The separation of phosphocreatine from creatine, and pH determination in frog muscle by natural abundance 13C-NMR

Phosphocreatine can be separated from creatine in superfused frog muscle by natural abundance 13C-NMR, based on the difference in resonance frequency of their guanidino carbons. After taking into account the longitudinal relaxation times and nuclear Overhauser enhancement factors, the integrated peak areas of the guanidino carbons could be used for determination of the phosphocreatine-to-creatine ratio in the muscle. The pH dependence of the chemical shift of the C-2 carbon in the histidine ring of carnosine was used for estimation of the intracellular pH in the intact muscle.     

Variation in proton affinity of the guanidino group between free and blocked arginine

Summary. In this paper, the analog of arginine residues in peptides was synthesized and characterized by ESI-MS/MS (electrospray ionization with tandem mass spectrometry), ³¹P NMR, ¹H NMR, IR and high-resolution mass spectrometry. When the Todd reaction activity of the guanidino group in free arginine and the arginine peptide analog were compared, it was found that the proton affinity of the guanidino group was decreased when both the N- and the C-terminal were blocked. As a result, the guanidino group of arginine residues in peptides could be phosphorylated under the Todd reaction condition, but not the free arginine. This result was further proved by the theoretical calculation of their proton affinity.     

Target protein for eucaryotic arginine-specific ADP-ribosyltransferase

Among ADP-ribosyltransferases reported in eucaryotes, arginine-specific transferases from turkey erythrocytes, chicken heterophils and rabbit skeletal muscle have been purified and extensively studied. They were reported to modify a number of proteinsin vitro. ADP-ribosylation of Ha-ras-p21 and transducin by the turkey erythrocyte transferase inhibits their GTPase and GTP-binding activities. Chicken heterophil enzyme modifies several substrate proteins for protein kinases and decreases the phosphate-acceptor activity. Rabbit skeletal muscle Ca²⁺-ATPase is inhibited by ADP-ribosylation catalyzed by the muscle transferase. Three transferases all ADP-ribosylate small molecular weight guanidino compounds such as arginine, arginine methylester and agmatine and poly-L-arginine and nuclear histones. However, the observation that muscle transferase did not ADP-ribosylate casein or actin, both of which can be modified by the heterophil transferase under the same conditions indicates that substrate specificity of these two enzymes are different. Substrate-dependent effects were observed with polyions of nucleotides such that polyanions stimulate the ADP-ribosylation of possible target protein, p33 by chicken heterophil transferase but has no effect on the modification of casein by the same enzyme.     

Mitochondrial intermembrane inclusion bodies: The common denominator between human mitochondrial myopathies and creatine depletion due to impairment of cellular energetics

Mitochondrial inclusion bodies are often described in skeletal muscle of patients suffering diseases termed mitochondrial myopathies. A major component of these structures was discovered as being creatine kinase. Similar creatine kinase enriched inclusion bodies in the mitochondria of creatine depleted adult rat cardiomyocytes have been demonstrated. Structurally similar inclusion bodies are observed in mitochondria of ischemic and creatine depleted rat skeletal muscle. This paper describes the various methods for inducing mitochondrial inclusion bodies in rodent skeletal muscle, and compares their effects on muscle metabolism to the metabolic defects of mitochondrial myopathy muscle. We fed rats with a creatine analogue guanidino propionic acid and checked their soled for mitochondrial inclusion bodies, with the electron microscope. The activity of creatine kinase was analysed by measuring creatine stimulated oxidative phosphorylation in soleus skinned fibres using an oxygen electrode . The guanidino propionic acid-rat soleus mitochondria displayed no creatine stimulation, whereas control soleus did, even though the GPA soled had a five fold increase in creatine kinase protein per mitochondrial protein. The significance of these results in light of their relevance to human mitochondrial myopathies and the importance of altered muscle metabolism in the formation of these crystalline structures are discussed. (Mol Cell Biochem 174: 283–289, 1997)     

Measurement of beta-guanidinopropionate and phosphorylated beta-guanidinopropionate in tissues.

