EPR signals of cytochrome b558 purified from porcine neutrophils.

Cytochrome b558 of pig blood neutrophils was partially purified, and its EPR spectra were measured. The cytochrome b558 was solubilized from membranes with the detergent n-heptyl-beta-thioglucoside and purified by DEAE-Sepharose and heparin-Sepharose chromatographies. The small and large subunits of cytochrome b558 were detected on gel by immunoblotting. A solution of the purified, undenatured cytochrome b558 at 85-108 microM concentration was obtained. The concentrated cytochrome b558 showed an EPR signal at a g value of 3.26 with a bandwidth of 100 G at 10 K. Addition of 2 mM KCN had no effect on the low spin signal at g = 3.26 but caused disappearance of a minor high spin signal. The cyanide-insensitive signal at g = 3.26 disappeared completely on reduction with Na2S2O4. These results suggest that the g = 3.26 signal is characteristic of the low spin heme in cytochrome b558 of neutrophils.     

Expression of human soluble tissue factor in yeast and enzymatic properties of its complex with factor VIIa.

The extracellular domain of human tissue factor (TF, amino acids 1-217) was expressed in Saccharomyces cerevisiae, using the inducible yeast acid phosphatase promoter and the yeast invertase signal sequence to direct its secretion into the culture broth. Two active soluble forms sTF alpha (high molecular weight form) and sTF beta (low molecular weight form) were purified, the yield being approximately 10 and 1 mg/liter of culture supernatant, respectively. sTF alpha had an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis and contained more than 200 residues of mannose/mol of protein. sTF beta had an apparent molecular mass of 37 kDa and contained 22 residues of mannose/mol of protein. N-Glycosidase F treatments of both rTFs reduced the apparent molecular mass to 35 kDa. The amino-terminal sequences and amino acid compositions of sTF alpha and sTF beta were consistent with those deduced from the cDNA sequence, thereby indicating that the difference in molecular mass is caused by heterogeneity of oligosaccharide structures. Of these recombinant TFs, sTF beta enhanced factor VIIa-amidolytic activity 40-fold toward the chromogenic substrate and 147-fold toward the fluorogenic substrate, affecting mainly the kcat value. The enhancement was comparable with that of TF purified from human placenta. The TF-mediated enhancement of factor VIIa-amidolytic activity was inhibited by heparin-activated antithrombin III, forming a high molecular weight complex. As treatment of sTF beta with denaturants such as guanidine hydrochloride or urea led to a biphasic loss of the activity, the extracellular domain of TF probably consists of two discrete domains. This expression system provides a significant amount of the extracellular domain of TF so that studies of interactions with factor VII are feasible.     

M ring, S ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF.

The flagellar basal body, a major part of the flagellar motor, consists of a rod and four rings. When the fliF gene of Salmonella typhimurium, which was previously shown to code for the component protein of the M ring, was cloned and overexpressed in Escherichia coli, the FliF subunits formed ring structures in the cytoplasmic membrane. Electron microscopic observation of the purified ring structures revealed that each was composed of two adjacent rings and a short appendage extending from the center of the rings. Antibodies raised against the purified FliF protein decorated both the M and S rings of the intact basal body. We conclude that the FliF protein is the subunit protein of the M ring, and of the S ring and of part of the proximal rod of the flagellar basal body.     

The effect of alpha-tocopherol as an antioxidant on the oxidation of membrane protein thiols induced by free radicals generated in different sites

Azo compounds enable us to generate peroxyl radicals by thermal decomposition at a constant rate and at a desired site, that is, water-soluble compounds produce initiating radicals in an aqueous phase and lipid-soluble compounds initiate the oxidation within the membrane-lipid layer. Using these radicals generated in different sites, we oxidized red blood cell ghost membranes to study the relationships between alpha-tocopherol depletion, initiation of lipid peroxidation, and protein damage. When radicals were generated in the aqueous phase, the loss of membrane protein thiols was observed concurrently with the consumption of membrane tocopherol and after tocopherol was exhausted the peroxidation of membrane lipids occurred. On the other hand, when radicals were initiated within the lipid region, the oxidation of thiols and the formation of thiobarbituric acid-reactive substances were suppressed to give an induction period until tocopherol fell below a critical level. Our results indicate that the surface thiols of extrinsic proteins may compete with alpha-tocopherol for trapping aqueous radicals and spare tocopherol to some extent, whereas the oxidation of intrinsic buried thiols may commence due to lipid-derived radicals produced after tocopherol was consumed. In conclusion, alpha-tocopherol in the membrane can break the free radical chain efficiently to inhibit the lipid peroxidation. However, the effect of tocopherol on the inhibition of membrane protein damage, exhibited by the loss of thiols and the formation of high-molecular-weight proteins, would be different depending on the site of initial radical generation.     

