Studies of 6-fluoropyridoxal and 6-fluoropyridoxamine 5'-phosphates in cytosolic aspartate aminotransferase

The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine, and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase, and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10 000-fold, the modified coenzymes bind predominately in their dipolar ionic ring forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate inhibitors normally and has about 42% of the catalytic activity of the native enzyme. The fluorine nucleus provides a convenient NMR probe that is sensitive to changes in the state of protonation of both the ring nitrogen and the imine or the -OH group of free enzyme and of complexes with substrates or inhibitors. The NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH, becomes protonated with a pKa of approximately 7.1, corresponding to the pKa of approximately 6.4 in the native enzyme. Below this pKa a single 19F resonance is seen, but there are two light absorption bands corresponding to ketoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio is altered markedly upon binding of dicarboxylate inhibitors. From the chemical shift values, we conclude that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine) onto the aspartate-222 carboxylate (in the enolimine). The possible implications for catalysis are discussed.     

Use of cell affinity chromatography for separation of lymphocyte subpopulations

We describe the use of affinity chromatography for separation of cell populations that do not differ significantly with respect to gross physical properties such as size, density, or charge. Cell affinity chromatography exploits differences in cell surface macromolecules by passage of mixtures of cell populations through a column containing beads to which are attached chemical ligands with specific binding affinity for particular cell surface receptors. In this article we focus on the application of this concept to separation of mature T lymphocytes from peripheral blood. This serves as a model for the separation of these cells from bone marrow in order to prevent graft-vs.-host disease in bone marrow transplantation. However, the concept of cell affinity chromatography should find more general widespread utility in a variety of biotechnological applications. Thus, we introduce a simple theoretical framework which is necessary in order to understand the results that might be expected in any given situation. Finally, we use this theory to provide a tentative explanation for experimental observation of the effects of temperature and flowrate on the degree of separation achieved for our current pplication.     

Isolation of Listeria monocytogenes from raw milk

During a recent outbreak of listeriosis, we examined 121 raw milk samples and 14 milk socks (filters). Listeria monocytogenes was recovered from 15 (12%) of 121 milk specimens and 2 (14%) of 14 milk socks. The optimal processing method consisted of cold enriching diluted milk for 1 month with culture to selective broth, followed by plating.     

Development of antibody to measles virus polypeptides during complicated and uncomplicated measles virus infections.

Immune precipitation of 181 sera from 152 patients with natural measles was studied to determine the temporal course and frequency of antibody responses to nucleocapsid, fusion, hemagglutinin, and matrix proteins of measles virus. Large amounts of antibody to nucleocapsid protein developed in all patients by day one of the rash. Antibody to hemagglutinin and fusion proteins developed in all patients over the next 3 weeks, the former to high levels and the latter to low levels. Antibody to matrix protein developed to very low levels and was detectable in only 41% of the patients; this poor response to matrix protein was not correlated with the age of the patient or the acute neurological complications of measles.     

Effect of oxyanions of the early transition metals on rabbit skeletal muscle phosphorylase

The differential effects of the oxyanions of the early transition metals ions V(V), W(VI), and Mo(VI) on the catalytic activity and coenzyme binding of rabbit skeletal muscle phosphorylase are studied. The oligoanions of V(V), W(VI), and Mo(VI) are potent inhibitors of phosphorylase. Kinetic studies revealed that oligovanadates inhibit pyridoxal-reconstituted phosphorylase b by competing with both the substrate, glucose 1-phosphate, and the activator, phosphite, with K1 values of 4 microM and 6 microM, respectively. Oligovanadates in the millimolar concentration range inhibit phosphorylases a and b by competing with glucose 1-phosphate binding. The polymeric decavanadate and paratungstates caused time-dependent inactivation of phosphorylase. Spectral studies with tungstate and phosphorylase b revealed that the inactivation is due to deformation of the coenzyme site. Kinetic studies and the protective effects of substrate and effectors on inactivation and deformation by tungstate or vanadate suggest that deformation and inactivation is caused by a primary binding of the oligoanions at the glucose 1-phosphate site. Nuclear magnetic resonance (NMR) studies of vanadate-phosphorylase complexes and vanadate solutions under different conditions were carried out to ascertain the nature of vanadate ions interacting with functional groups in phosphorylase. The results suggest that decavanadate is the major protein-bound species. NMR studies also showed that guanidino groups react with decavanadate and suggest that arginine residues in phosphorylase are potential functional groups that can interact with decavanadate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate in phosphorylase: a study using pyridoxal-reconstituted enzyme as a model system

