Studies on tyrosine residues in porcine muscle adenylate kinase. Circular dichroism spectra and chemical modification with tetranitromethane.

Substrate-induced conformational change of porcine muscle adenylate kinase (EC 2.7.4.3) is evidenced by a change in circular dichroism spectra in the near ultraviolet. In the absence of tryptophan in porcine muscle adenylate kinase, the spectral change may be assigned to a perturbation of tyrosine chromophore(s). The spectral change was specific for adenine nucleotide binding and was greater with ATP than with AMP. In the x-ray model, Tyr153 and Tyr154 are located at a hinge region of two domains which form a deep active site cleft and are therefore susceptible to conformational change on substrate binding. Adenylate kinase was treated with equimolar tetranitromethane. The yellow-colored product, separated from unmodified enzyme by substrate gradient elution on a phosphocellulose column, had about 1 mol of nitrotyrosine per mol of the enzyme by amino acid analysis and showed a slightly higher Km value than native enzyme for ADP (Km = 0.50 mM compared with 0.25 mM for native adenylate kinase). Spectrophotometric titration of nitroadenylate kinase gave pKa 8.4 for the dissociation constant of the nitrotyrosyl hydroxyl group. On binding ATP the pKa value increased to 9.0 while AMP binding caused very little change. By peptide mapping of the carboxypeptidase digestion product, 0.70 mol of nitro group per mol of adenylate kinase was detected on Tyr153 and a small amount of nitro group was also found on Tyr95. From these results it is proposed that Tyr153 is directly or indirectly involved in the binding of ATP.     

Proton magnetic resonance spectra of tRNA-Met-f from Thermus thermophilus.

220 MHz proton magnetic resonance spectra of tRNAs in bulk and tRNA-Met-f from Thermus thermophilus have been measured and compared with those of tRNAs from E. coli. Temperature dependences and chemical shift positions of the bulk tRNAs are well explained by the difference in their GC contents. It is known that the base sequence of the double helical regions in the cloverleaf structure of T. thermophilus tRNA-Met-f is different from that of E. coli tRNA-Met-f only at two positions in TpsiCarm; one more C:G pair is contained instead of a U:G pair of E. coli tRNA-Met-f and a C:G pair of E. coli is replaced by a G:C pair. In spite of the resembrance in the base sequences, nmr patterns around 13 ppm are fairly different from each other. The difference is discussed in relation with their tertiary structures and with the origin of chemical shift displacements.     

Two-dimensional NMR studies of the porcine muscle adenylate kinase

Porcine adenylate kinase was subjected to one- and two-dimensional proton NMR studies in order to identify amino acid spin systems and obtain sequence-specific resonance assignments. With a combination of results from a map of side-chain distances resulting from the refined X-ray crystallographic data and nuclear Overhauser effect spectroscopy (NOESY), assignments are suggested for all the aromatic spin systems.     

31P NMR studies of the structure of cation-nucleotide complexes bound to porcine muscle adenylate kinase

The paramagnetic effects on the spin-relaxation rates of 31P nuclei in complexes of porcine muscle adenylate kinase with ATP, GTP, GDP, and AMP were measured in the presence of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), to examine the structures of the enzyme-bound complexes. Experiments were performed exclusively on enzyme-bound complexes to limit contributions to observed relaxation rates to two exchanging complexes (with and without cation). Measurements were made at three frequencies, 81, 121.5, and 190.2 MHz, and as a function of temperature in the range 5-30 degrees C to determine the effect of exchange on the observed relaxation rates. Relaxation rates in the E.MnATP, E.MnGTP, and E.MnGDP complexes were shown to be exchange-limited and therefore without structural information. Relaxation rates for the complexes E.CoATP, E.CoGTP, and E.CoGDP were shown to depend on Co(II)-31P distances. Inability to precisely estimate spectral densities arising from electronic relaxation of Co(II) restricts calculations of Co(II)-31P distances in these complexes to upper and lower limits. At the center of these limits, the Co(II)-31P distances of beta-P and gamma-P in E.CoATP and E.CoGTP, and of beta-P (E.CoGDP), are in the range 3.1-3.5 A appropriate for the first coordination sphere. For all these complexes, the corresponding distance for alpha-P is appreciably larger in the range 3.9-4.5 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance of human muscle extracts

