cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

Microtubule destabilization by cdc2/H1 histone kinase: phosphorylation of a "pro-rich region" in the microtubule-binding domain of MAP-4.

Microtubule-associated protein-4 (MAP-4), a major MAP in proliferating cells, consists of a microtubule-binding domain and a projection domain protruding from the microtubule wall. The former contains a Pro-rich region and an assembly-promoting (AP) sequence region which is common to the neuron-specific MAPs, MAP-2 and tau1. In this paper, we describe the phosphorylation of the Pro-rich region of MAP-4 and the suppression of its assembly-promoting activity by cdc2/H1 histone kinase. This inactivation of MAP-4 may cause disassembly of the interphase microtubular network at the end of the G2 phase of the cell cycle.     

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.     

Purification of a K(+)-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system

A K(+)-channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K(+)-channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca(2+)-ATPase antibody did not recognize the protein. The characteristics of the K(+)-channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.     

Evidence for in vivo synthesis of thiamin triphosphate by cytosolic adenylate kinase in chicken skeletal muscle

We showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP) and ADP in addition to ATP formation from ADP [Shikata, H. et al. (1989) Biochem. Int. 18, 933-942]. To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching. Thiamin phosphate metabolism in chicken skeletal muscle was also studied. i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle. Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle. ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained. Both AK1 activity and TTP-synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum. iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant. In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of carbohydrate-mediated complement activation by the serum mannan-binding protein

Serum mannan-binding protein (S-MBP), a lectin specific for mannose and N-acetylglucosamine, was documented to activate complement through the classical pathway. In this study, we examined the mechanism that initiates this activation. By a passive hemolysis test using sheep erythrocytes coated with yeast mannan, the activation of complement by human S-MBP was shown to proceed in the absence of C1q. The following binding studies using 125I-labeled C1r2s2 and C1s indicated that the activated form of C1r2s2 bound to S-MBP located on the surface of the cells with high affinity. The binding of C1s to the cell-bound S-MBP require the presence of C1r, suggesting that C1r2s2 binds to S-MBP through C1r. The activation of C1s from a proenzyme to a protease was mediated by cell-bound S-MBP in the presence of C1r and the activated protease remained associated with the cells and was not released into the medium. The activation of complement with S-MBP was a solid phase event and did not proceed in a fluid phase. On the basis of these results, it was concluded that S-MBP is responsible for the initiation of carbohydrate-mediated complement activation as C1q does in immune complex-mediated complement activation.     

Two distinct cDNAs for human IMP dehydrogenase

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.     

Analysis of the ratio of alpha- to beta-globin and globin messenger ribonucleic acid content of fractionated rabbit erythroid bone-marrow cells.

The ratio of alpha- to beta-globin mRNA was measured by hybridization of a constant amount of highly purified alpha- or beta-globin cDNA (complementary DNA) with increasing amounts of RNA in the range up to 20% cDNA hybridization, where an essentially linear reaction is obtained. Statistical analysis indicates that the ratio of alpha- to beta-globin can be measured within a maximal error of +/- 0.3 and in most cases is better than +/- 0.15. Under these conditions there is no significant deviation from the ratio of 1.3 in the alpha- to beta-globin mRNA ratio of RNA isolated from erythroid cells rich in pronormoblasts through to reticulocytes. If the ratio of alpha- to beta-globin mRNA exceeded 1.7 or was less than 0.9 in pronormoblasts, it would be detected in these experiments. The overall globin mRNA content increases to a maximal value in the fractions rich in basophilic normoblasts of 30,000--50,000 molecules/cell. However, the accuracy of these determinations is not as great as for the ratio determinations, and no significant deviations were seen except in the cells rich in pronormoblasts, which contained less globin mRNA than the later stages.     

A change in membrane microviscosity of mouse neuroblastoma cells in association with morphological differentiation

Changes in membrane microviscosity as well as in membrane constituents of mouse neuroblastoma clone N-18 were studied in association with neurite formation. The membrane microviscosity studied by fluorescence technique increased with the formation of neurites. The concomitant increase increase in the ratio of cholesterol to phospholipids was also observed.