Phosphypyridoxyl trifluoroethylamine has been synthesized as an active site-directed 19F NMR probe for aspartate transaminase. This coenzyme derivative adds stoichiometrically to the apotransaminase as observed by both fluorescence and circular dichroism measurements. The fluorinated phosphypyridoxamine derivative, when bound to the apotransaminase, will not dissociate upon extensive dialysis or passage through Sephadex G-25. The compound behaves as a pyridoxamine phosphate derivative and not as a coenzyme-substrate complex, since both competing anions and dicarboxylic acid inhibitors still bind to the phosphopyridoxyl trifluoroethylamine enzyme. The 19F NMR spectra of the enzyme-bound phosphopyridoxyl trifluoroethylamine were measured as a function of pH, ionic strength, and temperature. The 19F MNR of the enzyme-bound coenzyme derivative revealed no predetermined asymmetry in the subunits of aspartate transaminase insolution in terms of differences in chemical shift or resonance line shape between the two environments. A pH-dependent chemical shift change of the single 19F resonance was observed, which is consistent with the influence of a single ionization with an apparent pKa of 8.4 in 0.10 M KCl at 30 degrees. Increasing the ionic strength resulted in increasing values for the observed pKa, the highest recorded value was 9.1 in 3.0 M KCl. The temperature dependence of the pH titration of the chemical shift gives deltaH' of ionization of 10.5 kcal/mol. The evidence suggests a possible epsilon-amino group, electrostatically affected by positive charges, being responsible for the titration effect of the active site-bound fluorine derivative of pyridoxamine phosphate.

Animals↗

PubMed1976

The origin of serum protein, A, B, and H blood group, and Gm and Inv antigens in house dust.

Materials precipitated from an aqueous extract of house dust by saturation with ammonium sulphate showed immunological reaction with antisera to human serum albumin, human alpha1-acid glycoprotein, human IgG, Gm and Inv antigens and to A, B and H antigens. It is concluded that the albumin and alpha1-acid glycoprotein are of human origin. It seems that the apparent IgG activity is due to cross-reactivity since, from the anticipated specificities of the Fab and Fc fragments of human IgG, the former could not be detected, and since Gm (6) activity was present, unexpectedly, in Swiss house dust. The A, B and H blood group antigenic activities were detected in relative concentration different from those expected if they were solely of human origin. It is concluded that they are from both human and non-human sources.

ABO Blood-Group System↗

PubMed1976

Specificity and possible origin of anti-N antibodies developed by patients undergoing chronic haemodialysis.

Studies were performed on two anti-N sera from patients who had formed anti-N antibodies during their period of haemodialysis. No specific inhibition of these antibodies was obtained by extracts prepared from a new and a used dialysis unit, and it was concluded that there was no evidence of a compound in the units which would stimulate production of the anti-N. The specificity of the anti-N's was shown to be against a precursor of MN antigens, since all activity in the sera could be removed by neuraminidase-treated M cells, which expressed MN precursor. Since the anti-N's would react with untreated N cells only at 4 degrees C, but with formaldehyde-treated cells at 4, 25 and 37 degrees C, it was concluded that the specificity of the anti-N's was directed against an antigen developed by red cells after formaldehyde treatment. It is proposed that the production of the anti-N antibodies is stimulated by formaldehyde-treated red cells passing into the patient during re-use of a dialysis unit sterilized with formaldehyde.

Antibody Specificity↗

PubMed1975

Fluorescence energy transfer between active sites in aspartate transaminase.

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Aspartate Aminotransferases↗

PubMed1975

Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction.

Glutamate aspartate transaminase (EC 2.6.1.1) is a dimeric enzyme with identical subunits with each active site containing pyridoxal 5'-phosphate linked via an internal Shiff's base to a lysine residue. It is not known if these sites interact during catalysis but negative cooperativity has been reported for binding of the coenzyme (Arrio-Dupont, M. (1972), Eur. J. Biochem. 30, 307). Also nonequivalence of its subunits in binding 8-anilinonaphthalene-1-sulfonate (Harris, H.E., and Bayley, P. M. (1975), Biochem. J. 145, 125), in modification of only a single tyrosine with full loss of activity (Christen, P., and Riordan, J.F. (1970), Biochemistry 9, 3025), and following modification with 5,5'-dithiobis(2-nitrobenzoic acid) (Cournil, I., and Arrio-Dupont, M. (1973), Biochemie 55, 103) has been reported. However, steady-state and transient kinetic methods as well as direct titration of the active site chromophore with substrates and substrate analogs have not revealed any cooperative phenomena (Braunstein, A. E. (1973), Enzymes, 3rd Ed. 9, 379). It was therefore decided that a more direct approach should be used to clarify the quistion of subunit interaction during the covalent phase of catalysis. To this end a hybrid method was devised in which a hybrid transaminase was prepared which contained one subunit with a functional active site while the other subunit has the internal Shiff's base reduced with NaBH4. The specific activities and amount of "actively bound" pyridoxal 5'-phosphate are both in a 2:1 ratio for the native and hybrid forms. Comparison of the steady-state kinetic properties of the hybrid and native enzyme forms shows that both forms gave parallel double reciprocal plots which is characteristic of the Ping-Pong Bi-Bi mechanism of transamination. The Km values for the substrates L-aspartic acid and alpha-ketoglutaric acid are nearly identical while the Vmax value for the hybrid is one-half the value of the native transaminase. It therefore appears that the active sites of glutamate aspartate transaminase function independently and a compulsory flip-flop mechanism is not involved.

Animals↗

PubMed1975