The Sakaguchi color reaction for monosubstituted guanidino compounds was applied to the measurement of β-guanidinopropionate and phosphorylated β-guanidinopropionate. The phosphorylated derivative was measured as an increase in β-guanidinopropionate following incubation with 0.1n HCl in a boiling-water bath for 10 min. After feeding rats 1% of β-guanidinopropionic acid in their diet for 69 days, skeletal muscle, heart, liver, kidney, and spleen contained 5–10 μmoles of a monosubstituted guanidino compound per gram wet weight of tissue. No β-guanidinopropionate was detected in brain or testes. Phosphorylated β-guanidinopropionate was found only in skeletal muscle (27 μmoles/g) and in heart (7 μmoles/g). Creatine hydrate (2%) added to the diet containing β-guanidinopropionic acid inhibited the accumulation of phosphorylated β-guanidinopropionate in the heart and partially inhibited its accumulation in skeletal muscle.     

Purification and Characterization of a Cardioexcitatory Neuropeptide from the Central Ganglia of a Bivalve Mollusc

Abstract

We have purified a cardioexcitatory substance, previously designated peak C, from ganglia of the Sunray Venus clam, Macrocallista nimbosa. Low concentrations (10^?9 -10^?8M) of this substance not only excite the isolated clam heart, but also produce tonic contractions of the isolated radula protractor muscle of the whelk, Busycon contrarium. These two muscle preparations have therefore been used as a parallel bioassay for peak C. Peak C is inactivated by proteolytic enzymes, has an isoelectric point greater than pH 10 and has an ultraviolet absorption spectrum similar to that of phenylalanine. On thin layer chromatograms, peak C separates into two components; one of these is probably a partially oxidized form produced during purification. Both components react with ninhydrin and with the Sakaguchi reagent for guanidino groups. The amino acid composition of peak C is Phe_2.00 Met_0.81 Arg_1.12. N-terminal analysis, one round of Edman-dansyl degradation, and tryptic digestion are consistent with the identification of Macrocallista peak C as a tetrapeptide amide: Phe-Met-Arg-Phe-NH₂ (FMRFamide).     

N- and [C]NMR determination of utilization of glycine for synthesis of storage protein in the presence of glutamine in developing cotyledons of soybean

Solid-state ¹⁵N- and [¹³C] NMR have been used to measure quantitatively the utilization of glycine in the presence of glutamine for the synthesis of storage protein in immature cotyledons of soybean (Glycine max L. cv. Elf) in culture. The presence of an equal molar amount of glycine in the medium causes a decrease in the use of glutamine-amide nitrogen. Glycine nitrogen is incorporated extensively into peptide bonds (in amounts greater than what would be expected if it appeared solely in glycine residues), but is used sparingly for synthesis of histidine ring residues, guanidino nitrogen residues of arginine, and lysine residues. The modest use of glycine carbon in protein synthesis does not parallel the use of glycine nitrogen.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Magnetic resonance study of the three-dimensional structure of creatine kinase-substrate complexes. Implications for substrate specificity and catalytic mechanism.

