Proton-metal distance determination in cobalt(II) stellacyanin by 1H nuclear magnetic resonance relaxation measurements including Curie-spin effects: a proposed structure of the metal-binding region

1H nuclear magnetic resonance (1H NMR) experiments on Co(II)-substituted stellacyanin have been performed. Large paramagnetic hyperfine shifts are observed, the whole spectrum covering a range of 190 ppm. Experiments were mainly performed at 270 MHz from which temperature and pH* dependencies of the out-shifted resonances were reported, as well as determinations of the longitudinal (T1) and transverse (T2) relaxation times. These relaxation times are among other things, dependent on the individual proton-metal distance, and the aim of this work has been to determine these distances, by use of the Solomon-Bloembergen equations modified to include the so-called "Curie spin". The application of this method to a protein has not been reported earlier. Experiments were also performed at 100, 400, and 500 MHz in order to estimate the size of the Curie spin from the field dependence of the line widths. Furthermore, determination of the values for the rotational correlation time, tau r, and the effective magnetic moment, mu eff, was necessary for the present approach. With apostellacyanin, tau r was found to be (6.0 +/- 0.4) X 10-8 s. From the paramagnetic susceptibility of Co(II) stellacyanin, the value (4.53 +/- 0.03)beta was determined for mu eff. The proposed assignments of several paramagnetically out-shifted resonances. the proton-metal distances obtained, and the known peptide sequence of stellacyanin have allowed us to build a three-dimensional model of the metal site and its surrounding structure consistent with all the experimental data. It is revealed that both histidine ligands bind the metal with their 3-nitrogens. Also we find strong indications that a second sulfur atom is actually binding the metal, this being the long-sought-after fourth ligand. The model suggests that this sulfur belongs to Cys-59, which together with Cys-93 constitutes the disulfide bridge known to be present in the structure. A potential fifth ligand, an amide oxygen from Asn-47, is also found.     

Platelet receptor recognition domain on the gamma chain of human fibrinogen and its synthetic peptide analogues

We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767]. To study the correlation between the structure of this segment of the gamma chain and its reactivity toward receptors on ADP-activated human platelets, we designed a series of analogues containing replacements at 9 out of 12 positions. A double substitution of the normal His400-His401 sequence by Ala-Ala reduced the inhibitory potency of the dodecapeptide 3-fold. When Lys406 was replaced by Arg, the inhibitory potency of the dodecapeptide decreased 15 times. On the other hand, substitution of Ala408 with Arg increased the inhibitory potency of the dodecapeptide 6-fold. A drastic decrease in the reactivity of the dodecapeptide toward platelet receptors was observed when Val411 was replaced by leucine or cysteine or tyrosine. A 3-fold decrease in reactivity was noted when Val411 was substituted with phenylalanine. Amidation of the carboxy-terminal Val411 also produced a significant decrease in dodecapeptide reactivity. With seven residues (His400, His401, Leu402, Lys406, Gln407, Asp410, and Val411) preserved, substitution of the intervening five amino acids with nonpolar leucine or polar serine, increasing or decreasing the hydrophobicity of the dodecapeptide, reduced more than 16-fold its inhibitory potency. Rabbit antibody Fab fragments directed against the human fibrinogen gamma-chain peptide encompassing residues 385-411 inhibited 50% of 125I-fibrinogen binding at a 2:1 stoichiometry with regard to 125I-fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis, processing and half-life of P-glycoprotein in a human multidrug-resistant KB cell

