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Biomedical subjects

C B Klee

Publications and source records attributed to C B Klee.

At least 73 records · Page 4Linked to original sources
PubMed1984

The effect of oncomodulin on cAMP phosphodiesterase activity.

Oncomodulin was purified from Morris rat hepatoma according to the procedure of Durkin, J.P., Brewer, L.M. and MacManus, J.P. (1983) Cancer Res. 43, 5390-5394. The preparation, in general, had the properties and amino acid composition of the material which they described. However, we were unable to confirm the reported stimulation of cyclic nucleotide phosphodiesterase under conditions where calmodulin gave the usual stimulation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Agonist and antagonist properties of calmodulin fragments.

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

The structure of the B subunit of calcineurin.

The complete primary structure of the B subunit of calcineurin (protein phosphatase 2B) has been determined by automated sequence analysis. The protein consists of a single polypeptide chain of 168 residues, relative molecular mass 19200. The structure shows 35% identity with the sequence of calmodulin and 29% with troponin C. Homology is mainly confined to the regions of the four putative Ca2+-binding loops. The results demonstrate that the B subunit is a new member of this family of Ca2+-binding proteins. The N-terminal glycine residue is blocked with the C14-saturated fatty acid myristic acid and the first four residues are very similar to those of the catalytic subunit of cyclic-AMP-dependent protein kinase which also contains a myristoyl blocking group.

Amino Acid Sequence↗
PubMed1984

Purification and characterization of a novel Ca2+-binding protein (CBP-18) from bovine brain.

A novel Ca2+-binding protein (CBP-18) has been identified and purified from bovine brain. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein consists of a single band of apparent Mr 18,000 in the presence of Ca2+ or 20,000 in the presence of EGTA. CBP-18 contains one high affinity Ca2+-binding site, measured at 10(-5) M Ca2+ in the presence of 1 mM Mg2+ and 0.1 M K+. The amino acid composition and UV absorption spectrum distinguish CBP-18 from other Ca2+-binding proteins identified in brain. The protein has an extinction coefficient epsilon 1% 279 nm = 4.9 and contains 1 tryptophan/mol, 5 tyrosines/mol, and no trimethyllysine. CBP-18 does not interact with or activate calmodulin-stimulated phosphodiesterase. However, available evidence suggests that CBP-18 binds to other component(s) present in the brain extract in a Ca2+-dependent manner.

Amino Acids↗
PubMed1984

CAPP-calmodulin: a potent competitive inhibitor of calmodulin actions.

A covalent adduct of norchlorpromazine (CAPP) and calmodulin is a very potent antagonist of calmodulin activation of several enzymes. The phenothiazine-calmodulin complex (CAPP-calmodulin) acts as a pure antagonist with phosphodiesterase and myosin kinase or a partial agonist with the phosphoprotein phosphatase, calcineurin. Because of its potency and the selectivity inherent to its calmodulin moiety, CAPP-calmodulin should be a uniquely useful probe of calmodulin actions.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Calcineurin: a member of a family of calmodulin-stimulated protein phosphatases.

Calcineurin, a major calmodulin-binding protein of brain, is a heterodimer composed of a 61,000 Mr calmodulin-binding subunit, calcineurin A, and a 19,000 Mr Ca2+-binding subunit, calcineurin B. The discovery of a calmodulin-regulated protein phosphatase in rabbit skeletal muscle with a similar subunit structure led to the identification of calcineurin as a protein phosphatase (AA Stewart, TS Ingebritsen, A Manalan, CB Klee, P Cohen (1982) FEBS Lett 137:80-84). Using rabbit polyclonal antibodies to bovine brain calcineurin, both subunits of calcineurin can be identified in crude homogenates of bovine brain by an immunoblotting technique. In crude homogenates of bovine skeletal and cardiac muscle, a 59,000-61,000 Mr doublet and a 15,000 Mr species (the electrophoretic mobility of calcineurin B) are also detected by this technique. The cross-reactivity of these species with antibodies to brain calcineurin indicates antigenic similarity between the muscle proteins and calcineurin, and suggests the existence of a family of structurally related calmodulin-stimulated protein phosphatases. Like calcineurin, the 61,000 Mr subunits in skeletal and cardiac muscle bind calmodulin and are detected in crude tissue extracts by 125I-calmodulin gel overlay. Thus, both the 125I-calmodulin gel overlay method and the immunoblotting technique are useful in screening crude preparations, in which detection of calmodulin-stimulated protein phosphatase activity may be complicated by the many phosphatases present.

