Sequence of insulin effects on cytoskeletal and cytosolic phosphofructokinase, mitochondrial hexokinase, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels, and the antagonistic action of calmodulin inhibitors, in diaphragm muscle.

1. Time-curves of insulin effects on energy-producing systems in different cellular compartments of rat diaphragm muscle have revealed: (a) a rapid (within minutes) and transient stimulatory effect of insulin on cytoskeletal phosphofructokinase and aldolase and mitochondrial hexokinase. (b) A slower and consistent stimulatory effect on glucose 1,6-bisphosphate level, with concomitant gradual activation of cytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin. (c) Lactate concentration correlated with the stimulation of cytoskeletal and cytosolic glycolysis. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source of ATP for the rapid actions of insulin, whereas cytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.     

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Expression of bovine lung prostaglandin F synthase in Escherichia coli

The full-length bovine lung prostaglandin(PG) F synthase cDNA was constructed from partial cDNA clones and ligated into bacterial expression vector pUC8 to develop expression plasmid pUCPF1. This plasmid permitted the synthesis of bovine lung PGF synthase in Escherichia coli. The recombinant bacteria overproduced a 36-KDa protein that was recognized by anti-PGF synthase antibody, and the expressed protein was purified to apparent homogeneity. The expressed protein reduced not only carbonyl compounds including PGD2 and phenanthrenequinone but also PGH2; and the Km values for phenanthrenequinone, PGD2, and PGH2 of the expressed protein were 0.1, 100, and 8 microM, respectively, which are the same as those of the bovine lung PGF synthase. The protein produced PGF2 alpha from PGH2, and 9 alpha, 11 beta-PGF2 from PGD2 at different active sites. Moreover, the structure of the purified protein from Escherichia coli was essentially identical to that of the native enzyme in terms of C-terminal sequence, sulfhydryl groups, and CD spectra except that the nine amino acids provided by the lac Z' gene of the vector were fused to the N-terminus. These results indicate that the expressed protein is essentially identical to bovine lung PGF synthase. We confirmed that PGF synthase is a dual function enzyme catalyzing the reduction of PGH2 and PGD2 on a single enzyme and that it has one binding site for NADPH.     

Pseudomonas aeruginosa cytochrome oxidase. Product inhibition by low thermodynamic driving force

The steady-state kinetics of Pseudomonas aeruginosa cytochrome oxidase were studied. Reduced cytochrome c551 and azurin from the same bacteria were used as the electron-donating substrates, while dioxygen served as the electron acceptor. Oxidized cytochrome c551 and azurin exhibited product inhibition of the reaction. However, apo-azurin and azurin derivatives in which the copper was substituted by the redox-inert ions Ni2+, Co2+, Cd2+ and Zn2+, did not show any effect on the kinetics. These observations implied that complex formation between the substrates or the products and the enzyme is not a rate-limiting step and is not the cause for product inhibition. The integrated rate law for a reaction scheme in which we assumed that complex formation was not rate limiting was fitted to the complete reaction traces. The results suggested that it is the low thermodynamic driving force, expressed in the small differences in redox potential between the substrates and heme c of the enzyme, which cause the observed product inhibition.     

Membrane dynamics of lymphocyte interaction with antigen and tolerogen.

The principle of hapten-specific carrier-dependent immunologic tolerance was used to study the in vivo and in vitro interaction of lymphocyte membrane receptors with antigen (DNP-KLH) and tolerogen (DNP-MGG). Direct fluorescent techniques were employed to illustrate the binding of tolerogeu and antigen to the same population of lymphoid cells and the subsequent in vivo and in vitro events related to capping and regeneration of membrane receptors.