The biosynthesis, processing, and half-life of the drug efflux pump, P-glycoprotein, were studied in human multidrug-resistant KB (KB-C2) cells selected for resistance to colchicine. An antibody directed against a synthetic oligopeptide corresponding to the amino-acid sequence (Glu-393-Lys-408) of P-glycoprotein from human mdr1 cDNA was prepared in rabbits. With immunoblotting and immunoprecipitation, we detected a 140-170 kDa protein in KB-C2 cells but not in parental sensitive KB cells. KB-C2 cells made a 125 kDa precursor that was slowly processed (t1/2 = 45 min) to the mature form of 140-150 kDa. The processing rate of P-glycoprotein was slower than that of low-density lipoprotein receptor. We detected another 160-180 kDa smear band, which might be a completely denatured form of P-glycoprotein. With immunoblotting, a minor band of high molecular mass (greater than 500 kDa) was also detected and this form increased after the cells were treated with chemical cross-linker, 1,5-difluoro-2,4-dinitrobenzene. The half-life of P-glycoprotein was long; no significant loss of P-glycoprotein was observed within 24 h after synthesis. Cells treated with tunicamycin produced a 120 kDa form of P-glycoprotein which was no longer processed but showed stability similar to that of the mature 140-150 kDa form. Agents that reverse multidrug resistance, phorbol ester and transport substrate did not affect the stability of P-glycoprotein.     

Lysophosphatidylcholine as an intermediate in phosphatidylcholine metabolism and glycerophosphocholine synthesis in cultured cells: an evaluation of the roles of 1-acyl- and 2-acyl-lysophosphatidylcholine

Previous studies in our laboratory have shown that the principal pathway of phosphatidylcholine (PtdCho) degradation in cultured mouse N1E-115 neuroblastoma, C6 rat glioma, primary rat brain glia and human fibroblasts is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----glycerophosphate plus choline (Morash, S.C. et al. (1988) Biochim. Biophys. Acta 961, 194-202). GroPCho is the first quantitatively major degradation product in this pathway, and could be formed by phospholipases A1 or A2, followed by lysophospholipase, or by a co-ordinated attack releasing both fatty acids by phospholipase B. The quality and quantities of lysoPtdCho present in cells reflect the nature of the initial hydrolysis step (A1 or A2), specificities of the lysophospholipases, and activities of acyltransferases that form PtdCho from lysoPtdCho. The present study was undertaken to elucidate the relative importance of these pathways by examining the fate of exogenous 1-acyl and 2-acyl-lysoPtdCho incubated with N1E-115 and C6 cells in culture. By fatty acid composition, endogenous lysoPtdCho was found to be mainly 1-acyl in both cell types based on a predominance of saturated acyl species; this suggested either preferential further deacylation or reacylation of 2-acyl-lysoPtdCho, or that 2-acyl-lysoPtdCho was not formed. Exogenous 1- and 2-acyl-lysoPtdCho specifically radiolabelled with choline and/or fatty acid were incubated either singly or as equimolar mixtures with cells. Cell association was rapid and not reversible by washing and both species were taken up at similar rates. The 2-acyl species was acylated to PtdCho faster than the 1-acyl species in both cell lines. Acylation of both lyso species was higher in C6 compared to N1E-115 cells. Hydrolysis of lysoPtdCho to GroPCho was higher in N1E-115 cells and with 1-acyl-lysoPtdCho. Transacylation between two molecules of lysoPtdCho was a minor pathway. These results document the variety and relative importance of reactions of lysoPtdCho metabolism; under similar conditions, 1- and 2-acyl-lysoPtdCho are handled differently. Both species turn over actively, but only the 1-acyl species accumulates while 2-acyl-lysoPtdCho is likely to be reacylated to form PtdCho.     

Molecular aspects of the removal of ferritin-bound iron by DL-dihydrolipoate

The removal of ferritin-bound iron by the physiologic dithiol DL-dihydrolipoate was studied over the pH range 5.5-9.0. A novel method was devised for the determination of iron removal, making it possible to study the actual release of iron from ferritin, regardless of the oxidation state or complexation form. The overall iron-removal process appears to depend upon a balance between the deprotonation of the dithiol and the protolytic dissolution of the iron core inside the ferritin molecule. The amount of iron removed at equilibrium increases with the pH, at any of the dihydrolipoate/ferritin iron ratios tested. The formation of the binuclear iron-dithiol complex [Fe2(dihydrolipoate)3]-3 is not strictly required for iron mobilization, but it seems to affect the efficiency of the dithiol in iron mobilization by providing a stable complexation form for the released iron outside the ferritin protein shell. Comparison of the release of ferritin-bound iron by free and immobilized dihydrolipoate indicates that mobility of the dithiol is mandatory for the removal process to take place.     

