Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme.

The mechanism for the generation of the Ca2+/calmodulin (CaM)-independent activity of calmodulin-dependent protein kinase II (CaM-kinase II) by autophosphorylation was studied by characterizing the autothiophosphorylated enzyme, which is resistant to hydrolysis. When CaM-kinase II was incubated with adenosine 5'-O-(thiotriphosphate) at 5 degrees C, the incorporation of thiophosphate into the enzyme occurred rapidly, reaching a maximum level within a few minutes, in parallel with increase in Ca2+/CaM-independent activity. The maximum level was 1 mol of thiophosphate per mol of subunit of the enzyme, and the thiophosphorylation occurred exclusively at Thr286 in the alpha subunit and Thr287 in the other subunits of the enzyme. These results, taken together, indicate that the autothiophosphorylation of Thr286/Thr287 of each subunit is involved in the generation of the Ca2+/CaM-independent activity. The activity of the autothiophosphorylated enzyme, when assayed in the presence of Ca2+/CaM, showed the same kinetic properties as did the Ca2+/CaM-dependent activity of the original non-phosphorylated enzyme, but when assayed in the absence of Ca2+/CaM, it showed the same Vmax as the Ca2+/CaM-dependent activity but higher Km values for protein substrates. Thus, the phosphorylation of Thr286/Thr287 of the subunit of the enzyme by autophosphorylation appears to not only enhance the affinity of its substrate-binding site for the protein substrate, although it is lower than that of the enzyme activated by the binding of CaM, but also convert the active site to the fully active state.     

Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity.

For Escherichia coli, growth on acetate requires the induction of the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase. The branch point between the glyoxylate bypass and the Krebs cycle is controlled by phosphorylation of isocitrate dehydrogenase (IDH), inhibiting that enzyme's activity and thus forcing isocitrate through the bypass. This phosphorylation cycle is catalyzed by a bifunctional enzyme, IDH kinase/phosphatase, which is encoded by aceK. We have employed random mutagenesis to isolate novel alleles of aceK. These alleles were detected by the loss of ability to complement an aceK null mutation. The products of one class of these alleles retain IDH kinase activity but have suffered reductions in IDH phosphatase activity by factors of 200 to 400. Selective loss of the phosphatase activity also appears to have occurred in vivo, since cells expressing these alleles exhibit phenotypes which are reminiscent of strains lacking IDH; these strains are auxotrophic for glutamate. Assays of cell-free extracts confirmed that this phenotype resulted from nearly quantitative phosphorylation of IDH. The availability of these novel alleles of aceK allowed us to assess the significance of the precise control which is a characteristic of the IDH phosphorylation cycle in vivo. The fractional phosphorylation of IDH was varied by controlled expression of one of the mutant alleles, aceK3, in a wild-type strain. Reduction of IDH activity to 50% of the wild-type level did not adversely affect growth on acetate. However, further reductions inhibited growth, and growth arrest occurred when the IDH activity fell to 15% of the wild-type level. Thus, although wild-type cells maintain a precise effective IDH activity during growth on acetate, this precision is not critical.     

Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity.

Mutations in aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase, which selectively inhibit phosphatase activity have been isolated. These mutations yield amino acid substitutions within a 113-residue region of this 578-residue protein. These mutations may define a regulatory domain of this protein.     

Identification of a novel transthyretin variant (Val30----Leu) associated with familial amyloidotic polyneuropathy.

A novel variant transthyretin which contains a leucine-for-valine substitution at position 30 was isolated and identified in the serum of a patient with familial amyloidotic polyneuropathy (FAP). The amino acid substitution was proven to result from a guanine-to-cytosine change at the first base of codon 30 located in exon 2 in the mutated transthyretin gene by restriction fragment length analysis on the amplified transthyretin gene using Cfr13 I. The study indicates that the point mutation of the transthyretin gene is a cause of the disorder.     

Initiation of transcription by T7 RNA polymerase as its natural promoters.

A kinetic assay has been developed to measure the strength of natural T7 promoters. By determining the rate of appearance of initiation products in the presence of constant concentrations of T7 RNA polymerase, an incomplete mixture of ribonucleoside triphosphates, and increasing promoter concentrations, a maximum rate of product formation (Vmax) and a promoter concentration giving half of the maximal activity ([P]Vmax/2) can be determined for any cloned T7 promoter. On supercoiled plasmids, it was found that the [P]Vmax/2 measured for the six promoters phi 1.1B, phi 1.3, phi 3.8, phi 6.5, phi 10, and phi 13 ranged from 3.4 +/- 1.1 to 12.0 +/- 2.4 nM while the Vmax values showed no significant trends. On plasmids that had been linearized by cleavage at a single site with a restriction endonuclease, the cloned T7 promoters assayed fell into two broad classes that appear to be characterized by the T7 class II and III promoters. Generally, the class II promoters required higher promoter concentrations to produce half of the maximum rates of initiation ([P]Vmax/2 values) than the class III promoters. The [P]Vmax/2 values for the class II promoters ranged from 20 +/- 2.7 to 23 +/- 3.6 nM, while the [P]Vmax/2 values for the class III promoters phi 10 and phi 13 were 13 +/- 1.6 nM and 7.8 +/- 1.4 nM. The one exception is the class III promoter phi 6.5 whose [P] Vmax/2 (17 +/- 5 nM) falls between the [P]Vmax/2 values of the class II promoters and the strong class III promoters. The Vmax values measured on linear templates are variable, but it appears that phi 10 is more active than the other five promoters.     

APGW-amide as an inhibitory neurotransmitter of Achatina fulica Ferussac

APGWamide (L-Ala-L-Pro-Gly-L-Trp-NH2) was purified from the ganglia of an African giant snail (Achatina fulica Ferussac). This peptide inhibited (hyperpolarized) more than half of the Achatina neurone types tested. This produced an outward current with the membrane conductance increase of RAPN (right anterior pallial neurone) under voltage clamp. The ED50 of the peptide was 6.2 x 10(-6) M (95% confidence limit: 5.0-7.8 x 10(-6) M) and the Emax was 3.9 +/- 0.2 nA. The effects were due to a membrane permeability increase to K+. The peptide is proposed as an inhibitory neurotransmitter of the Achatina neurones.     

The B subunit of cholera toxin enhances DNA synthesis in rat hepatocytes induced by insulin and epidermal growth factor.

The B subunit of cholera toxin, which binds to ganglioside GM1, enhanced DNA synthesis in rat hepatocytes in primary culture induced by insulin and/or epidermal growth factor. The effect was dose-dependent, and whole cholera toxin, activating adenylate cyclase, showed a higher effect than the B subunit alone. The B subunit acted additively with other agents that also increase cyclic AMP levels. A competitive antagonist of cyclic AMP could not suppress the effect of the B subunit completely. These data suggest that the effect is independent of the cyclic AMP signal pathway, and that GM1 plays a role in hepatocyte proliferation.     

Down-regulation of prostaglandin E2 receptors in regenerating rat liver and its physiological significance.

The properties of prostaglandin (PG) E2 receptors in regenerating liver were studied using rat hepatocytes in primary culture. The control cells possessed stereo-specific PGE2 receptors with Bmax and Kd values, at 4 degrees C, of 526 fmol/mg protein and 6.5 nM respectively. In cells from regenerating liver after 70% hepatectomy, Bmax was reduced to 42-43% that of the controls; Kd did not change. Administration of indomethacin before surgery prevented Bmax reduction. These results indicate that PGE2, produced during the regeneration process, evoked cellular events and regulated the density of its receptors.     

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.