High resolution proton nuclear magnetic resonance (NMR) spectra of normal and diseased human muscle extracts were recorded at 470 MHz. Resonances from lactic acid, creatine, glucose, ribose, purine and pyrimidine bases were identified. The longitudinal relaxation times of these resonances were determined to allow quantitation of muscle metabolites. With aid of a standardized reference capillary, inserted into the NMR tube containing the muscle extracts, the lactic acid and total creatine content of the extracts was determined. After 5 h of incubation at 37 degrees C, normal muscles contained on average 103 mumol lactic acid and 36 mumol creatine/173 mg of noncollagenous protein, equivalent to 1.0 g of fresh muscle. The lactic acid and creatine contents decreased slightly in scoliosis and idiopathic scoliosis and they decreased significantly in cerebral palsy. In an extract of a patient whose illness was diagnosed as 'scoliosis' no creatine was present, and in an extract of a patient with unknown diagnosis the creatine content was reduced to 2 mumol/173 mg of noncollagenous protein. The short time (1.7 sec to 6.5 min) and the small amount of tissue (300 mg) needed for an analysis add to the potential of proton NMR as a new technique for the characterization of muscular diseases.     

Comparison of adenylate kinase from normal and malignant hyperpyrexic porcine muscle

Adenylate kinase has been implicated as a key factor in malignant hyperpyrexia, a complication of general anaesthesia which is usually triggered by the anaesthetic drug, halothane. Because of this, the enzyme was purified from both malignant hyperpyrexia susceptible and control porcine muscle. Electrophoretic studies, amino acid analysis, and peptide mapping of the purified enzymes revealed no significant differences between the two preparations. Both enzymes responded similarly to halothane and to the three sulfhydryl reacting reagents which were tested and they also showed an identical affinity for the substrate AMP. It is concluded that porcine MH is not due to an abnormality in the enzyme AK.     

High-resolution proton magnetic resonance spectra of muscle

High-resolution proton magnetic resonance spectra of intact muscles of frog and rat were obtained with selective saturation of the water signal. The spectra consisted of the superposition of a broad component and a high-resolution portion. The line width of the former was about 5 ppm and is assumed to originate from the protons of the macromolecules in muscle. The high-resolution portion showed well-resolved signals arising from creatine phosphate, creatine, carnosine, lactate and lipids. It is suggested that this technique could be used to monitor the intracellular pH by measuring the chemical shift of carnosine and the lipid consumption due to muscular contraction. When the spectrum of ³¹P-NMR is prepared simultaneously, the ratio of creatine phosphate to total creatine can also be determined.     

Proton magnetic resonance spectroscopy of leech muscle and nervous system

1. Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to measure the major intracellular metabolites in perchloric acid extracts of the Macrobdella decora muscle and nervous systems and the Oryctolagus cuniculus cerebrum. 2. Acetate, alanine, choline, glutamate, inositol, and lactate were assigned in the spectrum of leech ventral cord, leech muscle, and rabbit cerebrum. 3. Hirudonine and propionate were clearly observed only in the spectrum of leech muscle. 4. Creatine, N-acetyl aspartate, gamma aminobutyric acid, aspartate, and taurine, distinctive components of spectra of the mammalian cerebrum, were not seen in the invertebrate spectra. 5. 1H NMR spectroscopy provides a simple and rapid means of characterizing the major organic metabolites found in leech muscle and nervous tissues.     

Proton nuclear magnetic resonance spectra of excised rat brain. Assignment of resonances

We have recorded 1H NMR spectra of excised rat brain at 361 MHz using two different water suppression pulse sequences. The assignment of the resonances has been carried out in perchloric acid extracts and subcellular fractions. Our results show that cytosolic proteins, membrane phospholipids and 16 different metabolites contribute to the observed spectra. The new resonances assigned allow the direct observation of myo-inositol and urea. Moreover, changes in the spectral pattern upon anesthesia, ischemic exposure of the brain and age of the rat have been recorded and correlated with the compounds producing the spectra.     