Influence of lactate on isoproterenol-induced lipolysis and beta-adrenoceptors distribution in human fat cells

The influence of lactate on human adipocytes lipolysis and the possible relationship between lactate-induced metabolic effects and beta-adrenoceptor binding sites were investigated. beta-sites were identified in membranes with (125I)-cyanopindolol and in intact cells with (125I)-cyanopindolol and (3H)-CGP 12177. Lactate reduced isoproterenol-induced lipolysis in a dose-response fashion and such inhibition became significant only at 16 mmol/l lactate. Exposure of human fat cells to 16 mmol/l lactate significantly reduced beta-adrenoceptors density on crude membranes. When the binding assay was performed on intact cells using (125I)-cyanopindolol at 37 degrees C, the radioligand identified the same number of receptors, regardless of the presence of lactate in the preincubation medium. When (3H)-CGP 12177 was used, it bound to about 35% less receptors in lactate pre-treated cells than in control. Seemingly, at 37 degrees C, because of its lipophilicity, (125I)-cyanopindolol can cross the plasma membrane and bind to intracellular sites whereas, (3H)-CGP 1277, due to its hydrophilicity, identifies surface receptors only. Thus, the present in vitro study provides evidence that high levels of lactate, similar to the concentrations usually achieved in overt lactic acidosis, are able per se to inhibit human lipolysis and to redistribute beta-adrenoceptors from cell surface to a domain not accessible to hydrophilic ligands.     

Ca2(+)-dependent glucose transport in chicken intestine

Transport of glucose was measured in the intestine of white leghorn layers in vivo using ligated upper small intestinal segment in the presence of Ca2+ and other ions either singly or in combination. Transport of glucose across the intestine was very significantly increased with Ca2+ than Na+, K+ and Po4(3-) individually, but when Ca2+ was combined with Na+, K+ and PO4(3-), the glucose absorption increased significantly over that achieved by Na+ ions alone. These data revealed that Ca2+ ions might be exerting the major influence on glucose transport processes of the chicken intestine.     

The rapid detection of direct-acting DNA mutagens by electrical impedence with a DNA repair-deficient strain of Escherichia coli

A differential killing assay using Escherichia coli WP2 (wild type) and WP67 (uvrA, polA) was combined with impedence microbiology to produce a rapid screening method for direct-acting mutagenic compounds. The assay showed that mitomycin C, N-nitroso guanidine, potassium dichromate, sodium azide and acridine orange were direct-acting mutagens. With this method results can be obtained within hours, as compared with two days for the standard Salmonella/microsome test.     

Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum

Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.     

Microgeographic distribution of immature Ixodes dammini ticks correlated with that of deer

In order to determine whether the small-scale distribution of immature Ixodes dammini Spielman et al. corresponds closely to the activity patterns of white-tailed deer, Odocoileus virginianus (Zimmerman), these relationships were examined in a site on Long Island, New York, U.S.A. We first determined the extent and temporal pattern of adult ticks feeding on deer by examining twenty-three resident deer tranquilized during September-December 1985. I. dammini adults infested deer throughout this fall period, most abundantly during October and November. With radio-telemetry collars attached to deer we determined the relative frequency that they occupied 0.25 ha quadrats of the study site. During the following summer, we examined white-footed mice, Peromyscus leucopus (Rafinesque), that inhabited these quadrats and removed immature ticks from each. 8975 larval and 163 nymphal I. dammini were removed from 208 mice trapped in forty-three such quadrats. The frequency of deer using these quadrats was positively correlated with both the number of larval and of nymphal ticks per mouse. These results suggest that risk of I. damminiborne zoonotic disease may be decreased by locally reducing deer density in sites that experience intense human activity.