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It is our purpose to isolate and characterize the putative "Natriuretic Hormone", ostensibly responsible for ECF homeostasis, as well as identify endogenous pressors and compounds that induce prolonged natriuresis; we report here our initial progress in this area. Large volumes of pooled urine collected from uremic patients were fractionated, and the resulting isolates were evaluated for in vivo natriuretic and pressor effects and Na+/K(+)-ATPase inhibitory activity in renal cells. The purification steps involved ultrafiltration to obtain materials of less than 3000 da, gel filtration, and sequential reversed-phase high performance liquid chromatography (HPLC). After each HPLC step, the fractions were evaluated for their ability to elicit significant natriuresis and/or influence mean arterial pressure in the normal conscious female rat. Each fraction was also assayed for its ability to inhibit Na+/K(+)-ATPase as determined by the inhibition of 86Rb+ uptake into MDBK renal cells. While several of the fractions elicited profound natriuresis and/or pressor activity and other fractions inhibited Na+/K(+)-ATPase, there was no correlation among the activities in individual fractions. We have concluded that this plethora of bioactivities is responsible for much of the confusion and multiplicity of crude isolates claimed to be the putative hormone. Presently we are attempting to purify each of these activities to chemical homogeneity for structure determination.

Mouse monoclonal antibody 2H7 recognizes the CD20 cell surface phosphoprotein that is expressed in normal as well as malignant B cells. CD20 may be a useful target for therapy of B cell lymphomas, since damaged normal B cells can be replaced by their antigen-negative precursors. Monoclonal antibody 2H7 is an IgG2b (kappa) immunoglobulin which cannot mediate antibody-dependent cellular cytotoxicity with human lymphocytes or complement-dependent cytotoxicity with human serum. We have now generated a chimeric 2H7 antibody by substituting the mouse constant domains of 2H7 with the human gamma 1 and kappa domains. This new antibody has the same binding specificities as 2H7 but is highly effective in mediating antibody-dependent cellular cytotoxicity with human effector cells and complement-dependent cytotoxicity with human complement.

The synthetic peptide, L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala, is a substrate for the erythrocyte and brain protein carboxyl methyltransferases. These enzymes catalyze the methyl esterification of the free alpha-carboxyl group of the isoaspartyl residue, to which the glycyl residue is linked through the side chain beta-carboxyl group. In this work, we show that the alpha-methyl ester of this peptide was rapidly demethylated (t1/2 = 4 min at 37 degrees C, pH 7.4) in erythrocyte cytosolic extracts and that the product of this reaction appears to be the succinimide ring derivative of the peptide. The rate of demethylation, measured at either pH 6.0 or 7.4, was the same in buffer and erythrocyte extracts, suggesting that succinimide formation was a nonenzymatic reaction. The L-succinimide is more stable than the ester, but can be hydrolyzed in buffer at pH 7.4 (t1/2 = 180 min at 37 degrees C) to give a mixture of about 75% isoaspartyl peptide and 25% normal aspartyl peptide. The metabolism of the succinimide hexapeptide in erythrocyte extracts appears to be more complex, however. The implications of this work for the methylation and demethylation of cellular proteins containing structurally altered aspartyl residues are discussed.

Four hexapeptides of sequence L-Val-L-Tyr-L-Pro-(Asp)-Gly-L-Ala containing D- or L-aspartyl residues in normal or isopeptide linkages have been synthesized by the Merrifield solid-phase method as potential substrates of the erythrocyte protein carboxyl methyltransferase. This enzyme has been shown to catalyze the methylation of D-aspartyl residues in proteins in red blood cell membranes and cytosol. Using a new vapor-phase methanol diffusion assay, we have found that the normal hexapeptides containing either D- or L-aspartyl residues were not substrates for the human erythrocyte methyltransferase. On the other hand, the L-aspartyl isopeptide, in which the glycyl residue was linked in a peptide bond to the beta-carboxyl group of the aspartyl residue, was a substrate for the enzyme with a Km of 6.3 microM and was methylated with a maximal velocity equal to that observed when ovalbumin was used as a methyl acceptor. The enzyme catalyzed the transfer of up to 0.8 mol of methyl groups/mol of this peptide. Of the four synthetic peptides, only the L-isohexapeptide competitively inhibits the methylation of ovalbumin by the erythrocyte enzyme. This peptide also acts as a substrate for both of the purified protein carboxyl methyltransferases I and II which have been previously isolated from bovine brain (Aswad, D. W., and Deight, E. A. (1983) J. Neurochem. 40, 1718-1726). The L-isoaspartyl hexapeptide represents the first defined synthetic substrate for a eucaryotic protein carboxyl methyltransferase. These results demonstrate that these enzymes can not only catalyze the formation of methyl esters at the beta-carboxyl groups of D-aspartyl residues but can also form esters at the alpha-carboxyl groups of isomerized L-aspartyl residues. The implications of these findings for the metabolism of modified proteins are discussed.

Differential scanning calorimetry was used as a probe to follow structural disturbances in cytochrome c with electrostatic modification. At 51.7% maleylation, the Td decreased 14.1 degrees C; however, relatively stable delta H values reflected minor structural variations. With 77.5 and 96.4% modification, a significant decrease in delta H was more indicative of major conformational change. On this basis, a critical labelling point was considered. Extensive maleylation (96.4%) did not result in complete cytochrome denaturation. In general, assessment of cytochrome thermal parameters by DSC provided a conformational perspective for the influence of specific electrostatic parameters on molecular integrity.

Prolonged incubation of L-isoaspartate-containing forms of lactate dehydrogenase (231-242), sperm activating peptide, and adrenocorticotropin (22-27) at 37 degrees C, pH 7.4, with S-adenosyl-L-methionine and protein carboxyl methyltransferase from bovine brain leads to extensive conversion of the atypical isopeptide bond to a normal peptide bond. For the lactate dehydrogenase-related peptide, conversion was 80% complete after 24 h. For the other two peptides, conversion reached a level of approximately 65% after 48 h. The mechanism of conversion involves (i) rapid enzymatic methylation of the alpha-carboxyl of the L-iso-Asp residue; (ii) nonenzymatic demethylation resulting in formation of an L-aspartyl cyclic imide; and (iii) a slow, nonenzymatic hydrolysis of the cyclic imide to form a mixture of 15-25% normal L-Asp peptide and 75-85% L-iso-Asp peptide. The regenerated L-iso-Asp peptide is remethylated and the cycle is repeated. The extent of conversion is limited by a competing side reaction wherein the L-imide slowly racemizes, leading to the formation of mainly D-iso-Asp peptide, which is not a substrate for the methyltransferase. The ability of protein carboxyl methyltransferase to initiate conversion of L-iso-Asp residues to normal L-Asp suggests a possible role for this enzyme in facilitating the repair or degradation of deamidated proteins in vivo.

A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C gamma 2a and C kappa, were substituted by the human C gamma 1 and C kappa by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man.