Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.     

Biosynthesis of plasma-membrane proteins during myogenesis of skeletal muscle in vitro.

1. Surface labelling of plasma-membrane proteins with 125I, catalysed by lactoperoxidase, and radioactive l-fucose incorporation into glycoprotein were used as plasma-membrane markers for skeletal-muscle cells in culture. 2. Plasma membranes were prepared at various stages of myogenesis in vitro and rates of synthesis and accumulation of proteins in the membranes were compared. 3. Increased synthesis and accumulation of a protein of apparent mol.wt. 70000 occurred in the plasma-membrane fraction concomitant with the onset of myoblast fusion. 4. In cultures in which fusion of myoblasts was inhibited by 5'-bromo-2-deoxyuridine, synthesis and accumulation of the protein of apparent mol.wt. 70000 was selectively inhibited. 5. It is suggested the protein of apparent mol.wt. 70000 may be involved in the process of myoblast fusion.     

Specific protein synthesis in isolated rat testis leydig cells. Influence of luteinizing hormone and cycloheximide.

The effect of luteinizing hormone (luteotropin) and cycloheximide on specific protein synthesis in rat testis Leydig cells has been investigated. Proteins were labelled with either I114C]leucine, [3H]leucine or [35S]methionine during incubation with Leydig-cell suspensions in vitro. Total protein was extracted from the cells and separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. No detectable increase in the synthesis of specific proteins could be observed after incubation of Leydig cells with luteinizing hormone for up to 1 h. However, after a 2h incubation period, an increase in [35S]methionine incorporation was observed in a protein with an apparent mol.wt. of 21000 (referred to as 'protein 21"). When, after labelling of this protein with [35S]-methionine, Leydig cells were incubated for another 30min with cycloheximide, no decrease in radioactivity of this protein band was observed, indicating that it does not have a short half-life. However, another protein band was detected, which after incubation with cycloheximide disappeared rapidly, the reaction following first-order kinetics, with a half-life of about 11 min. This protein, with an apparent mol.wt. of 33000 (referred to as "protein 33"), was found to be located in the particulate fraction of the Leydig cell, and could not be demonstrated in other rat testis-cell types or blood cells. No effect of luteinizing hormone on molecular weight, subcellular localization or half-life of protein 33 was observed. A possible role for protein 33 and protein 21 in the mechanism of action of luteinizing hormone on testosterone production of Leydig cells is discussed.     

Two types of receptor for insulin-like growth factors in mammalian brain.

Two types of receptor for insulin-like growth factors (IGFs) have been identified on adult rat and human brain plasma membranes by competitive binding assay, affinity labelling, receptor phosphorylation and interaction with antibodies to insulin receptors. The type I IGF receptor consists of two species of subunits: alpha-subunits (mol. wt. approximately 115 000), which bind IGF I and IGF II with almost equal affinity and beta-subunits (mol. wt. approximately 94 000), the phosphorylation of which is stimulated by IGFs. The alpha-subunits of type I IGF receptors in brain and other tissues differ significantly (mol. wt. approximately 115 000 versus 130 000), whereas the beta-subunits are identical (mol. wt. approximately 94 000). The type II IGF receptor in brain is a monomer (mol. wt. approximately 250 000) like that in other tissues. Two antibodies to insulin receptors, B2 and B9, interact with type I but not with type II IGF receptors. B2 is more potent than B9 in inhibiting IGF binding and in immunoprecipitating type I IGF receptors, in contrast to their almost equal effects on insulin receptors. This pattern is characteristic for IGF receptors in other cells. The presence of two types of IGF receptor in mammalian brain suggests a physiological role of IGFs in regulation of nerve cell function and growth. Since IGF II, but not IGF I, is present in human brain, we propose that IGF II interacts with both types of IGF receptor to induce its biological actions.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia VI. Endogenous protein substrates for in vitro and in vivo phosphorylation in cilia and ciliary membranes

