Site-specific phosphorylation by protein kinase C inhibits assembly-promoting activity of microtubule-associated protein 4

We have examined the phosphorylation of bovine microtubule-associated protein 4 (MAP4), formerly named MAP-U, by protein kinase C (PKC). When MAP4 was incubated with PKC, about 1 mol of phosphate was incorporated/mol of MAP4. Phosphorylation of MAP4 caused a remarkable decrease in the ability of the MAP to stimulate microtubule assembly. MAP4 consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain is subdivided into a Pro-rich region and an assembly-promoting (AP) sequence region containing four tandem repeats of AP sequence that is conserved in MAP4, MAP2, and tau [Aizawa et al. (1990) J. Biol. Chem. 265, 13849-13855]. In order to identify the site of MAP4 phosphorylated by PKC, a series of expressed MAP4 fragments was prepared and treated with the kinase. A fragment corresponding to the Pro-rich region (P fragment) was phosphorylated, while fragments corresponding to the projection domain and the AP sequence region were not. In addition, chymotryptic digestion of an authentic MAP4 prephosphorylated by PKC revealed that phosphate was incorporated almost exclusively into a 27-kDa fragment containing the carboxyl-terminal half of the Pro-rich region. We investigated the phosphorylation site in MAP4 using the P fragment and found that Ser815 was phosphorylated almost exclusively. We conclude that the phosphorylation of a single Ser residue in the Pro-rich region negatively regulates the assembly-promoting activity of MAP4.     

Simple in vivo bioassay without radioisotopes for recombinant human erythropoietins.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHuEPO) analogues was developed. The assay took four days, normal mice were used and radioactive compounds were not needed. EPO activity was measured by the increased number of some part of reticulocytes which increased specifically and dose-dependently by the injection of rHuEPO. They were considered to be mostly immature reticulocytes and were counted as the residual particles from blood cells after treatment with a hemolysing reagent. These particles could be counted by conventional automated microcell counters. The assay procedure was simple and easy. The sensitivity, reliability and reproducibility of this method were acceptable for routine in vivo bioassay of rHuEPOs. This method was economical, and can be used instead of the existing bioassays for rHuEPOs.     

Purification and characterization from bovine brain cytosol of proteins that regulate the GDP/GTP exchange reaction of smg p21s, ras p21-like GTP-binding proteins

Novel regulatory proteins for smg p21A and -B, ras p21-like GTP-binding proteins (G proteins) having the same putative effector domain as ras p21s, were purified to near homogeneity from bovine brain cytosol and characterized. These regulatory proteins, designated as GDP dissociation stimulator (GDS) 1 and -2, stimulated the dissociation of both [3H]GDP and [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) from smg p21s to the same extent. smg p21 GDS1 and -2 also stimulated the binding of [35S]GTP gamma S to the GDP-bound form of smg p21s but not that to the guanine nucleotide-free form. These actions of smg p21 GDS1 and -2 were specific for smg p21s and inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, rhoB p20, and smg p25A. Neither smg p21 GDS1 nor -2 stimulated the GTPase activity of smg p21s and by itself showed [35S]GTP gamma S-binding or GTPase activity. smg p21 GDS1 and -2 showed very similar physical and kinetic properties and were indistinguishable by peptide map analysis. The Mr values of smg p21 GDS1 and -2 were estimated to be about 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S values, indicating that smg p21 GDS1 and -2 are composed of a single polypeptide without a subunit structure. smg p21 GDS1 and -2 were distinguishable from GTPase activating proteins (GAPs) for the ras and rho proteins, and smg p21B, and GDP dissociation inhibitors for smg p25A and the rho proteins previously identified in bovine brain cytosol. These results indicate that bovine brain contains regulatory proteins for smg p21s that stimulate the dissociation of GDP from and thereby the subsequent binding of GTP to smg p21s in addition to smg p21 GAP. It is likely that the conversion from the GDP-bound inactive form of smg p21s to the GTP-bound active form is regulated by smg p21 GDS and that its reverse reaction is regulated by smg p21 GAP.     

Super-high-affinity binding site for [3H]diazepam in the presence of Co2+, Ni2+, Cu2+, OR Zn2+

Chloride salts of Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Cr³⁺, Mn²⁺, Fe²⁺, and Fe³⁺ had no effect on [³H]diazepam binding. Chloride salts of Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺ increased [³H]diazepam binding by 34 to 68% in a concentration-dependent fashion. Since these divalent cations potentiated the GABA-enhanced [³H]diazepam binding and the effect of each divalent cation was nearly additive with GABA, these cations probably act at a site different from the GABA recognition site in the benzodiazepine-receptor complex. Scatchard plots of [³H]diazepam binding without an effective divalent cation showed a single class of binding, with a Kd value of 5.3 mM. In the presence of 1 mM Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺, two distinct binding sites were evident with apparent Kd values of 1.0 nM and 5.7 nM. The higher-affinity binding was not detected in the absence of an effective divalent cation and is probably a novel, super-high-affinity binding site.     

Acid protease expression and regulation in the embryonic proventriculus of birds

Normal embryonic proventriculi and the heterospecific recombinants of proventricular endoderm and mesenchyme were transplanted onto the chorio-allantoic membrane, and electrophoretic patterns of acid proteases in the explants were analyzed. The results demonstrated that the 6-day chick and 5-day quail proventricular endoderm produces acid proteases according to its own genetic information even under the influence of heterospecific mesenchyme, and that the production of acid proteases is regulated by some humoral factors of the hosts.     

Purification and characterization of embryonic chicken pepsinogen, a unique pepsinogen with large molecular weight

An embryo-specific pepsinogen was isolated from the proventriculi of 15-day-old chicken embryos and purified by means of fractionation with ammonium sulfate, filtration on Sephadex G-100, and chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The properties of this pepsinogen and pepsin derived from it were compared with those of an adult-specific chicken pepsinogen and its pepsin. Though the optimal pH and alkali-stability were similar in the two pepsinogens, molecular weight, sensitivity to pepstatin, and antigenicity were quite different. Among the properties of this embryo-specific pepsinogen, the large molecular weight (56,000 for pepsinogen and 53,000 for pepsin) is especially noteworthy, since the molecular weights of the known pepsinogens of mammals and birds fall into the range of 35,000-48,000.     

In vivo degradation of rat globin messenger RNA during maturation of reticulocytes.

The degradation of globin mRNA in rat reticulocytes maturing in the peripheral blood was investigated. Poly(A) and non poly(A) portions of mRNA molecules were determined quantitatively by hybridization with radioactive poly(U) and complementary DNA, respectively. During the degradation of mRNA in vivo, it was shown that (1) globin mRNA and the bulk of RNA decrease in parallel, (2) the average chain length of poly(A) segments in the mRNA does not change, (3) the percentage of poly(A) (-) globin mRNA in total globin mRNA does not change, and (4) fragments of large molecular weight do not accumulate. Possible mechanisms of degradation of globin mRNA in the reticulocytes are discussed on the basis of these observations.