The paramagnetic effects of the bound manganese ion and of a covalently attached spin label on proton nuclear spin relaxation rates have been used to calculate distances for a structural model of the MnADP and creatine complexed to creatine kinase from rabbit muscle. The nucleotide and guanidino substrates are so aligned on the enzyme that the transferable phosphoryl group on one substrate is in apposition to the acceptor moiety on the second substrate. The divalent metal ion is most probably liganded to the alpha and beta phosphates of the nucleotide substrate, both in the abortive MnADP-creatine-enzyme complex and in the active MnATP-creatine-enzyme complex. The metal ion-formate distance approximately 5 A in the Mn(II)ADP-formate-creatine-enzyme complex and less than 5 A in the Co(II)ADP-formate-creatine-enzyme complex is consistent with the suggestion that the monovalent anion is binding at the site normally occupied by the transferable phosphoryl group, thus producing a complex which mimics the transition state. Although only an upper limit of the distance from Mn(II) to the guanidino substrate could be determined in the presence of formate, it could be concluded that the disposition of the guanidino substrate changes upon addition of formate, since the relative distances of the methyl and methylene group are inverted. The effect of formate and nitrate on increasing the residence time of creatine in the MnADP-creatine-enzyme complex as determined by NMR provides evidence that the complexes observed by NMR are identical with those involved in the catalytic mechanism, since a parallel effect of formate and nitrate is observed in the kinetics of the enzymatic reaction, where the dissociation constant of creatine from the abortive quaternary complex decreases in the presence of the anions as had been determined from their inhibition of the forward reaction (Milner-White, E.J., and Watts, D.C. (1971) Biochem. J. 122, 727-740). Although the guanidino substrate is not directly liganded to the divalent metal ion, the electron paramagnetic resonance spectrum of manganese in the transition state analog complexes, i.e. nitrate-ADP-guanidino substrate-enzyme, is strongly dependent on catalytic activity of the guanidino substrate. The structural differences observed by EPR among transition state analog complexes with various guanidino substrates were not reflected in distances from Mn(II) to the guanidino substrate, which were 10% and 0.3% as active as creatine. Within the experimental error of 1 A, the distances were the same. The enzyme or the enzyme-substrate complexes may be considered to exist in a number of structurally distinct conformations in equilibrium based on the EPR spectra and on the anomalous temperature-dependence of the relaxation rates of the formate proton of the transition state analog complexes...     

Effect of ammonium acetate-induced hyperammonemia on metabolism of guanidino compounds.

Guanidino compounds are synthesized from arginine in various tissues such as liver, kidney, brain, and skeletal muscle. Guanidino compounds such as arginine and creatine play an important role in nitrogen metabolism, whereas other guanidino compounds such as guanidinosuccinic acid and alpha-N-acetylarginine are known toxins. In order to understand the changes in the metabolism of guanidino compounds during ammonia toxicity, we investigated the effect of hyperammonemia induced by an ammonium acetate injection on the levels of guanidino compounds in plasma, liver, kidney, and brain of rats. Control animals were injected with an equal volume of saline. Blood and tissues were removed 1 h following ammonium acetate or saline injection and guanidino compounds were analyzed by high-performance liquid chromatography. Plasma and kidney levels of guanidinosuccinic acid were significantly elevated in rats challenged with ammonium acetate. Brain alpha-N-acetylarginine levels were also significantly higher in rats injected with ammonium acetate as compared to those in controls. Our results suggest that guanidinosuccinic acid and alpha-N-acetylarginine may play an important role in hyperammonemia.     

Sequence homology and structure predictions of the creatine kinase isoenzymes

Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guamidino kinases. A CK framework is defined, consisting of the most conserved sequence blocks, and diagnostic boxes are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis.Abbreviations GuaK guanidino kinase - CK creatine kinase - B-and M-CK brain and muscle cytosolic CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - ArgK arginine kinase - Cr creatine - PCr phosphorylcreatine - PArg phosphorylarginine     

Possible incorporation of petroleum-based carbons in biochemicals produced by bioprocess

The biomass carbon ratios of biochemicals related to biomass have been reviewed. Commercial products from biomass were explained. The biomass carbon ratios of biochemical compounds were measured by accelerator mass spectrometry (AMS) based on the ¹⁴C concentration of carbons in the compounds. This measuring method uses the mechanism that biomass carbons include a very low level of ¹⁴C and petroleum carbons do not include ¹⁴C similar to the carbon dating measuring method. It was confirmed that there were some biochemicals synthesized from petroleum-based carbons. This AMS method has a high accuracy with a small standard deviation and can be applied to plastic products.     

Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-¹³C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-¹³C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA–CO coupling in the backbone should improve the resolution of NCACX spectra.     

Methylthioadenosine metabolism in malignant murine cells

An ether-extractable product formed from 5′-methylthioadenosine by extracts of malignant murine lymphocytic cells is shown to be 2-keto-4-methylthiobutyric acid. When 5′-methylthio [U-¹⁴C]adenosine was used as substrate, the product was labelled, confirming earlier reports that carbons of the keto acid are derived from carbons of the ribose. When hydroxylamine was added to the reaction mixture, the ketomethylthiobutyric acid was trapped as the oxime. When glutamine was added, the main product was methionine.     

Neurotensin Binding to Dopamine

Rotating disk electrode voltammetry at glassy carbon electrodes and ultraviolet/visible spectroscopy were used to demonstrate that dopamine binds to neurotensin with a dissociation constant of 7.5 × 10^-8. By measuring the binding constants of various neurotensin analogs, it was found that the -Arg⁸-Arg⁹-portion of the neurotensin sequence is critical for binding dopamine. Neurotensin also formed a complex with 4-ethylcatechol, 4-methylcatechol, 3-methoxytyramine, and norepinephrine. Although changes in the side chain did not alter the binding constant, methoxylation of the catechol moiety significantly increased the dissociation constant. These data along with additional studies of dopamine interactions with arginine derivatives suggest that the guanidino groups of arginine and the catechol hydroxyls of dopamine are responsible for mediating the observed binding. It is hypothesized that the capacity of neurotensin to bind directly to dopamine may be partly responsible for its previously observed antagonism of dopamine-induced locomotor activity.     

Insulin as a probe of mitochondrial metabolism in situ

Our previous studies of insulin action have led us to the finding that insulin acts specifically on the mitochondrial Krebs cycle to stimulate, by 30%, the oxidation of carbons 2 and 3 of pyruvate to CO₂. Insulin also stimulates the oxidation of both carbons of acetate. These carbons can be converted to CO₂ only after passing through all of the reactions of the Krebs cycle more than once. Carboxyl groups, such as number 1 of pyruvate, are oxidized to CO₂ without any effect of insulin, and can be converted to CO₂ by extramitochondrial enzyme. We conclude that insulin must act on the complete intramitochondrial cycle and not on the four enzymes of the Krebs cycle which are present in the cytoplasm. The path taken by those carbons affected by insulin is traced through the complete Krebs cycle, and the necessity for this effect to be mitochondrial has been verified by demonstration of the same specific effect of insulin on the oxidation of the 2 and 3 carbons of succinate. The use of this phenomenon is proposed for the study not only of human diabetes, but of all mitochondrial disorders, by using ¹⁴C specifically labeled tracers in culture or biopsy material, or ¹³C labeled tracer material in vivo. (Mol Cell Biochem 174: 91–96, 1997)     

13C NMR studies on fluorescent probes: 13C chemical shifts and longitudinal relaxation times of n-hydroxy-fatty (n=2,6,9,12) acids and n-(9-anthroyloxy)-stearic (n=6,12) acids

¹³C NMR has been used to confirm the structure of two fluorescent probes, n-(9-anthroyloxy)-stearic acids (n=6,12), and the series of n-hydroxy-fatty acids (n=2,6,9,12) from which the set of fluorescent fatty acids may be synthesised. ¹³C longitudinal relaxation times and correlation times of the individual carbon atoms in 12-hydroxy- and 6- and 12-(9-anthroyloxy)-stearic acids show differences in motional properties between these derivatives and the parent stearic acid in chloroform(d) solution. The correlation times of the substituted carbons in 6-, 9-, and 12-hydroxy-stearic acids are longer than the corresponding carbons in stearic acid. The change in correlation times at the substituted carbons reflects the increase in motion along the acyl chain. Attachment of the bulky anthracene ring causes greater restriction of motion at the substituted carbon atom but the gradient of motion along the chain is preserved. These results are discussed in terms of the types of motion which lead to fluorescence depolarization when the fluorescent fatty acids are used as fluidity probes in biomembranes.     