The endogenous protein kinases of isolated Paramecium tetraurelia cilia phosphorylated approximately 30 ciliary polypeptides in vitro. Labeling with [gamma-32P]ATP was not proportional to the amount of each protein in cilia; some minor polypeptides (e.g., 67,000 and 180,000 mol wt) were more heavily labeled than some major polypeptides. Certain of the endogenous substrates for protein kinase were localized in the ciliary membrane (130,000, 86,000, 67,000, and 45,000 mol wt); others were found in axonemes or in both fractions. With cilia from bacterized cultures in the undefined Cerophyl medium, the labeling of specific endogenous phosphate acceptors was altered by pH, cyclic AMP, and cyclic GMP, but the labeling pattern was not affected by the presence of Na+ or K+ (15 mM), Ba++ (5 mM), Ca++ (10(-5) or 10(-4) M), or EGTA. Very similar results were obtained with cilia from cells grown axenically in a semidefined medium; the molecular weights and the extent of phosphorylation of the phosphopolypeptides were comparable to those of cilia from bacterized Cerophyl cultures, although no significant cyclic nucleotide effects were observed in the axenic cilia. Most of the phosphopolypeptides labeled in vitro also turned over rapidly in vitro. The phosphoprotein phosphatase responsible for turnover was partially inhibited by 5 mM NaF. The pattern of ciliary polypeptides labeled in vivo was similar to that observed in the in vitro experiments, although the relative intensities of labeling differed. Six behavioral mutants of Paramecium, known to have defects in the excitable membrane that regulates the ciliary beat, showed normal patterns of ciliary protein phosphorylation in vitro, with and without added cyclic nucleotides, at both pH 6.0 and pH 8.0. The mutants also had apparently normal phosphoprotein phosphatase. The Paranoiac A mutant, however, showed a reduction in cyclic GMP-stimulated protein kinase activity.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

Apparent molecular weight of substance P/neurokinin-1 receptors determined using a photoaffinity labelled probe, [(3'-125I) D-Tyro, (4'-N3)Phe8, Nle11]-substance P

A photoreactive derivative, [(3'-125I) D-Tyro, (4'-N3)Phe8, Nle11]-substance P (SP) was prepared and iodinated using carrier-free [125I] to determine the apparent molecular weight of one sub-type of neurokinin (NK) receptor, the SP/NK-1 type. The unlabelled analogue competed for [3H]-SP sites with an IC50 of 10 nM. The radioactive photoprobe (KD approximately 0.17 nM, Bmax = 15.6 fmol/mg protein) was used to photoaffinity label membranes prepared from rat brain. Autoradiographs revealed that a single band with an apparent molecular weight of 46,000 daltons was specifically labelled. This labelling was inhibited by non-radioactive SP in a concentration-dependent manner (1.0 nM-0.1 mM) suggesting that the observed labelling represents the SP/NK-1 receptor type.     

Occurrence of protein phosphorylation in various bacterial species.

1. The occurrence of protein phosphorylation in Escherichia coli B, Bacillus megaterium, Bacillus sphaericus, Pseudomonas fluorescens and Arthrobacter S1-55, was investigated by means of both in vivo and in vitro experiments. 2. In each bacterial species the presence of several phosphorylated proteins was evidenced by gel electrophoresis and autoradiography after either labelling of growing cells with [32P]orthophosphate or incubating cellular extracts with radioactive ATP. 3. The analysis of the radioactive moiety of proteins showed that they contained phosphoserine, phosphothreonine and phosphotyrosine. These three phosphoamino acids were found in varying proportions depending both on the bacterial species and, within the same species, on the conditions used for labelling proteins, either in vivo or in vitro. 4. By measuring the effect of cyclic nucleotides on the extent of protein phosphorylation in cellular extracts, it was observed that, in all five bacterial species analyzed, neither cyclic AMP nor cyclic GMP was able to stimulate the activity of protein kinases. 5. All together these results bring evidence that protein phosphorylation catalyzed by protein kinases is a post-translational modification widespread among prokaryotes.     

Phosphorylation of hormone sensitive phosphodiesterase in isolated adipocytes.

Cyclic AMP phosphodiesterase in rat adipocytes is stimulated by insulin and also by agents that increase cyclic AMP levels. When the enzyme is immunoprecipitated from a solubilised microsomal preparation from adipocytes prelabelled with radioactive phosphate and separated on SDS polyacrylamide gels, label is found in a protein band at the expected Mr for adipose tissue phosphodiesterase. Treatment of the adipocytes with isoproterenol or methyl isobutylxanthine increased the labelling of this band. Insulin alone had no effect on its labelling but did decrease the incorporation of label caused by isoproterenol.     

