Differential reactivities of lysines in calmodulin complexed to phosphatase

Calmodulin and calmodulin complexed with calcineurin phosphatase were trace labeled with [3H]acetic anhydride and the incorporation of [3H]acetate into each epsilon-amino lysine of calmodulin was measured. The relative reactivities of calmodulin lysines were higher in the presence of Ca2+ than in the presence of EGTA, and the order was: Lys-75 greater than Lys-94 greater than Lys-148 greater than or equal to Lys-77 greater than Lys-13 greater than or equal to Lys-21 greater than Lys-30. The changes in relative reactivity implied a change in conformation. When calmodulin was complexed with the phosphatase, Lys-21, Lys-77, and Lys-148 were most protected, implying that these residues are at or near the interaction sites or are conformationally perturbed by the interaction. Lys-30 and Lys-75 were slightly protected, lysine 13 showed no change, while lysine 94 significantly increased in reactivity. Comparison with results obtained from myosin light chain kinase using a similar technique (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232) reveals that calmodulin may interact with each of the two enzymes similarly at or near Lys-21, Lys-75, and Lys-148; one difference with phosphatase is that complex formation also involved Lys-77. These findings suggest that calmodulin interacts differently with its target enzymes.     

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.     

Purification and characterization of aquacobalamin reductase (NADPH) from Euglena gracilis

Euglena aquacobalamin reductase (NADPH: EC 1.6.99.-) was purified, and its subcellular distribution was studied to elucidate the mechanism of the conversion of hydroxocobalamin to 5'-deoxyadenosylcobalamin. The enzyme was found in the mitochondria. It was purified about 150-fold over the Euglena mitochondrial extract in a yield of 38%. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis. Spectra of the purified enzyme showed that it was a flavoprotein. The molecular weight of the enzyme was calculated to be 66,000 by Sephadex G-100 gel filtration and 65,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific to NADPH with an apparent Km of 43 microM and to hydroxocobalamin with an apparent Km of 55 microM. The enzyme did not require FAD or FMN as a cofactor. The optimum pH and temperature were 7.0 and 40 degrees C, respectively.     

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.     

Binding of linker histones to the core nucleosome

Binding of chicken erythrocyte linker histones H1/H5 to the core nucleosome has been studied. Histones H1/H5 bind very efficiently to the isolated core nucleosome in vitro. The binding of linker histones to the core nucleosome is associated with aggregation of the particles. Approximately one molecule of linker histone binds per core nucleosome in the aggregates, irrespective of the concentration of the linker histones and the salt used. Histone H5 shows greater binding affinity to the core nucleosome as compared to H1. The carboxyl-terminal fragment of the linker histones binds strongly to the core nucleosome while the binding of the central globular domain is weak. Each core nucleosome is capable of binding two molecules of carboxyl-terminal fragment of linker histone. The core nucleosome containing one molecule of carboxyl-terminal fragment of linker histone requires higher salt concentration for aggregation while the core nucleosome containing two molecules of carboxyl-terminal fragment of linker histone can self-associate even at lower salt concentrations. On the basis of these results we are proposing a novel mechanism for the condensation of chromatin by linker histones and other related phenomena.     

Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.     

A cellular 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line. Purification and characterization

A cellular binding protein for 3,3',5-triiodo-L-thyronine (T3) was solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) from A431 human epidermoid carcinoma cells. The binding activity is T3 specific. Analysis of the equilibrium binding data indicated that the binding protein has one class of binding sites for T3 with a Kd of (17 +/- 3) nM and Bmax of (1.8 +/- 0.6) pmol/50 micrograms of protein. The pH optimum for binding is 6.8. The T3 binding protein elutes from Sephadex G-200 in an included peak which has a Stokes radius of 40 A and sediments on glycerol gradients at 3.7 S. By affinity labeling with [3,5-125I]thyroxine a protein with a molecular weight of 58,000 was specifically labeled. Its isoelectric point was determined to be 7.1, which is different from the reported pIs of other thyroid hormone binding proteins. p58 was successively purified to apparent homogeneity by chromatography on Sephadex G-200, QAE-Sephadex, SP-Sephadex, and hydroxylapatite. Approximately 50 micrograms of purified protein was obtained from 2.5 X 10(9) cells with a yield of 1.1%. The purified protein retains its binding activity. The specific binding activity is enriched by approximately 1000-fold. With the availability of a purified protein with T3 binding activity, it becomes possible to study its cellular function.     

Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes

Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of the relationship between regional variations in cell shape and sites of motive force generation

Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed.     

Photoaffinity labeling of T4 bacteriophage 32 protein

With a view toward the determination of nucleic acid binding domains and sites on nucleic acid helix-destabilizing (single strand-specific) proteins (HDPs), we have studied the interactions of the copolymer polynucleotide photoaffinity label, poly(adenylic, 8-azidoadenylic acid), (poly(A,8-N3A] with the T4 bacteriophage HDP, 32 protein. Poly(A,8-N3A) quenched the intrinsic tryptophan fluorescence of 32 protein in a manner similar to that observed with other polynucleotides, and the effect could be reversed by addition of sufficient NaCl. The binding affinity and site size of this noncovalent interaction of poly(A,8-N3A) with 32 protein are similar to the values obtained for poly(A) and this protein. When [3H]poly(A,8-N3A)/32 protein mixtures were irradiated at 254 nm, fluorescence quenching was not reversed by NaCl, suggesting that the label was covalently bound to the protein. Mixtures of photolabel and protein subjected to short periods of irradiation (generally 1 min, 2000 erg mm-2) formed high molecular weight complexes, which when electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels were radioactive and stained with Coomassie Blue R. Under the same conditions, [3H]poly(A) failed to label 32 protein. The radioactivity of [3H]poly(A,8-N3A)-labeled complexes subjected to micrococcal nuclease after irradiation was seen to migrate just behind the free 32 protein monomer on SDS-polyacrylamide gels, indicating that portions of the photolabel not in direct contact with protein were accessible to this enzyme. By several criteria, we conclude that 32 protein was photolabeled specifically at its single-stranded nucleic acid binding site. Single-stranded nucleic acids with affinities for protein greater than that of poly(A,8-N3A) effectively inhibited photolabeling. The [NaCl] dependence of photolabeling monitored on SDS gels paralleled the NaCl reversal of (noncovalent) poly(A,8-N3A)-32 protein binding. Photolabeling reached a plateau after 1-2 min. The formation of high molecular weight complexes with increasing [poly(A,8-N3A)] paralleled the disappearance of free protein on SDS gels, and reached a saturation level of about 75% labeling. Several chromatographic procedures appear to be useful for the separation of the photolabeled complexes from free protein and photolabel. Limited trypsin hydrolysis of photolabeled 32 protein indicated that all the label was within the central ("III") portion of the protein. This approach should have general applicability to the identification of nucleic acid binding sites on helix-destabilizing proteins.