Pyridoxal-reconstituted phosphorylase was used as a model system to study the possible functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate (PLP) in rabbit muscle glycogen phosphorylase. Kinetic study was conducted by using competitive inhibitors of phosphite, an activator, and alpha-D-glucopyranose 1-phosphate (glucose-1-P) to study the relationship between the PLP phosphate and the binding of glucose-1-P to phosphorylase. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy of fluorophosphate bound to pyridoxal phosphorylase showed that its ionization state did not change during enzymatic catalysis. Evaluation of the apparent kinetic parameters for the activation of pyridoxal phosphorylase with different analogues having varied pKa2 values demonstrated a dependency of KM on pKa2. Molybdate, capable of binding as chelates in a trigonal-bipyramidal configuration, was tested for its inhibitory property with pyridoxal phosphorylase. On the basis of the results in this study, several conclusions may be drawn: (1) The bound phosphite in pyridoxal phosphorylase and, possibly, the 5'-phosphoryl group of PLP in native phosphorylase do not effect the glucose-1-P binding. (2) One likely function of the 5'-phosphoryl group of PLP in native phosphorylase is acting as an anchoring point to hold the PLP molecule and/or various amino acid side chains in a proper orientation for effective catalysis. (3) The force between the PLP phosphate and its binding site in phosphorylase is mainly electrostatic; a change of ionization state during catalysis is unlikely. (4) Properties of the central atoms of different anions are important for their effects as either activators or inhibitors of pyridoxal phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex chromosome homology and incomplete, tissue-specific X-inactivation suggest that monotremes represent an intermediate stage of mammalian sex chromosome evolution

Female mammals have two X chromosomes and males have a single X and a smaller, male-determining Y chromosome. The dosage of X-linked gene products is equalized between the sexes by the genetic inactivation of one X chromosome in females. The characteristics of the mechanism of X-chromosome inactivation differ in eutherian and metatherian mammals, and it has been suggested that the metatherian system represents a more primitive stage. The present study of monotreme sex chromosomes and X-chromosome inactivation suggests that the prototherian mammals may represent an even more primitive stage. There is extensive G-band homology between the monotreme X and Y chromosomes, and differences in the patterns of replication of the two X chromosomes in females suggest that X inactivation is tissue specific and confined to the unpaired segment of the X. On the basis of these results, we propose a model for the differentiation of mammalian sex chromosomes and the evolution of the mechanism of X-chromosome inactivation. This model involves a gradual reduction of the Y chromosome and an accompanying gradual recruitment of (newly unpaired) X-linked loci under the control of a single inactivation center.     

Characterization of a high-molecular-weight protein immunoprecipitated by platelet-derived growth factor antisera

We previously demonstrated that a high-molecular-weight glycoprotein could be immunoprecipitated from metabolically labeled U-2 OS cells with platelet-derived growth factor (PDGF) antiserum and that it appears to be derived from a different precursor than is the 30 kD PDGF-like mitogen produced by these cells. These findings were unexpected, since the molecular weight of this glycoprotein is too large to be encoded by the PDGF structural genes. From experiments with metabolically labeled U-2 OS human osteosarcoma, fibroblasts, and NRK cells, we report here that a 185 kD protein immunoprecipitated with PDGF antiserum has the following characteristics. 1) It is a PDGF binding protein that is unrelated to alpha 2-macroglobulin. 2) It is phosphorylated in response to PDGF stimulation. 3) It is immunoprecipitated by phosphotyrosine antibodies. 4) It is not a substrate of epidermal growth factor-induced tyrosine kinase activity. These studies indicate that high-molecular-weight proteins immunoprecipitated by antiserum to PDGF represent a complex between PDGF and a binding protein capable of being phosphorylated by a PDGF-induced tyrosine kinase. These characteristics are identical to those of the PDGF receptor.