Structural characterization of the 9-10S estradiol receptor from calf uterus

An affinity chromatography-based method has been developed for estrogen receptor isolation which requires the inclusion of sodium molybdate in purification buffers for maintaining the large 9-10S form of the receptor. The protein products obtained from affinity chromatography of calf uterine receptor extracts or from extracts presaturated with estradiol have been analyzed by gel electrophoresis under denaturing conditions. Major estrogen sensitive proteins were peptides with Mr approximately 90,000, 65,000 and 50,000. Two additional proteins (60,000 and 53,000) of lower abundance and with demonstrated estrogen sensitivity were also observed. Affinity labeling with [3H]tamoxifen aziridine identified the Mr 65,000 protein as the estrogen receptor and suggested that the Mr 60,000, 53,000 and 50,000 peptide components were derived proteolytically from this parent unit. The 90,000 mol. wt component was readily dissociated from heparin-sepharose immobilized estrogen receptor by elution with low salt buffers without molybdate. Peptide mapping experiments indicated that the 90,000 mol. wt component was not related to the Mr 65,000 and 50,000 estrogen receptors, but confirmed the smaller binding unit to be a proteolytic fragment of the 65,000 mol. wt receptor. The results suggest that the 90K protein associates non-covalently with the Mr 65,000 estrogen binding unit as a nonhormone binding component of the 9-10S receptor.     

Dunce mutants of Drosophila melanogaster: mutants defective in the cyclic AMP phosphodiesterase enzyme system

The cyclic AMP and cyclic GMP phosphodiesterase activities present in flies of six mutant strains of the dunce gene and in the parent wild-type strains are characterized. All of the mutants exhibit aberrant cyclic AMP metabolism. The mutant strains dunceM14, dunceM11, and dunceML appear to be amorphic, because they completely lack the cAMP-specific phosphodiesterase normally present in adult flies. These strains exhibit extremely high levels of cAMP. The mutant strains dunce1, dunce2, and dunceCK are hypomorphic and exhibit reduced levels of the cAMP-specific phosphodiesterase. These strains exhibit less marked increases in cAMP content compared with the three amorphic strains. The dunce2 strain possesses a residual enzyme activity that exhibits anomalous kinetics compared with those of the normal enzyme. The possibility that the dunce locus is the structural gene for the cAMP-specific phosphodiesterase is discussed.     

Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.

The non-stimulated (basal) adenylate cyclase from bovine brain cortical membranes was purified 10 000-fold to apparent homogeneity by Lubrol PX extraction and two cycles of affinity chromatography on forskolin-agarose. The final product appears as one major band (mol. wt. 115 000) on SDS-polyacrylamide gels. Further identification was achieved by affinity cross-linking using Gs (stimulatory GTP-binding protein) that was [32P]ADP-ribosylated by cholera-toxin/[32P]NAD: cross-linking with disuccinimidyl suberate gave products with mol. wts. of 160 000, approximately 270 000 and higher. The distribution of these products was dependent on the concentration of cross-linker, suggesting aggregation of two or more adenylate cyclase complexes. In contrast, photo-affinity cross-linking with 4-azidobenzoyl-[32P]Gs yielded a single product with a mol. wt. of 160 000. Purified adenylate cyclase was completely unresponsive towards stimulators (GTP-analogs, NaF) acting via Gs suggesting that this component was removed during purification. On the other hand, stimulation by forskolin and by added activated Gs was preserved but to a smaller degree as compared with the crude enzyme. In contrast, the stimulation of Ca2+/calmodulin was only marginal. Purified adenylate cyclase reversibly bound to wheat germ agglutinin-Sepharose. This suggests that bovine brain adenylate cyclase is a glycoprotein.     

Labelling by axonal transport of myelin-associated proteins in the rabbit visual pathway.

