The separation of phosphocreatine from creatine,and pH determination in frog muscle by natural abundance13C-NMR

Phosphocreatine can be separated from creatine in superfused frog muscle by natural abundance¹³C-NMR, based on the difference in resonance frequency of their guanidino carbons. After taking into account the longitudinal relavation times and nuclear Overhauser enhancement factors, the integrated oak 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 carboon in the histtdine ring of carnosine was used for estimation of the intracellular pH in the intact muscle.     

Exploring the role of the active site cysteine in human muscle creatine kinase

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.     

Blood drugs and other analytical challenges (Methodological Surveys in Biochemistry,Vol. 7) : Edited by E. Reid,Ellis Horwood (Wiley), Chichester, 1978, X + 355 pp., price £ 19.50, ISBN 0-85312-124-9

A high-performance liquid chromatographic procedure has been developed for the separation and fluorometric detection of guanidino compounds in physiologic fluids. All guanidino compounds were separated on a 17 × 0.46 cm cation-exchange column using a stepwise pH gradient. The chromatographic system was designed to enable the use of the specific reagent 9,10-phenanthrenequinone as a means of monitoring the guanidino compounds of physiologic fluids. This new analytical method is so sensitive that it enables the analysis at the picomole level. Our automatic guanidino-compound analyzer was successfully applied to the quantitative determination of all guanidino compounds in physiologic fluids from normal controls and uremic patients.     

Comparative studies on the structure and aggregative properties of the myosin molecule. III. The in vitro aggregative properties of the lobster myosin molecule.

The solubility of rabbit skeletal and lobster abdominal muscle myosin has been studied in monovalent salt solutions as a function of pH (over the range 4.75 to 8.5) and ionic strength (50-500 mM). Rabbit skeletal muscle myosin was found to precipitate over a narrower pH range than the lobster abdominal muscle myosin but at equivalent pH values and ionic strengths the former exhibited greater solubility. Comparison of the solubility of rabbit myosin, per se with that of light meromyosin and lobster myosin with its equivalent proteolytically produced fragment (fraction B1) showed that both rod fragments were more soluble than their parent molecules. Under conditions of low solubility (low ionic strength and pH) the quantitiy of protein in solution remained essentially constant with increasing total protein, thus suggesting that the aggregation phenomenon is of a phase transition type. Examination of the aggregates by electron microscopy revealed that rabbit myosin formed classical, elongate, spindle-shaped filaments similar to those previously observed by others. In contrast lobster myosin only formed short, dumbbell-shaped filaments 0.2-0.3 mum long. Consideration of the pH ranges over which aggregation occurred suggests that protonation of histidine residues may be involved in rabbit myosin filament formation while for lobster myosin, aggregation may involve protonation of epsilon-amino or guanidino groups. The possible relationship between the distribution of these groups along the rod portion of the myosin molecule and the formation of elongate filaments has been explored.     

Synthesis and characterization of biodegradable cationic poly(propylene fumarate-co-ethylene glycol) copolymer hydrogels modified with agmatine for enhanced cell adhesion

We synthesized positively charged biodegradable hydrogels by cross-linking of agmatine-modified poly(ethylene glycol)-tethered fumarate (Agm-PEGF) and poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) to investigate the effect of the guanidino groups of the agmatine on hydrogel swelling behavior and smooth muscle cell adhesion to the hydrogels. The weight swelling ratio of these hydrogels at pH 7.0 increased from 279 +/- 4 to 306 +/- 7% as the initial Agm-PEGF content increased from 0 to 200 mg/g of P(PF-co-EG), respectively. The diffusional exponents, n, during the initial phase of water uptake were independent of the initial Agm-PEGF content and were determined to be 0.66 +/- 0.08, 0.71 +/- 0.07, and 0.60 +/- 0.05 for respective initial Agm-PEGF contents of 0, 100, and 200 mg/g. The heat of fusion of water present in the hydrogels increased from 214 +/- 11 to 254 +/- 4 J/g as the initial Agm-PEGF content increased from 0 to 200 mg/g. The number of adherent smooth muscle cells increased dose-dependently from 15 +/- 6 to 75 +/- 7% of the initial seeding density as the initial Agm-PEGF content increased from 0 to 200 mg/g. These results suggest that the incorporation of the guanidino groups of agmatine into P(PF-co-EG) hydrogels increases the hydrogel free water content and the total water content of the hydrogels and also enhances cell adhesion to the hydrogels.     