Proton and C-13 nuclear magnetic resonance spectra of some benzoylated aldohexoses

Chemical shifts and coupling constants of ¹H-n.m.r. spectra of the perbenzoates of α-d-glucopyranose (1), β-d-glucopyranose (2), α-d-galactopyranose (3), α-d-mannopyranose (4), β-d-mannopyranose (5), and α-d-galactofuranose (6) are reported. The ¹³C-n.m.r. chemical shifts of compounds 1-3 and 6, and of penta-O-benzoyl-β-d-galactofuranose (7) are given. Mass spectra were used to differentiate the furanoses 6 and 7 from the pyranose 3.     

Proton nuclear magnetic resonance spectra of Pseudomonas aeruginosa ferricytochrome cd1

Proton nuclear magnetic resonance spectra of ferricytochrome cd1 from the denitrifying bacterium Pseudomonas aeruginosa have been obtained. The normal 0-10-ppm chemical shift range shows many overlapping and nonresolvable peaks, as would be expected for a dimeric protein of molecular weight approximately 120,000. In the downfield region between 10 and 50 ppm, and in the upfield region between 0 and -20 ppm, resolvable resonances corresponding to a small number of protons are observed. The temperature and pH behavior of these resonances have been examined. For some of the resolved resonances, the pH behavior of chemical shifts and intensities indicates that the oxidized form of the enzyme undergoes a structural transition with a pK of 5.8 +/- 0.3. On the basis of several lines of evidence, some assignments are proposed in which resolvable resonances are assigned as originating from either the heme c or the heme d1 prosthetic groups of the enzyme.     

Crystalline adenylate kinase from carp muscle.

1. The concentration of adenylate kinase in carp muscle is about 0.3 mg/g. An improved isolation procedure makes use of a dilute solution of the substrates, ATP and AMP, to elute the enzyme from a phosphocellulose column in overall yields of 60% before crystallization. By the hexokinase--pH-stat assay the specific activity is 3550 units/mg. The preparation has been found to be essentially homogeneous by dodecylsulfate gel electrophoresis, isoelectrofocusing and gel filtration. 2. The molecular weight has been determined to be 22000 by several methods. The absorbance of a 1% solution at 280 nm is 6.9 and the isoelectric point by electrofocusing is pH 5.9. 3. The crystals of carp adenylate kinase have the space group P4-1-22 or P4-3-22. 4. The amino acid composition has been determined. There is no tryptophan, no cystine. There is one amino acid residue each of cysteine and histidine which are at or close to the catalytic center. 5. Several peptides derived by tryptic hydrolysis have been isolated and identified with corresponding peptides of porcine adenylate kinase. Consideration is given to histidine and cysteine being a part of the active site.     

Proton magnetic resonance of exercise-induced water changes in gastrocnemius muscle

Exercise-induced water concentration changes have been determined in the medial gastrocnemius muscle noninvasively with image-guided in vivo proton magnetic resonance spectroscopy (MRS). These measurements were performed on seven normal male volunteers during and after isometric and ischemic-isometric exercise at 5, 10, and 20% maximum voluntary contraction (MVC). The observed water flux changes during different phases of exercise have been interpreted in terms of fluid transfer between the vasculature and exercising muscle. Good correlation between individual MVC and changes in water fluxes and the decay of water content was observed after 20% MVC exercise level. MRS results have been found to be consistent with those reported in the literature based on invasive biopsy techniques.     

Hydrogen-bonded complexes of the ribodinucleoside monophosphates in aqueous solution. Proton magnetic resonance studies.

A proton magnetic resonance study of the chemical shifts of a series of ribodinucleoside monophosphates in neutral H2O solution has been recorded in the 1-100 mM concentration range. The self-complementary dinucleoside monophosphates CpG and GpC and the complementary mixture GpU + ApC form intermolecular hydrogen-bonded complexes at low temperatures. The amino proton chemical shifts in the CpG and GpC spectra are consistent with the formation of a miniature double helical dimer in neutral aqueous solution at low temperatures (approximately 2 degrees C). The complementary mixture of dinucleosides GpU + ApC formed much less stable complexes than either GpC or CpG, while UpA did not show any indication of the formation of intermolecular hydrogen-bonded complexes. This result is consistent with the well-known observation that the stability of a double helix is proportional to the percent of G-C base pairs present.