Anti-phosphocholine hybridoma antibodies. II. Functional analysis of binding sites within three antibody families.

The present investigation extends our immunochemical characterization of binding site heterogeneity among a large series of monoclonal anti-phosphocholine (PC) antibodies. Hybridoma proteins (HP) from eight genetically distinct strains are included in this study, yet no strain specific characteristics are observed. These HP, as previously shown (5), are divided into three well-defined families based on public or family-specific Id and L chain isotypes characteristic of three PC-binding myeloma proteins: T15, M603, and M511. All antibodies exhibited some degree of inter- or intra-family heterogeneity, or both. Some of this intra-family diversity was reflected by differential reactivity for PC when attached to three different carriers. In spite of this, the specificity profiles for hapten analogues of PC, as measured by hapten inhibition of binding, were the same for all members of the T15 family. Altering the carrier had no effect, thus suggesting that the binding site pocket for PC is essentially preserved, whereas that for carrier is variable. Similar conclusions were reached for most of the M603 HP, although the binding site is different from the T15 HP. The M511 HP stand in sharp contrast to the HP in the other two families because their binding sites exhibit extensive variability. The independence in reactivity for PC and PC plus carrier offers a rational explanation for idiotypic and/or structural heterogeneity within a family. More importantly it suggests interesting strategies for diversification within one group of antibodies.

Human placental adenosine kinase. Kinetic mechanism and inhibition.

The kinetic properties of human placental adenosine kinase, purified 3600-fold, were studied. The reaction velocity had an absolute requirement for magnesium and varied with the pH. Maximal activity was observed at pH 6.5 with a Mg2+:ATP ranging from 1:1 to 2:1. High concentrations of Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both adenosine and MgATP2-. The Michaelis constant was 0.4 micro M for adenosine and 75 micro M for MgATP2-. Inhibition by adenosine was observed at concentrations greater than 2.5 micro M. AMP was a competitive inhibitor with respect to adenosine and a noncompetitive inhibitor with respect to ATP. ADP was a noncompetitive inhibitor with respect to adenosine and ATP. Hyperbolic inhibition was observed during noncompetitive inhibition of adenosine kinase by AMP and ADP. Other purine and pyrimidine nucleoside mono-, di-, and triphosphates were poor inhibitors in general. S-Adenosylhomocysteine and 2'-deoxyadenosine inhibited adenosine kinase. The data suggest that (a) MgATP2- is the true substrate of adenosine kinase, and both pH and [Mg2+] may regulate its activity; (b) the kinetic mechanisms of adenosine kinase is Ordered Bi Bi; and (c) adenosine kinase may be regulated by the concentrations of its products, AMP and ADP, but is relatively insensitive to other purine and pyrimidine nucleotides.

Human placental adenosine kinase: purification and characterization.

Human placental adenosine kinase has thus been purified 3600-fold and characterized with respect to molecular weight, substrate specificity, divalent cation requirements, pH optimum, isoelectric pH, and kinetic properties. These data contribute to the information currently available about the regulation of adenosine metabolism, information critical for an understanding of the biological properties of adenosine.

Purine nucleoside phosphorylase deficiency: altered kinetic properties of a mutant enzyme.

Erythrocyte purine nucleoside phosphorylase from two brothers had 0.5% of normal activity. It differed from the normal enzyme by a tenfold increase in the Michaelis constant for inosine, an inability of inosine to protect against thermal lability, and a more positive net charge. The altered kinetic properties may account for the milder disease in the patients compared to the previously described cases. The data provide evidence for a structural gene mutation and genetic heterogeneity in the new disease of purine nucleoside phosphorylase deficiency and T cell dysfunction.