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.     

The purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl-Sepharose 4B

The pH-dependent interaction between the cyclic guanidino analog of biotin, 2-iminobiotin, and avidin has been used in the design of an efficient affinity isolation system for avidin and its fluorescent and iodinated derivatives. Avidin and its derivatives are retained by a column of 2-iminobiotin-6-aminohexyl-Sepharose 4B at pH values between 9 and 11 and are specifically eluted from the column at pH 4. This affinity isolation procedure overcomes the harsh conditions, i.e., 6 m guanidine-HCl, pH 1.5, required to dissociate avidin from an immobilized biotin column.     

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...     

Chemical modification of canavanine with p-nitrophenylglyoxal. Factors influencing the chemistry and reactivity of alpha-dicarbonyl-guanidino reactions

The role of structural features and deprotonation of guanidino derivatives on chemical reactions with p-nitrophenylglyoxal has been investigated. Canavanine, an arginine analog, reacts to form a yellow product, which absorbs maximally at 350 nm (epsilon = 6500) and at 278 nm (epsilon = 14 500). Elemental analysis, fast atom bombardment mass spectral analysis, n.m.r. and i.r. studies suggest that the product is a 5-(p-nitrophenyl)4-oxo-2 imidazoline derivative of canalaline. Kinetic studies show that the second order rate constant for the reaction increases with increasing pH in the range of pH 7-11.0. It is concluded that the pH dependence of the reaction can be explained by general base catalysis and not simply by a deprotonation of the guanidinoxy side chain. The reaction of arginine, polyarginine, and other derivatives differs markedly from that of canavanine. The results suggest that change in the tautomeric equilibria between the imino and amino forms of the guanidino group may partly account for differences in reaction of canavanine and arginine and the reactions of specific arginyl residues in proteins.     

Decolorization of sugar syrups using commercial and sugar beet pulp based activated carbons

Sugar syrup decolorization was studied using two commercial and eight beet pulp based activated carbons. In an attempt to relate decolorizing performances to other characteristics, surface areas, pore volumes, bulk densities and ash contents of the carbons in the powdered form; pH and electrical conductivities of their suspensions and their color adsorption properties from iodine and molasses solution were determined. The color removal capabilities of all carbons were measured at 1/100 (w/w) dosage, and isotherms were determined on better samples. The two commercial activated carbons showed different decolorization efficiencies; which could be related to their physical and chemical properties. The decolorization efficiency of beet pulp carbon prepared at 750 degrees C and activated for 5h using CO2 was much better than the others and close to the better one of the commercial activated carbons used. It is evident that beet pulp is an inexpensive potential precursor for activated carbons for use in sugar refining.     

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)     

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.     

Synthesis of nitric oxide from the two equivalent guanidino nitrogens of L-arginine by Lactobacillus fermentum.

Ten strains of Lactobacillus fermentum that differed in origin converted metmyoglobin to nitrosylmyoglobin [a pentacoordinate nitric oxide (NO) complex of Fe(II) myoglobin] in MRS broth at pH 4.3. Of the 10 strains, L. fermentum IFO 3956 possessed the strongest capacity to convert metmyoglobin to nitrosylmyoglobin. This strain synthesizes NO enzymatically from the two equivalent guanidino nitrogens of L-arginine. To our knowledge, this demonstrates for the first time the production of NO synthesized from the guanidino nitrogens of L-arginine by lactic acid bacteria. IFO 3956 may possess a bacterial NO synthase.     