A novel brain-specific mRNA encoding nuclear protein (necdin) expressed in neurally differentiated embryonal carcinoma cells

A novel DNA sequence has been isolated from a subtraction cDNA library of P19 embryonal carcinoma cells treated with retinoic acid which induces neural differentiation of the stem cells. The cDNA insert (4B) hybridized with a single 1.7 kb mRNA, whose abundance was markedly increased in P19 cells after retinoic acid treatment. The 1.7 kb mRNA was also expressed in the brain, but not in other non-neuronal tissues. A 1.6 kb cDNA insert (4BFL), which was cloned by screening another cDNA library with the 4B probe, encodes a novel protein sequence of 325 amino acids (Mr 36,831). The protein expressed in 4BFL-transfected COS cells was translocated into the nuclei as detected with antibodies against subsequences of the predicted protein. The antibodies stained the nuclei of neurally differentiated P19 cells but not of the undifferentiated stem cells. This novel mRNA encoding the nuclear protein, termed necdin, may represent a useful marker for the differentiation and development of brain cells.     

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.     

Effect of dehydration on thirst and drinking during immersion in men.

The mechanism for reduced voluntary water intake during water immersion was studied in eight men (19-25 yr of age) immersed to the neck while sitting for 3 h at 34.5 degrees C or in air at 28 degrees C when euhydrated (Eu-H2O and Eu-air, respectively) and hypohydrated (Hypo-H2O and Hypo-air) by 3.6% body weight loss. Thirst sensations (degree of thirst, mouth dryness and taste, drinking desirability, and stomach fullness) were similar at the beginning of Hypo-air and Hypo-H2O test periods. Initial drinking of tap water (15 degrees C) was 216 +/- 30 ml/7 min (P less than 0.05) with Hypo-air, decreased to 108 +/- 28 ml/7 min (P less than 0.05) with Hypo-H2O, and was 10-50 ml/10-30 min thereafter. Intake was less than 10 ml/10-30 min in Eu-air, and there was no drinking in Eu-H2O. Within the first 10 min of immersion, compared with Hypo-air findings, the significant reduction in drinking in the Hypo-H2O experiment was associated with unchanged plasma Na+, plasma osmolality, heart rates, and mean arterial pressures; the different responses were increased cardiac output, plasma volume, and atrial natriuretic peptides and decreased plasma renin activity and arginine vasopressin. Thus the extracellular pathway, as opposed to the osmotic pathway, appears to be the major mechanism for immersion-induced suppression of drinking.     

Genetic and behavioral analysis of flagellar switch mutants of Salmonella typhimurium.

At the interface between the sensory transduction system and the flagellar motor system of Salmonella typhimurium, the switch complex plays an important role in both sensory transduction and energy transduction. To examine the function of the switch complex, we isolated from 10 cheY mutants 500 pseudorevertants with a suppressor mutation in one of the three genes (fliG, fliM, and fliN) encoding the switch complex. Detailed mapping revealed that these suppressor mutations were localized to several segments of each switch gene, suggesting localization of functional sites on the switch complex. These switch mutations were introduced into the wild-type background and into a chemotaxis deletion background. Behavior of the pseudorevertants and their derivatives (1,500 strains in all) was observed by light microscopy. In the chemotaxis deletion background, about 70% of the switch mutants showed smooth swimming and the rest showed more or less tumbly swimming. There was some correlation between the mutational sites and the swimming patterns in the chemotaxis deletion background, suggesting that there is segregation of functional sites on the switch complex. The interaction of the switch complex with the chemotaxis protein, CheY, and the stochastic nature of switching in the absence of CheY are discussed.