Animals↗
PubMed1984

Calmodulin.

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Animals↗
PubMed1983

Calcium ion dependent covalent modification of calmodulin with norchlorpromazine isothiocyanate.

Calmodulin forms a covalent, one to one, complex with 3H-labeled norchlorpromazine isothiocyanate. Complex formation was monitored by high-performance liquid chromatography using a CN reverse-phase column which resolves calmodulin, the calmodulin-norchlorpromazine adduct, and norchlorpromazine isothiocyanate. Formation of the adduct requires Ca2+ and is not observed with norchlorpromazine. The one to one calmodulin-norchlorpromazine complex does not activate phosphodiesterase but can interact with the enzyme and competitively inhibit its stimulation by calmodulin. High concentrations of trifluoperazine inhibit whereas low concentrations stimulate complex formation. This apparent potentiation of the interaction of calmodulin with norchlorpromazine by another phenothiazine suggests that calmodulin contains at least two phenothiazine binding sites and that the binding of phenothiazine to calmodulin is cooperative.

Animals↗
PubMed1983

Activation of calcineurin by limited proteolysis.

Calcineurin, a heterodimer of calcineurin B, a 19,000 Mr Ca2+-binding subunit, and calcineurin A, a 61,000 Mr calmodulin-binding subunit, was previously proposed to be a calmodulin- and Ca2+-regulated protein phosphatase. Like other calmodulin-stimulated enzymes, calcineurin can be activated and rendered calmodulin- and Ca2+-independent by limited proteolysis. By glycerol gradient centrifugation, the native enzyme has a s20,w of 4.5 S in EGTA and 5 S in the presence of Ca2+-calmodulin. Under the same conditions, the s20,w of the trypsin-activated enzyme (4.3 S) is not affected by Ca2+ and calmodulin. The trypsin-treated enzyme is a heterodimer of calcineurin B and a 45,000 Mr fragment of calcineurin A that has lost its ability to interact with calmodulin. Phosphatase activity sediments with calcineurin or its proteolytic fragments, providing further evidence that calcineurin is indeed a protein phosphatase. Calmodulin protects calcineurin against tryptic digestion; proteolysis occurs more slowly, yielding fragments with Mr 57,000, 55,000, and 54,000 that have preserved their ability to interact with calmodulin. After trypsin treatment in the presence of calmodulin, the protein phosphatase activity of calcineurin is still regulated by calmodulin. Prolonged trypsin treatment in the presence of calmodulin produces a 46,000 Mr fragment. Unlike the fragments generated in the absence of calmodulin, this 46,000 Mr fragment still interacts weakly with calmodulin. Thus, calcineurin, like other calmodulin-regulated enzymes, consists of a catalytic domain resistant to proteolysis and a calmodulin-binding regulatory domain susceptible to protease action in the absence of calmodulin but not in its presence. In the absence of calmodulin, the regulatory domain exerts an inhibitory effect on the catalytic domain; the inhibition is relieved upon calmodulin binding to or tryptic degradation of the regulatory domain.

Animals↗
PubMed1983

Ultrastructural immunocytochemical localization of calmodulin in cultured cells.

