Chromatographic analysis of the enantiomers of ifosfamide and some of its metabolites in plasma and urine

The enantiomers of the cytostatic drug ifosfamide and the two metabolites 2- and 3-dechloroethylifosfamide were isolated from plasma and urine by liquid-liquid extraction with ethyl acetate, resolved on a Chirasil- -val gas chromatographic column and detected by a nitrogen-phosphorus-selective flame ionisation detector. Resolution of the racemic compounds for identification purposes was also accomplished with high-performance liquid chromatography on a chiral column. The validated gas chromatographic method was suitable to determine the total concentrations and the enantiomeric composition of ifosfamide and its dechloroethylated metabolites in plasma and urine samples from treated patients. Some metabolic preferences in the metabolism of ifosfamide were found.     

A large-subunit mitochondrial ribosomal DNA sequence translocated to the nuclear genome of two stone crabs (Menippe)

Two DNA sequences that appear to be homologous to large-subunit mitochondrial ribosomal RNA genes have been identified in the stone crabs Menippe mercenaria and M. adina. Amplification from whole genomic DNA by polymerase chain reaction (PCR) with oligonucleotide primers based on conserved portions of large-subunit mitochondrial rRNA genes consistently amplified two products of similar length (565 and 567 bp). These products differed at 3% of their nucleotide bases, and could be distinguished by a HindIII site. Only one of these sequences (designated the A sequence) was detected by PCR in purified mitochondrial DNA. The other (designated the B sequence) hybridized to total genomic DNA at a level consistent with a nuclear genome location. It is unlikely that the type B product would have been recognized as a nuclear copy by examination of its sequence alone. This is the first report of a mitochondrial gene sequence translocated into the nuclear genome of a crustacean.      

Conditional probability analysis for a domain model of Ca(2+)-inactivation of Ca2+ channels.

The domain model of Ca2+ inactivation of Ca2+ channels, which has been used to explain rapid inactivation of whole cell Ca2+ currents in pancreatic beta cells, is applied to single-time and conditional open probability measurements on guinea pig ventricular myocyte Ca2+ channels. These two measurements greatly constrain the choice of kinetic constants in the model. Calculations with the model provide a simple quantitative explanation of recent experimental results, including a slow increase in the inactivation rate.     

Two roles of Ca2+ in agonist stimulated Ca2+ oscillations.

We propose a mechanism for agonist-stimulated Ca2+ oscillations that involves two roles for cytosolic Ca2+: (a) inhibition of inositol-1,4,5-trisphosphate (IP3) stimulated Ca2+ release from the endoplasmic reticulum (ER) and (b) stimulation of the production of IP3 through its action on phospholipase C (PLC), via a Gq protein related mechanism. Relying on quantitative experiments by Parker, I., and I. Ivorra (1990. Proc. Natl. Acad. Sci. USA. 87:260-264) on the inhibition of Ca2+ release from the ER using caged-IP3, we develop a kinetic model of inhibition that allows us to simulate closely their experiments. The model assumes that the ER IP3 receptor is a tetramer of independent subunits that can bind both Ca2+ and IP3. Upon incorporation of the action of Ca2+ on PLC that leads to production of IP3, we observe in-phase-oscillations of Ca2+ and IP3 at intermediate values of agonist stimulation. The oscillations occur on a time scale of 10-20 s, which is comparable to the time scale for inhibition in Xenopus oocytes. Analysis of the mechanism shows that Ca(2+)-inhibition of IP3-stimulated Ca2+ release from the ER is an essential step in the mechanism. We also find that the effect of Ca2+ on PLC can lead to an indirect increase of cytosolic Ca2+, superficially resembling "Ca(2+)-induced Ca(2+)-release." The mechanism that we propose appears to be consistent with recent experiments on REF52 cells by Harootunian, A. T., J. P. Y. Kao, S. Paranjape, and R. Y. Tsien. (1991. Science [Wash. DC]. 251:75-78.) and we propose additional experiments to help test its underlying assumptions.     

Nonequilibrium chemical potentials and free energies for enzyme-catalyzed reactions

Using the statistical theory of nonequilibrium thermodynamics we explore the nature of nonequilibrium corrections to chemical potentials in simple enzyme-catalyzed reactions. The statistical definition of the chemical potential, which pertains to systems that are at stable steady states, is applied to the Michaelis-Menten reaction scheme in a cellular-sized compartment that communicates with out-side reservoirs. Calculations based on the kinetic parameters for hexokinase and triose phosphate isomerase show that substantial corrections to the chemical potential of product (the order of 25 mV) are possible if the reaction is sufficiently far from equilibrium. The dependence of the corrections to the chemical potentials on the size of the cellular compartment are explored, and the relevance of the corrections for understanding the thermodynamics of metabolites is discussed.     

Kinetics and thermodynamics of metabolite transfer between enzymes

Based on experimental evidence put forward by Bernhard and others, we explore the kinetics and thermodynamics of the proposed direct and diffusional transfer mechanisms of enzymatic catalysis. Data for transient transfer of NADH between two cognate dehydrogenases (E1 and E2) are combined with steady-state catalytic data to quantify the kinetics of the two transfer mechanisms. In order to rationalize these data we find: (1) that the rate constants for direct transfer of NADH from E1-NADH to E2 must be much larger when a reactive metabolite, M, is bound to E2, (2) that a significant amount of noncatalytic complex E1-E2-M must be formed, and (3) that dissociation constants of the order of 1 microM are required for the ternary complexes involved in direct transfer (E1-NADH-E2). Using values of rate parameters similar to those assumed in these calculations, we proceed to explore the kinetics and thermodynamics of a hypothetical two-enzyme segment of a metabolic pathway that involves direct and diffusional metabolite transfer operating in parallel. Under steady-state conditions, we conclude from our calculations: (1) that the flux through the direct transfer branch would be comparable to or greater than that through the diffusional transfer branch under physiological conditions, (2) that activity effects resulting from physiological concentrations of inert protein enhance the predominance of the direct transfer flux, (3) that significant concentrations of ternary transfer complexes are formed, (4) that changes in the catalytic mechanism involving the ternary complex have little effect, (5) that direct transfer significantly moderates the reduction in metabolic flux caused by protein complexation, and (6) that direct transfer greatly alters the thermodynamics and kinetics of our hypothetical pathways. We conclude, therefore, that direct transfer--when it exists--would have important kinetic, thermodynamic, and physiological consequences in vivo.