After intraocular injections of [3H]leucine, six regions of the visual pathway of adult rabbit were used to study the spatio-temporal pattern of the slow anterograde axonal transport of radioactive proteins associated with the particulate fraction, the water-soluble fraction and the myelin fraction. Unlike other fractions, myelin-associated labelled proteins represented a time-constant (for a given region) percentage of total tissue radioactivity. This percentage increased from the first half to the second half of the optic nerve and remained high in the chiasma and tract. The peak specific radioactivity of myelin decreased in the same direction. Myelin proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the labelling patterns obtained in different regions and at different survival times were compared. At the peak of myelin radioactivity of a given region the label was typically associated with four protein bands, L1, L2, L3 and L4, of 40000, 44000, 62000, and 68000 mol.wts. respectively. The basic protein, the proteolipid protein and the W1 component (mol.wt. 51000-53000) of the Wolfgram proteins were not significantly labelled. The radioactivity associated with the W2 component (mol.wt 60000) of the Wolfgram proteins could be derived from the closely migrating L3 component. At shorter survival times no clear labelling pattern could be detected. At longer survival times radioactivity was almost totally localized around band L3. The results presented underline the importance of choosing appropriate experimental conditions to obtain a consistent labelling pattern of myelin-associated proteins and to investigate the possible mechanism responsible for this phenomenon.     

Identification of immunodominant Trypanosoma musculi antigens recognized by monoclonal antibody and curative immunoglobulin G2a antibody.

Trypanosoma musculi obtained from normal or irradiated (900 rad) hosts or from in vitro cultures were lysed and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar protein banding patterns with a molecular weight (mol. wt) range from 34 to 68 kDa were observed between the two bloodstream forms. In comparison, lysates of cultured parasites showed a unique banding pattern of antigens within the same mol. wt range. Western blot of bloodstream form lysates, probed with immune plasma (IP), revealed a wide range of parasite proteins. However, when probed with the IgG2a-enriched fraction of IP, a major band of approximately 66 kDa was detected on the blot. Several bands of higher mol. wt were also observed. When anti-T. musculi monoclonal antibodies were used to probe the blot, the 66 kDa protein was again recognized. Using indirect fluorescence, live bloodstream form parasites were analysed by flow cytometry and the p66 protein was determined to be a surface molecule. Finally, lysates of 35S-methionine-labelled trypanosomes were immunoprecipitated with Sepharose linked anti-T. musculi monoclonal antibodies and the eluted ligand analysed by SDS-PAGE and autoradiographed. The 66 kDa band was identified, therefore confirming that this protein was of parasite origin.     

Isolation of the Drosophila melanogaster dunce chromosomal region and recombinational mapping of dunce sequences with restriction site polymorphisms as genetic markers.

Using the method of chromosomal walking, we have isolated a contiguous region of the Drosophila melanogaster X chromosome which corresponds to salivary gland chromosome bands 3C12 to 3D4. This five-band region contains approximately 100 kilobases of DNA, including those sequences comprising dunce, a gene which functions in memory and cyclic nucleotide metabolism. Genome blots of DNA from flies carrying several different chromosomal aberrations with breakpoints in the region have been probed with the isolated clones to map the breakpoints on the cloned DNA and to delimit dunce sequences. This has localized dunce to a 50-kilobase region. In addition, we have searched this 50-kilobase region for restriction site polymorphisms between X chromosomes from different Drosophila strains by genome blotting experiments, and we have followed the segregation of detected polymorphisms and dunce alleles after meiotic recombination. The data map one dunce mutation between two polymorphisms located 10 to 12 kilobases apart.     

Thermodynamic stabilities of yeast iso-1-cytochromes c having amino acid substitutions for lysine 32

Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.     

Protein phosphorylation in normal and neoplastic development. Phosphorylation of proteins endogenous to foetal tissues and tumours.

The abilities of proteins endogenous to normal and neoplastic tissues to serve as substrates in a protein-phosphorylation reaction in vitro were compared. After the tissue extracts were incubated with [gamma-32P]ATP, the phosphorylated proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the dried gels were subjected to radioautography. Considerable incorporation of 32P into a protein of mol.wt. 135000 was observed with extracts from foetal tissues and tumours, but only minimal incorporation into this protein occurred when extracts from adult tissues were used. The ability of this protein to become phosphorylated in vitro may be related to cell proliferation. When ascites cells were incubated with [32P]Pi, one of the major phosphoproteins migrated on sodium dodecyl suphate/polyacrylamide gels at mol.wt. 135000, suggesting that this protein can be phosphorylated both in intact cells and broken-cell preparations. A protein of mol.wt. 87000 was highly phosphorylatable in extracts from solid tumours, but was not phosphorylated in extracts from ascites tumours, foetal or adult tissues. The phosphorylation pattern of these two proteins can thus distinguish solid neoplasms and normal adult tissues from ascites tumours and from foetal tissues. A protein of mol.wt. 49000, which was the most labelled protein in adult tissues, was also one of the major phosphoproteins in foetal and neoplastic tissues. Numerous mechanisms are postulated to explain how the extent of 32P incorporation into a protein could vary as a function of biological state.     