Using an antibody prepared against performic acid-treated calmodulin, we have localized calmodulin in cultured fibroblastic cells by immunofluorescence and immunoelectron microscopy. In interphase cells, calmodulin was found to be diffusely distributed throughout the cytosol. An increased amount of calmodulin was found in the pericentriolar region of interphase cells. No significant aggregation of calmodulin was found in association with microfilaments, peripheral cytoplasmic microtubules or clathrin-coated structures. Calmodulin was present in moderate amounts in microvilli, ruffles, and zeiotic blebs of the cell surface. In motitic cells, calmodulin was found concentrated in the pericentriolar region, and appeared to concentrate along radiating spindle microtubules proximal to the centrioles. Redistribution of calmodulin was seen between early and late telophase, in which the pericentriolar pattern of calmodulin in early telophase shifted to an aggregation on the intercellular bridge, with a large part of the midbody portion of the bridge being devoid of calmodulin. These results show that calmodulin is distributed throughout the cytosol, but is markedly concentrated in the region of the microtubule organizing center in interphase cells, as well as in elements of the mitotic spindle apparatus. This distribution suggests that calmodulin has a regulatory role in the organization and function of microtubules during interphase, as well as during mitosis.

Actins↗
PubMed1982

Identification of the NH2-terminal blocking group of calcineurin B as myristic acid.

The NH2-terminal blocking group of the Ca2+-binding B-subunit of calcineurin (protein phosphatase-2B) has been identified as myristic acid by fast atom bombardment mass spectrometry and gas chromatography. The sequence, myristyl-Gly-Asn-Glu-Ala-, is very similar to that of the catalytic subunit of cyclic AMP-dependent protein kinase, the only other protein known to contain this fatty acid. This finding, and the elution of all myristyl peptides at 57% acetonitrile on reverse phase HPLC, may facilitate the identification of other proteins with this blocking group.

Amino Acids↗
PubMed1982

Characterization of polynucleotide phosphorylase from Micrococcus luteus and isolation of the 13,000 base poly(A) product of the polymerization reaction.

A new purification procedure for polynucleotide phosphorylase from freeze-dried Micrococcus luteus cells gives approximately 20% yield of nearly homogeneous, primer-independent enzyme which is free of nucleic acid. The physicochemical properties of M. luteus polynucleotide phosphorylase are similar to those previously described for the enzyme from Escherichia coli in terms of Mr, subunit structure, and amino acid composition. The purified enzyme appears to be a trimer composed of three identical subunits (Mr 92,000), but it probably does not exist as such in the cell. Ferguson plot analyses of enzyme in cell extracts indicate that prior to purification the enzyme exists in oligomeric forms characterized by both higher charge and greater Mr. Changes in size and charge of oligomers which occur during purification are probably due to the dissociation of proteins and/or nucleic acids. Dissociation of the oligomers is achieved by dilution and electrophoresis, but reassociation does not occur after concentration. The poly(A) product of the initial polymerization stages migrates as a single band on both nondenaturing and urea-agarose gels. It is 13,000 +/- 2,000 nucleotides long, as measured by electron microscopy, and 8,000 nucleotides long by gel electrophoretic analysis. This poly(A) product remains bound to the enzyme after synthesis, yet can be easily obtained free of protein by proteinase K digestion.

Amino Acid Sequence↗
PubMed1982

Calmodulin.

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Amino Acid Sequence↗
PubMed1981

Calmodulin activates the isolated catalytic unit of brain adenylate cyclase.

The catalytic and guanine nucleotide regulatory (G/F) units of solubilized bovine brain adenylate cyclase were separated by gel filtration as described by Strittmatter, S., and Neer, E. J. ((1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6344-6348). The isolated catalytic unit is activated 4 +/- 1-fold (n = 11) by pure bovine brain calmodulin and is stabilized by calmodulin against thermal inactivation. The separated G/F unit can be freed of endogenous calmodulin by gel filtration in buffer containing 1 mM EDTA and no divalent cations. The calmodulin-free G/F unit still activates the catalytic unit. Re-addition of calmodulin does not affect the rate or extent of activation of the G/F unit by guanosine 5'-(beta, gamma-imino)triphosphate. The activation by calmodulin and the G/F unit together is additive, not synergistic. These studies show that calmodulin interacts with the adenylate cyclase catalytic unit but does not seem to affect the function of the G/F unit.

Adenylyl Cyclases↗