Faculty Bibliography

The murine macrophage-like cell line J774.16 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin (CaM)-binding protein (CaMBP) which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and peritoneal macrophages elicited with concanavalin A, lipopolysaccharide, proteose peptone, or Bacillus Calmette-Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein, as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed with the use of a rabbit antiserum raised to the partially purified CaM-binding protein and [125I]CaM covalently cross-linked to the principal CaM-binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein, suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared, and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of Mr 50,000 to 60,000 was isolated. The protein could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property, plus the sensitivity of the protein to endoglycosidase F, led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization, and evidence for glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for Ca2+-CaM

A calmodulin-binding protein is present in extracts of the macrophage-like mouse cell line J774 and in extracts of thioglycollate-stimulated mouse peritoneal macrophages; it is deficient in variants of J774 resistant to trifluoperazine and in resident peritoneal macrophages. The calmodulin-binding protein [CaMBP (J7)0.5] was purified from J774 and resolved from endogenous cyclic nucleotide phosphodiesterase and protein kinase activities. The protein has an apparent native Mr of 125,000-150,000 and binds calmodulin in a calcium-dependent manner with a Kd of 20 nM. It inhibits the ability of calmodulin to activate phosphodiesterase. Its sedimentation constant in glycerol gradients containing calmodulin was dependent upon the relative concentrations of calmodulin and the calmodulin-binding protein

Two forms of the extracellular cyclic nucleotide phosphodiesterase (EC 3.1.4.17) of Dictyostelium discoideum have been purified. One species has a molecular weight of about 55,000 measured by gel filtration and has been purified to apparent homogeneity. This monomeric form of the enzyme can be resolved by isoelectrofocusing into 4 bands with isoelectric points between 7.5 and 9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands with molecular weights of 50,000 and 48,000. The second form of the enzyme has been partially purified and has a higher molecular weight (150,000-200,000) and an isoelectric point of about 5. The high molecular weight form of the enzyme is converted to the low molecular weight monomer by isoelectrofocusing in the presence of 6 M urea. Under these conditions the isoelectric point is shifted to that of the monomeric form of the enzyme. The two species are immunologically indistinguishable after urea treatment, and react identically with the cyclic nucleotide phosphodiesterase inhibitor (see the accompanying paper: Franke, J., and Kessin, R. H. (1981) J. Biol. Chem. 256, 7628-7637). The two forms have the same Km and show equal sensitivity to a number of ions, chelators, and low molecular weight cyclic nucleotide phosphodiesterase inhibitors. Tryptic maps of the purified monomeric enzyme and the urea dissociated high molecular weight form revealed no differences

The extracellular adenosine 3',5'-cyclic monophosphate phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) produced by Dictyostelium discoideum has two kinetic forms. The free enzyme has a Km of approximately 10 microM. The second form is the result of a complex formed with a heat-stable inhibitor and has a Km in the millimolar range. Treating the enzyme-inhibitor complex with dithiothreitol stimulated enzyme activity 20- to 100-fold and changed in Km from millimolar to micromolar. Dithiothreitol inactivated the inhibitor. Reconstituting purified enzyme with excess inhibitor returned the Km to the millimolar range. Under conditions known to inhibit the production of extracellular inhibitor or in mutants that lack it, extracellular phosphodiesterase activity was already high and could not be increased by dithiothreitol. The phosphodiesterase and inhibitor sedimented at 6 S and 3 S, respectively; the enzyme-inhibitor complex sedimented at 6.7 S