A gene encoding for an alpha-amylase from thermophilic Bacillus sp. strain TS-23 and its expression in Escherichia coli

An alpha-amylase gene from Bacillus sp. strain TS-23 was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli. A cell fractionation experiment revealed that approximately 60% of the amylase activity was in the periplasmic space. Analysis and activity staining of the concentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a mol. wt of approximately 65,000. The amylase gene (amyA) consisted of an open reading frame of 1,845 bp encoding a protein of 613 amino acids with a calculated mol. wt of 69,543. The predicted amino acid sequence showed high homology with Bacillus species, E. coli and Salmonella typhimurium alpha-amylases. Deletion of 96 amino acids from the C-terminal portion of the amylase did not result in the loss of amylolytic activity. The truncated amylase, deletion of the first 50 amino acids from the N-terminus, was overexpressed in E. coli system and refolded to yield an activable enzyme.     

Beta-adrenergic agents increase the phosphorylation of phosphofructokinase in isolated rat epididymal white adipose tissue.

Pieces of rat epididymal adipose tissue were incubated in medium containing [32P]phosphate for 2 h to achieve steady-state labelling of intracellular phosphoproteins and then with or without hormones for a further 15 min. Phosphofructokinase was rapidly isolated from the tissue by use of either Blue Dextran-Sepharose chromatography or immunoprecipitation with antisera raised against phosphofructokinase purified from rat interscapular brown adipose tissue. Similar extents of incorporation of 32P into phosphofructokinase were measured by both techniques. Exposure of the tissue to adrenaline or the beta-agonist isoprenaline increased phosphorylation by about 5-fold (to about 1.4 mol of phosphate/mol of enzyme tetramer). No change in phosphorylation was detected with the alpha-agonist phenylephrine, but exposure to insulin resulted in an approx. 2-fold increase. The increased phosphorylation observed with isoprenaline was found to be associated with a decrease in the apparent Ka for fructose 2,6-bisphosphate similar to that observed on phosphorylation of phosphofructokinase purified from rat epididymal white adipose tissue with the catalytic subunit of cyclic AMP-dependent protein kinase. These results support the view [Sale & Denton (1985) Biochem. J. 232, 897-904] that an increase in cyclic AMP in adipose tissue may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.     

An electrophoretic study of endogenous phosphorylation in vitro of the polypeptides of microsomal membrane fractions of mouse liver

1. The patterns of phosphopolypeptides produced by endogenous phosphorylation in vitro of rough- and smooth-membrane fractions of the microsomal fraction of mouse liver were studied by radioautographic analysis of sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms. 2. A minimum of 17 polypeptides of both rough- and smooth-microsomal-membrane fractions were phosphorylated by using [gamma-(32)P]-ATP as the phosphate donor; only minor differences in phosphorylation pattern between the two membrane fractions were detected. 3. Phosphorylation in vitro by [gamma-(32)P]ATP was markedly stimulated by Mg(2+), but not by cyclic AMP, cyclic GMP or Ca(2+). The phosphorylation of certain polypeptides was preferentially stimulated by Mg(2+). Addition of cyclic AMP resulted in a decrease in the amount of (32)P detected in one polypeptide of mol.wt. approx. 56000, present in both the rough- and smooth-membrane fractions. 4. [gamma-(32)P]GTP was found to be a relatively poor donor of (32)P as compared with [gamma-(32)P]ATP. However, incubation of rough- and smooth-membrane fractions with this compound resulted in the phosphorylation of one polypeptide of mol.wt. approx. 96000 that was scarcely or not at all phosphorylated by [gamma-(32)P]ATP. 5. Under the conditions of incubation used, appreciable incorporation of (32)P from [gamma-(32)P]ATP occurred into products migrating at the front of the electrophoretograms; these products were identified as being principally comprised of 1-phosphatidylinositol 4-phosphate. Incorporation of (32)P into this lipid was also markedly stimulated by Mg(2+). 6. The overall results show that a considerable number of polypeptides of the rough- and smooth-microsomal-membrane fractions of mouse liver may be phosphorylated in vitro and indicate that the enzymes responsible are principally non-cyclic AMP-dependent protein kinases.