i.e., newly synthesized) transport system was degraded with a half-life of about 10 min.     

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.     

D2O-alanine exchange reactions catalyzed by alanine racemase and glutamic pyruvic transminase

NMR studies in D₂O (>90%) reveal that Alanine Racemase (5.1.1.1.) from $\text{B. subtilis}$ catalyzes the exchange of the α hydrogen of $\text{D}$- and $\text{L}$-alanine with D₂O. Glutamic Pyruvic Transaminase (2.6.1.2.) and Glutamic Oxaloacetic Transaminase (2.6.1.1.) catalyze the exchange of α and β hydrogens of $\text{L}$-alanine. The rates of exchange of α and β hydrogens appear to be of the same order of magnitude. The transaminase catalyzed exchange is enhanced by catalytic amounts of pyruvate. The side chain of $\text{L}$-alanine is held more rigidly at the active site of transaminase so that the planar conjugated system can be extended to include the α and β carbons. A generalized mechanism is proposed for the action of pyridoxal phosphate dependent transaminases which extends Braunstein and Snell mechanism to include the structures which contribute to the labilization of β hydrogens of amino acids by the transaminases that have been studied.     

The anal sac secretion of the red fox (Vulpes vulpes); its chemistry and microbiology. A comparison with the anal sac secretion of the lion (Panthera leo)

Phenylacetic, 3-phenylpropionic, $\text{p}$-hydroxyphenylacetic and 3 ($\text{p}$-hydroxyphenyl) propionic acids together with the series of C₂ to C₆ saturated fatty acids previously reported in the anal sac secretion of the red fox ($\text{Vulpes vulpes}$) are identified as constituents of the anal sac secretion of the lion (Panthera leo). All these compounds are also observed in the anal sac secretion of the red fox using gas chromatography. The aerobic microflora of red fox and domestic dog ($\text{Canis familiaris}$) anal sac secretion samples invariably consisted predominantly of $\text{Streptococcus faecium}$ and $\text{Streptococcus faecalis}$. The hypothesis that the secretion volatiles so far identified may be microbiologically produced is examined.     

Stoichiometry versus coupling ration in the cotransport of Na and different neutral amino acids

The stoichiometry of Na coupling to amino acid movement across the brush border membrane of the rabbit distal ileum has been determined under initial rate conditions.The coupling ratio, defined as the amino acid-dependent Na influx/the Na-dependent amino acid influx, was equal to unity for alanine, measured over a 10-fold range of Na and alanine concentrations. Coupling ratio values determined under a single set of conditions for a number of amino acids varied from 1 for serine to 4.6 for methionine. Reducing the methionine concentration from 12.5 to 1.5 mM caused the coupling ratio value to fall from 4.6 to 1.2.These results are explained by assuming a fixed stoichiometry of 1 : 1 under all conditions, with initial binding of the amino acid (A) to the Na-dependent carrier (E) but with some amino acids being able to cross on the Na-dependent carrier in the absence of Na.The variation in coupling ratio values can be used to calculate K_A, the apparent dissociation constant of amino acid from the Na-dependent carrier in the absence of Na, and the ratio $\text{k}{}_1\text{k}{}_2$, where k₁ and k₂ are first-order rate constants for translocation of the complexes EA and EANa, respectively. This method of processing results has been defined as delta analysis. The value of K_A for methionine is 3.6 ± 1.1 mM and the $\text{k}{}_1\text{k}{}_2$ ratio is 1.01 ± 0.07. The constant coupling ratio value of 1 for alanine indicates that the value for K_A is extremely high or that the k₁ value is extremely low.     

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 $\text{80}\text{20}$).     

Chiroptical properties of fluorescamine condensation compounds with dipeptides in situ

The free amino group of a dipeptide reacts efficiently with fluores-camine (FLURAM^®) to form pyrrolinone-type chromophores with long wavelength absorption maxima in the 380 nm region. A simple test tube procedure is described which allows $\text{in situ}$ determination of the absolute configuration of the NH₂-terminal amino acids of the dipeptides based on the chiroptical properties of their chromophoric derivatives.     

Identification of cis-5-methylproline in hydrolysates of actinomycin Z 5

Actinomycins of the Z series, synthesized by $\text{Streptomyces}$$\text{fradiae}$, contain the unusual amino acid, N-methylalanine, but no proline. Hydrolysates of actinomycin Z₅ were investigated using paper, gas and ion-exchange chromatographic procedures. Identification of an unknown amino acid in actinomycin Z₅ as 5-methylproline was confirmed by mass spectrometry. Configuration of the imino acid was defined as $\text{cis}$.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

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.     

Secondary structure of peptides: 15. 13C n.m.r. CP/MAS study of solid elastin and proline-containing copolyesters

In order to study the chemical shifts and the cis—trans isomerism of prolyl units neighbouring glycine or other amino acids, 75.4 MH_z¹³C nuclear magnetic resonance (n.m.r.) cross-polarization/magic angel spinning (CP/MAS) spectra of the following solid oligopeptides and sequence polypeptides were measured: Z-Gly-Pro-OH,Z-Gly-Pro-Gly-Gly-OEt,Z-Gly-Pro-Ala-Ala-OMe,(Gly-Pro-Gly)_n,(Gly-Pro-Ala)_n,(β-Ala-Pro)_n and $\text{(δ-Ava-Pro)}{}_{\mathrm{<mtext>n</mtext>}}\text{(δ-Ava=δ-}\text{aminovaleric acid}$). Whereas all these oligo- and polypeptided contain exclusively trans X-Pro bonds, both cis and trans peptide bonds were found in a polypeptide prepared by copolymerization of glycine- and $\text{proline-}\text{N}\text{-carboxyanhydrides}$ in pyridine. On the basis of these model compounds, the ¹³C n.m.r. CP/MAS spectra of solid elastin allows the following conclusions. Almost all X-Pro bonds assume the trans conformation, most alanine and leucine units form α-helical chain segments, whereas only a small fraction of β-sheet structure is present. A 30.3 MHz ¹⁵N n.m.r. CP/MAS spectrum of solid elastin confirms that ～25% of all amino acids assume the α-helical structure. A model of elastin is discussed consisting of an amorphous phase, α-helical chain segments and helical segments of still unknown pitch.     

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells

F₁-ATPase was isolated from yeast $\text{S.}\text{cerevisiae}$. The constituent subunits 1 and 2 were purified by gel permeation chromatography, and their amino acid compositions determined. Both subunits have a similar composition except for $\text{1}\text{2}$ cystine, methionine, leucine, histidine, and tryptophan. When F₁ is treated for three hours with 5′-p-[³H]fluorosulfonylbenzoyl adenosine in dimethylsulfoxide, 90% of the activity is lost. Disc gel electrophoresis of the modified complex showed that over 90% of the label was associated with subunit 2. A labelled peptide from a $\text{S.}\text{aureus}$ digest of subunit 2 was isolated and sequenced. It had the following amino acid sequence: His-Try¹-Asp-Val-Ala-Ser-Lys-Val-Gln-Glu, whereby Tyr¹ is the modified amino acid residue. This sequence shows homology to other sequences obtained from maize, beef heart, and $\text{E.}\text{coli}$ F₁-ATPases.