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J L Markley

Publications and source records attributed to J L Markley.

At least 163 records · Page 9Linked to original sources
PubMed1984

Natural abundance 13C NMR spectra of human muscle, normal and diseased.

13C NMR spectra of human surgical muscle samples have been obtained at 50 and 118 MHz. Numerous sharp peaks in the 13C spectrum have been assigned to carbon atoms of soluble metabolites and fatty acyl chains of neutral fats and membrane-bound phospholipids. Comparisons have been made of 13C NMR spectra of normal and diseased muscles after removal of neutral fat by extraction with isopentane. Creatine, lactic acid, and phospholipids are not removed from muscles by isopentane. The most striking difference between 13C NMR spectra of isopentane-extracted normal and diseased muscle samples was the size of the residual 30.5 ppm methylene carbon resonance, that is, small in normal muscles and in nonspecific muscle diseases, but large in myogenic and neurogenic muscle diseases. In addition, differences were also found between normal and diseased muscles in their creatine content and their ability to produce lactic acid. By deoxycholate treatment of isopentane-extracted diseased muscle it is estimated that about one-fifth of the total phospholipids are highly mobile. T1 and NOE measurements indicated that the differences in peak height for the 30.5 ppm resonance between normal and diseased muscle are due only to difference in the amount of highly mobile fatty acyl chains in the muscle and not due to differences in relaxation parameters. 13C NMR of isopentane-extracted muscle appears to permit differentiation of normal from diseased muscle and, within diseased muscle, grading of the severity of the disease.

Biopsy↗
PubMed1984

Some plant leaves have orientation-dependent EPR and NMR spectra.

Proton nuclear magnetic resonance ((1)H NMR) spectra of leaves from 50 plant species were obtained at a spectrometer frequency of 470 MHz. Water present in leaf samples gives rise to characteristic spectral patterns. Most species show only one broad (1)H NMR peak; however, the leaves of some plants display complex, orientation-dependent spectra in which a common three-line pattern is discerned. The pattern varies with the angle between the leaf surface and the external magnetic field. Proton relaxation measurements show the presence of at least two water compartments in the leaves. The compartments are responsible for different components of the spectral pattern. EPR spectra, obtained at 35 GHz and at a temperature of -180 degrees C, of plant leaf sections are dominated by the strong signals of manganous ions. We find that most plant leaves have isotropic Mn(2+) EPR spectra. However, in some species (including ones that exhibit orientation-dependent (1)H NMR spectra) we detect orientation-dependent intensities in the forbidden lines; the spectra indicate that Mn(2+) ions occupy binding sites with axial or lower symmetry on nonrandomly oriented membranes. Both the NMR and the EPR results suggest that the chloroplasts of some plants are preferentially aligned with respect to the leaf surface.

Journal Article↗
PubMed1984

Detailed analysis of protein structure and function by NMR spectroscopy: survey of resonance assignments.

Techniques are now available for making extensive assignments in NMR spectra of proteins of moderate size (molecular weight 20,000 or less). Such assignments provide the first step for experiments designed to extract the full complement of NMR parameters for each group in a protein. The stage is set for exciting research scenarios in protein chemistry involving, for example, the determination of hydrogen exchange kinetics at all exchangeable positions whose half times are on the order of 100 ms (277) or longer than a few minutes (316, 569, 570); the characterization of intermediates in protein folding pathways (318); measurement of the distribution of internal motions within a protein molecule (573); a detailed description of the biophysical consequences of single amino acid replacements in small proteins (387); elucidation of the mechanisms of conformational transitions in proteins; and multiparametric characterization of the parts of an enzyme that participate in catalytic mechanisms. Small proteins for which extensive 1H NMR assignments have been made include lysozyme, several cytochromes, ferredoxins, myelin basic proteins, PTI and related proteinase inhibitors, proteinase inhibitors from seminal plasma and avian eggs, apamin, and several snake venom neurotoxins. (References are given in Table 1).

Animals↗
PubMed1984

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.

Cerebral Palsy↗
PubMed1984

Two-dimensional NMR approaches to the study of protein structure and function.