Despite its high similarity with monomeric arginine kinase, muscle creatine kinase is only enzymatically active as a dimer

Although having highly similar primary to tertiary structures, the different guanidino kinases exhibit distinct quaternary structures: monomer, dimer or octamer. However, no evidence for communication between subunits has yet been provided, and reasons for these different levels of quaternary complexity that can be observed from invertebrate to mammalian guanidino kinases remain elusive. Muscle creatine kinase is a dimer and disruption of the interface between subunits has been shown to give rise to destabilized monomers with slight residual activity; this low activity could, however, be due to a fraction of protein molecules present as dimer. CK monomer/monomer interface involves electrostatic interactions and increasing salt concentrations unfold and inactivate this enzyme. NaCl and guanidine hydrochloride show a synergistic unfolding effect and, whatever the respective concentrations of these compounds, inactivation is associated with a dissociation of the dimer. Using an interface mutant (W210Y), protein concentration dependence of the NaCl-induced unfolding profile indicates that the active dimer is in equilibrium with an inactive monomeric state. Although highly similar to muscle CK, horse shoe crab (Limulus polyphemus) arginine kinase (AK) is enzymatically active as a monomer. Indeed, high ionic strengths that can monomerize and inactivate CK, have no effect on AK enzymatic activity or on its structure as judged from intrinsic fluorescence data. Our results indicate that expression of muscle creatine kinase catalytic activity is dependent on its dimeric state which is required for a proper stabilization of the monomers.     

Improved NMR experiments with 13C-isotropic mixing for assignment of aromatic and aliphatic side chains in labeled proteins

Three improved ¹³C-spinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented. These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes. The first application, the H(C^aliC^aro)H-TOCSY, is an HCCH-TOCSY in which all CH_n moieties of a protein are detected in a single experiment, including the aromatic ones. This enables unambiguous assignment of aromatic and aliphatic amino acids in a single, highly sensitive experiment. In the second application, the ¹³C-detected C^all-TOCSY, magnetization transfer comprises all carbons—aliphatic, aromatic as well as the carbonyl carbons—making the complete carbon assignment possible using one spectrum only. Thirdly, the frequently used HC(CCO)NH experiment was redesigned by replacing the long C-carbonyl refocused INEPT transfer step by direct ¹³C–¹³C-TOCSY magnetization transfer from side chain carbons to the backbone carbonyls. The resulting HC(CCO)NH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins. The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDa.     

Molecular conformational changes in the triglyceride and methyl ester of stearic acid as studied by temperature dependent carbon-13 chemical shifts

The temperature dependence of the ¹³C chemical shifts in tristearin and methyl stearate has been investigated in both the melt and solution phases. Intramolecular conformational changes dominate the observed behaviour and there is little evidence for intermolecular interactions even in the melt phase of tristearin. The terminal methyl carbons of methyl stearate and tristearin and the C17, C2, and glyceryl carbons of tristearin exhibit a temperature dependence consistent with there being only two rotamers of significant population. The calculated enthalpy difference between the terminal methyl and C17 rotamers is of the same order of magnitude as would be expected for tt and tg^± rotamers in hydrocarbon chains. For the glyceryl carbons the rotamer energy difference is very large and only one of the rotamers is significantly populated at room temperature. The remaining carbons (C16, C17, C15, C6, C4 and C3) show a general drift to high fields with increasing temperature but the observed temperature dependence requires the existence of more than two rotamers. In the absence of an acceptable mechanism for the chemical shifts of ¹³C nuclei in hydrocarbon chains it is not possible to use this data to investigate conformational changes along the hydrocarbon chain.