Application of arylsulphonyl side-chain protected arginines in solid-phase peptide synthesis based on 9-fluorenylmethoxycarbonyl amino protecting strategy.

One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid liability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8- pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have liability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.     

The design of fluorescent probes which bind to the active centre of guanidinobenzoatase. Application to the location of cells possessing this enzyme

Cells possessing a known enzymic activity may be located by fluorescent probes designed to act as competitive inhibitors of this enzyme. We have prepared a series of dansyl N-substituted guanidino derivatives which bind to the active centre of guanidinobenzoatase. 9-Aminoacridine also acts as a competitive inhibitor and behaves similarly to these guanidino derivatives. These fluorescent probes have been used to locate tumour cells possessing this enzyme in thin sections of fixed tissue by employing fluorescent microscopy.     

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     

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.     

Hydrogen-ion titration curve of ovomucoid.

A systematic study of the H+ titration curve of purified ovomucoid was made at three temperatures (15, 25 and 35 degrees C) and three ionic strengths (0.05, 0.15 and 1.0). In all, 49 protons were dissociated reversibly in the pH range, 2.0-12.0. From the analysis of the results up to pH 12.0, the numbers of different dissociable groups per 28 300 g protein, together with their intrinsic pK values in parentheses were found tp be' 27 sode-chain carboxyl (pKint=4.0), four imidazole (pKint=6.5), one alpha-amino (pKint=7.5), 12 epsilon-amino (pKint=9.6), one guanidino (pKint=11.8) and one alpha-carboxyl group with abnormally low pK. The total number of basic nitrogens per mole of the protein was 22 so that four guanidino groups remained untitrated up to pH 12.0. Spectrophotometric titration showed that three out of five phenolic groups were titrated reversibly up to pH 11.9 with an intrinsic pK of 10.25; the remaining two groups became accessible only on protein denaturation. Viscosity results suggested absence of conformational change in the pH range 2.0-11.2. This explains the constancy of the pK values of carboxyl groups in the pH range 2.0-5.0. The empirical value of the electrostatic interaction factor, w, was 0.04, both in the carboxyl and phenolic regions.     

Occurrence of a new enzyme catalyzing the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline in rats

An enzyme concerned with the degradation of NG,NG-dimethyl-L-arginine to L-citrulline was investigated in rats. The enzyme purified from rat kidney catalyzed the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline with liberation of dimethylamine from the methylated guanidino moiety. The reaction required no co-factor and the maximum activity was obtained at pH 6.5. The enzyme was highly specific for NG,NG-dimethyl-L-arginine.     

Functional Groups Determine Biochar Properties (pH and EC) as Studied by Two-Dimensional 13C NMR Correlation Spectroscopy

While the properties of biochar are closely related to its functional groups, it is unclear under what conditions biochar develops its properties. In this study, two-dimensional (2D) ¹³C nuclear magnetic resonance (NMR) correlation spectroscopy was for the first time applied to investigate the development of functional groups and establish their relationship with biochar properties. The results showed that the agricultural biomass carbonized to biochars was a dehydroxylation/dehydrogenation and aromatization process, mainly involving the cleavage of O-alkylated carbons and anomeric O-C-O carbons in addition to the production of fused-ring aromatic structures and aromatic C-O groups. With increasing charring temperature, the mass cleavage of O-alkylated groups and anomeric O-C-O carbons occurred prior to the production of fused-ring aromatic structures. The regression analysis between functional groups and biochar properties (pH and electrical conductivity) further demonstrated that the pH and electrical conductivity of rice straw derived biochars were mainly determined by fused-ring aromatic structures and anomeric O-C-O carbons, but the pH of rice bran derived biochars was determined by both fused-ring aromatic structures and aliphatic O-alkylated (HCOH) carbons. In summary, this work suggests a novel tool for characterising the development of functional groups in biochars.