Two-dimensional Fourier transform methods for homonuclear proton NMR spectroscopy have been introduced by Wüthrich and Ernst as a means of extending assignments in spectra of proteins. Multinuclear two-dimensional approaches also appear promising. We are applying current one- and two-dimensional NMR methods to protein family members that differ from one another by one or more amino acid substitutions. The overall goal is to elucidate relationships among the sequences, structures, and functions of these proteins: for example, to delineate primary structural requirements for changes in observable properties such as conformation, amino acid side chain dynamics, hydrogen exchange dynamics, pK'a values, and oxidation-reduction potentials. The ovomucoids from a variety of species of birds, which include a single set of 12 pairs of third-domain proteins (Mr = 6062 for turkey third domain, similar for others) that differ by single amino acid substitutions, provide a favorable system for the study of the structural and dynamic effects of single amino acid replacements. X-ray crystallographic structures of two ovomucoid third domains are available. Other series of proteins being studied by these methods include the photosynthetic electron transport proteins ferredoxin and plastocyanin.

Amino Acid Sequence↗
PubMed1983

Imino proton assignments in the proton nuclear magnetic resonance spectrum of the lambda phage OR3 deoxyribonucleic acid fragment.

The 17 base pair duplex d(TATCACCGCAAGGGATAp) . d(TATCCCTTGCGGTGATAp) corresponding to the OR3 operator site of lambda phage has been synthesized and studied by 1H nuclear magnetic resonance spectroscopy at 470 MHz. The 13 imino proton resonances observed at 20 degrees C have been assigned to specific base pairs at positions 3-15 on the basis of nuclear Overhauser effect measurements and studies of the temperature dependence of peak intensities. Resonances from the A-T base pairs at positions 1, 2, 16, and 17 are assumed to be absent from the spectrum because of terminal fraying. Resonance from many of the base pairs suggested by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., & Matthews, B. W. (1982) Nature (London) 298, 718-723] to be involved in specific binding of the lambda phage cro repressor are well resolved.

Bacteriophage lambda↗
PubMed1982

High-resolution proton nuclear magnetic resonance studies of the nickel(II) derivative of azurin.

High-resolution (360 and 470 MHz) 1H NMR studies of Ni(II) azurin, the nickel(II) derivative of the blue copper protein azurin, are reported. The aliphatic resonances of Ni(II) azurin closely parallel those of apoazurin and Cu(I) azurin and indicate that no major structural changes are associated with the binding of nickel(II). The magnetic moment of Ni(II) azurin (mueff = 3.2 muB) is in keeping with a pseudotetrahedral coordination environment like that of Cu(I) azurin. Resonances of protons from the ligand moieties are shifted as far as 125 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonate and as far as 20 ppm upfield by internal fields due to the nickel center. One of these strongly shifted resonances is assigned to the methyl protons of the methionine ligand. From spectra of Ni(II) azurin as a function of pH, the pKa' values of histidine-35 and histidine-83 have been measured to be approximately 6.0 and 7.5, respectively. Histidine-35 titrates in a discontinuous fashion, and, significantly, so do several of the isotropically shifted ligand protons, also within experimental error with the same pHmid. This result reinforces the suggestion that the conformational change coupled to the protonation of histidine-35 plays an important role in regulating electron transfer reactions of native azurin [Silvestrini, M. C., Brunori, M., Wilson, M. T., & Darley-Usmar, V. M. (1981) J. Inorg. Biochem. 14, 327-338].

Azurin↗
PubMed1982

Phosphorus-31 nuclear magnetic resonance study of internal motion in ribonucleic acid of southern bean mosaic virus.

Phosphorus-31 nuclear magnetic resonance spectroscopy was used to study southern bean mosaic virus (SBMV) in its native and ethylenediaminetetraacetic acid treated forms at various pH values in the range 4.5-9.0. The degree of swelling in the latter form is a critical function of pH. Spin-lattice relaxation time (T1), line-width, and nuclear Overhauser effect (NOE) measurements were made at 40.51, 80.98, and 190.3 MHz. The results indicate that the RNA phosphate groups in native SBMV exhibit low-amplitude internal motions on a nanosecond time scale while the phosphate groups of swollen virus exhibit large-amplitude, more rapid, internal motions. A spread of T1 and NOE values is measured at intermediate stages in the swelling process. A simple explanation, consistent with the data, is that the RNA core of the virus undergoes a two-state phase transition from a relatively rigid, compact solid form in the native SBMV to a mobile, solvated state in the fully swollen particle.

Edetic Acid↗
PubMed1982

Structure and heme environment of ferrocytochrome c553 from 1H NMR studies.

