13C NMR investigations on Npi-[13C1]carboxymethyl-histidine-119 ribonuclease.

The ribonuclease A derivative Npi-[13C1]carboxymethyl-histine-119 ribonuclease prepared by using [13C1]bromoacetate as alkylating reagent has been investigated with high resolution 13C NMR spectroscopy. In the 13C NMR spectra two carbon resonances of relatively high intensity appear which can be assigned to carboxyl groups attached to His-119 and Met-30, their intensity ratio being 10 : 1. The pH dependence of the carbon resonance of the carboxy-methyl group bound to the Npi of His-119 differs in the absence and presence of Cyd-2'-P, thus indicating that the catalytically inactive derivative does bind nucleotides. A mechanism of the alkylation reaction at pH 5.6 is proposed in which the epsilon-amino group of Lys-41 acts as the binding site for the carboxyl group of bromoacetate pushing the bromomethylene group towards the Npi of His-119 or the Ntau of His-12.     

An in vivo 31P-NMR study of the possible regulation of glycogen phosphorylase a by phosphagen via phosphate in the abdominal muscle of the shrimp Crangon crangon

31P-NMR spectroscopy has been used to determine the concentrations of Pi and phosphagen in the abdominal muscle of the shrimp Crangon crangon at rest and to follow the changes during their recovery after exhaustive work in vivo. Additionally, the effect of physiological Pi concentrations to the phosphorylase a activity has been examined. At rest, the cytoplasmic Pi and phosphagen concentrations were 1 mM and 38 mM, respectively. After exhaustive work the concentrations of both metabolites were in the same range (20 mM). During recovery, the time course of phosphagen regeneration correlates with the decrease of Pi, as the sum of the concentrations of these metabolites is constant. The ATP level was stable throughout the experiment. The phosphorylase a, which was already present in the resting muscle, was virtually inactive at the resting Pi level (1 mM). During work, however, Pi, which is probably generated by phosphagen-ATP splitting, might ensure a sufficient activation of phosphorylase a.     

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.     

15N-NMR studies of living systems

15N-NMR spectroscopy has been applied to the study in vivo of nitrogen uptake during primary and secondary metabolism in Streptomyces parvulus. The nitrogen metabolism of Saccharomyces cerevisiae has been studied in a series of experiments in an effort to elucidate the flow of nitrogen along the various competing pathways as well as its dependence on culture conditions. The low NMR sensitivity of the 15N nucleus required, in spite of high isotopic enrichment, quite long acquisition times (10-20 min). Therefore, an indirect detection method using double quantum 1H-NMR spectroscopy was introduced allowing the selective detection of 15N-bound protons with excellent S/N-ratio in less than a minute.     

Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l⁻¹. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l⁻¹. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l⁻¹ has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l⁻¹ h⁻¹. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb⁻¹ by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291. Received 12 September 2000/ Accepted in revised form 27 January 2001     

The effect of diethyl- -fluoroglutarate on phosphates and glutamate in slices of rabbit cerebral cortex

Abstract— The diethyl ester of α-fluoroglutarate (DEFG), an inhibitor of glutamate dehydrogenase, was prepared, and its effect on glutamate and phosphates in slices of rabbit cerebral cortex was examined. The primary effect of the drug on cortical slices incubating in a Krebs-Ringer glucose medium was to decrease the tissue levels of glutamate in association with decreased levels and turnover of high-energy phosphates. Assimilation of exogenous glutamate by the slices was partially blocked in the presence of the drug and severely depressed oxidative phosphorylation resulted when glutamate and DEFG were both present in the incubation mixture. The results suggested a significant relationship between the activity of cerebral glutamate dehydrogenase and oxidative phosphorylation. During incubation in a Krebs-Ringer glucose medium the endogenous pool of free amino acids in the cortical slice partitioned with the medium. Little or no glutamate, aspartate or GABA was present in the medium after incubation, but glycine, alanine, threonine, serine and glutamine did partition to varying degrees, with over one-half of the glutamine present in the incubation medium. With the exception of ‘leakage’ of aspartate, the partitioning patterns were relatively unaffected by the presence of added glutamate or DEFG.