SummaryRecombinant monocyte chemotactic-activating factor (MCAF) has been shown to induce histamine release from human basophils with a dose response between 10 -9 and 10 -6 M. The peak of activity was reached at 10 -7 M. Histamine release by MCAF was rapid with an initial rate comparable with histamine release by an optimal dose of anti-IgE. MCAF led to peak histamine release within 1 min. 80% of the subjects tested were responsive to MCAF or anti-IgE, while all were responsive to FMLP. The percentage histamine release by MCAF was, however, less than that seen with anti-IgE or FMLP, but this was attributable to a lesser percent release in nonatopic subjects; atopic subjects responded similarly to all three agonists. MCAF was also shown to activate highly purified human basophils more readily than mixed leukocytes, and its activity was inhibited by a polyclonal rabbit antibody. At a suboptimal concentration (2.5 x 10-9 M), MCAF was unable to prime the basophil to histamine release by other secretagogues. However, interleukin 3 (I1:3) and I1:5 could each prime basophils for MCAF-induced secretion. Therefore, our results suggest that MCAF may be a major contributor to the histamine-releasing activity seen in peripheral blood mononuclear cell supernatants that has been designated histamine releasing factor(s).

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A rat liver DNA helix-destabilizing protein (HDP) that has previously been proposed to play a role in transcription has been identified as M chain lactate dehydrogenase (LDH-5; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). Tryptic peptides accounting for 157 amino acids in the rat liver HDP have been characterized and then matched to the published sequence for the M chain of porcine LDH. Based on amino acid compositions and direct solid-phase protein sequencing, at least 148 of the 157 residues that were compared are identical in both proteins. In addition, both porcine LDH and the rat liver HDP have blocked amino termini and similar amino acid compositions and molecular weights. Rat liver HDP and LDH-5 that were purified to molecular homogeneity had similar specific activities in both single-stranded DNA (ss DNA) binding and LDH assays. HPLC tryptic peptide maps were also identical for both the rat liver HDP and LDH proteins. Since preincubation of HDP in NADH prevents its binding to ss DNA, both NADH and ss DNA may be binding at the same site. Further support for this latter idea derives from chemicalmodification studies which demonstrate that tyrosine-238, which is located near the coenzyme binding site of LDH, seems to be essential for the ability of HDP to bind ss DNA. These results indicate caution in ascribing in vivo roles solely on the basis of binding to ss DNA. Alternatively, they suggest that a single protein may play more than one biological role.

A cDNA clone encoding the 3CD proteinase (3CDP"') of poliovirus type 2 (Sabin), the precursor to proteinase 3CPr' and RNA polymerase 3DP"', was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3CPr' and 3DP'. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDPr' T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDPl°M (M designates the cleavage site mutant 3CDPr' T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDPmM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDP1°M did not have any detectable RNA polymerase activity, whereas 3D°"', separated from 3CDPIOM by gel filtration in the last step of purification, did. We conclude that 3CDP°can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3D"°' is needed to activate the 3D RNA polymerase.