Cytochrome c553 is a photosynthetic electron transport protein found in algae and cyanobacteria. We have purified cytochromes c553 from five cyanobacteria and studied the structures of the ferrocytochromes by 1H NMR spectroscopy at 360 and 470 MHz. Using standard NMR techniques and by comparing the amino acid sequences of four cytochromes c553 with their 1H NMR spectra, we have assigned in the spectrum of the Aphanizomenon flos-aquae protein 18 resonances to specific amino acid residues and 12 resonances to specific heme protons. Steady state and truncated driven nuclear Overhauser enhancement experiments indicate that a tyrosine and methionine are located near pyrrole ring IV of the heme and that a phenylalanine ring is near the heme alpha-mesoproton. The general folding of the cytochrome c553 protein backbone appears to resemble that of Pseudomonas aeruginosa cytochrome c551, but the chirality of the cytochrome c553 axial methine sulfur is R, the same as that of horse heart cytochrome c.

Cyanobacteria↗
PubMed1982

Properties of conserved amino acid residues in tandem homologous protein domains. Hydrogen-1 nuclear magnetic resonance studies of the histidines of chicken ovomucoid.

Peaks corresponding to the C6 protons of the four histidine residues (positions 58, 111, 123, and 182) of chicken ovomucoid have been assigned in 1H NMR spectra (360 or 470 MHz) of the native single-chain protein and of fragments of the protein corresponding to its three homologous structural domains. Comparison of the 1H NMR pH titration behavior of these histidine residues and the deuterium exchange rates of their C6-H positions show the following: (1) The chemical shift properties of histidine residues 58, 123, and 182 differ despite the fact that the three residues are located in homologous positions in the three tandem domains. (2) The properties of three of the four histidine residues (58, 111, and 123) do not change appreciably when the domains in which they are located are isolated, indicating that their environments are similar in both the fragment and the native protein. (3) The properties of the fourth histidine (182) differ in the isolated domain and in the native protein. (4) The observed properties of the histidine residues stem primarily from intradomain interactions that remain constant in isolated domains rather than from interactions with neighboring domains; an interdomain interaction is required to explain the behavior of only histidine-182. (5) The chemical shift of histidine-111 is affected by the titration of the side chain of aspartate-98 with pHmid 2.6 in native ovomucoid but not in isolated second domain; the chemical shift of histidine-182 is perturbed by the titration of the carboxyl group of the C-terminal cysteine-186 with pHmid 2.4 in native ovomucoid and pHmid 2.6 in isolated third domain.

Animals↗
PubMed1982

Changes in the natural abundance 13C NMR spectra of intact frog muscle upon storage and caffeine contracture.

The carbons of phospholipids have limited mobility in fresh, resting, gastrocnemius frog muscle, but a population of phospholipids gains considerable mobility upon storage in the muscle or in contracture induced by caffeine. In parallel with the appearance of sharp phospholipid resonances, lactic acid also appears in the 13C NMR spectra. There is a correlation between the mobility of phospholipids and the depletion of phosphocreatine and ATP in muscle.

Animals↗
PubMed1982

Biosynthesis of aromatic compounds: 13C NMR spectroscopy of whole Escherichia coli cells.

13C and 31P NMR spectra of wild-type Escherichia coli showed resonances from metabolic intermediates of glycolysis and ATP formation but no detectable signals from aromatic amino acids. However, tyrosine biosynthesis from D-[l-13C]glucose was observed in cells harboring a feedback-resistant allele of aroF, the gene encoding tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC4.1.2.15], one of the isoenzymes that control carbon flow through the common aromatic biosynthetic pathway. A similar accumulation of tyrosine and phenylalanine is seen in cells carrying a multiple-copy plasmid that carries a wild-type aroF allele in addition to pheA and tyrA, the structural genes for controlling enzymes of the terminal pathways to phenylalanine and tyrosine biosynthesis. These in vivo measurements by a noninvasive probe suggest feedback inhibition as the quantitatively major mechanism controlling carbon flow in the common aromatic compound biosynthetic pathway. In strains accumulating aromatic amino acids, a transient accumulation of trehalose was detected, indicating that previously unknown changes in Escherichia coli metabolism accompany overproduction of aromatic compounds.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1981

Lack of evidence for a tetrahedral intermediate in the hydrolysis of nitroanilide substrates by serine proteinases. Subzero-temperature stopped-flow experiments.

We have used a stopped-flow apparatus to reinvestigate reports, based on the observation of "burst" kinetics, of an intermediate prior to the acyl-enzyme complex in hydrolysis reactions of anilides catalyzed by trypsin and elastase [M. W. Hunkapiller, M. D. Forgac and J. H. Richards (1976) Biochemistry 15, 5581-5588; D. D. Petkov (1978) Biochim. Biophys. Acta, 523, 538-541; A. L. Fink and P. Meehan (1979) Proc. Natl Acad. Sci. USA, 76, 1566-1569; P. Compton and A. L. Fink (1980) Biochem. Biophys. Res. Commun. 93, 427-431]. We studied the hydrolysis of several anilide substrates by bovine and porcine trypsin and porcine elastase between -30 degrees C and 20 degrees C. In no case did we record true "burst" kinetics. We show that confusion spectral changes can arise from incomplete mixing, thermal gradients, or heterogeneity of the substrate. We conclude that there is no solid spectroscopic evidence at present for the existence of a tetrahedral intermediate in the hydrolysis of amides by serine proteinases. The substrate N-acetyl-L-alanyl-L-prolyl-L-alanine 4-nitroanilide is a mixture of two isomers trans and cis about the L-alanyl-L-propyl peptide bond. It appears that elastase hydrolysis the cis isomer more rapidly than the trans isomer and this could lead to false "burst" kinetics. We describe the construction of the stopped-flow apparatus designed for cryoenzymology used for this work that has novel features and is adaptable to a variety of spectrophotometers. Solutions can be handled under anaerobic conditions. A window allows the drive syringes to be observed or exposed to light for photochemical experiments. The apparatus operates over the temperature range -35 degrees C to + 25 degrees C. The dead time is under 5 ms. A recording system is described that permits one to follow reactions over a wide time scale covering half-time of the order of several milliseconds to hours.

Anilides↗
PubMed1978

Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aalpha.

Reversible unfolding of bovine chymotrypsinogen A in 2H2O either by heating at low pH or by exposure to 6 M guanidinium chloride results in the exchange of virtually all the nitrogen-bound hydrogens that give rise to low-field 1H NMR peaks, without significant exchange of the histidyl ring Cepsilon1 hydrogens. These preexchange procedures have enabled the resolution of two peaks, using 250-MHz correlation 1H NMR spectroscopy, that are attributed to the two histidyl residues of chymotrypsinogen A. Assignments of the Cepsilon1 hydrogen peaks to histidine-40 and -57 were based on comparison of the NMR titration curves of the native zymogen with those of the diisopropylphosphoryl derivative. Two histidyl Cepsilon1 H peaks were also resolved with solutions of preexchanged chymotrypsin Aalpha. The histidyl peaks of chymotrypsin Aalpha were assigned by comparison of NMR titration curves of the free enzyme with those of its complex with bovine pancreatic trypsin inhibitor (Kunitz). The NMR titration curves of histidine-57 in the zymogen and enzyme and histidine-40 in the zymogen exhibit two inflections; the additional inflections were assigned to interactions with neighboring carboxyl groups: aspartate-102 in the case of histidine-57 and aspartate-194 in the case of histidine-40 of the zymogen. In bovine chymotrypsinogen A in 2H2O at 31 degrees C, histidine-57 has a pK' of 7.3 and aspartate-102 a pK' of 1.4, and the histidine-40-aspartate-194 system exhibits inflections at pH 4.6 and 2.3. In bovine chymotrypsin Aalpha under the same conditions, the histidine-57-aspartate-102 system has pK' values of 6.1 and 2.8, and histidine-40 has a pK' of 7.2. The results suggest that the pK' of histidine-57 is higher than the pK' of aspartate-102 in both zymogen and enzyme. A significant difference exists in the structure and properties of the catalytic center between the zymogen and activated enzyme. In addition to the difference in pK' values, the chemical shift of histidine-57, which is highly abnormal in the zymogen (deshielded by 0.6 ppm), becomes normalized upon activation. These changes may explain part of the increase in the catalytic activity upon activation. The 1H NMR chemical shift of the Cepsilon1 H of histidine-57 in the chymotrypsin Aalpha-pancreatic trypsin inhibitor (Kunitz) complex is constant between pH 3 and 9 at a value similar to that of histidine-57 in the porcine trypsin-pancreatic trypsin inhibitor complex [Markley, J.L., and Porubcan, M. A. (1976), J. Mol. Biol. 102, 487--509], suggesting that the mechanisms of interaction are similar in the two complexes.

Animals↗