Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase

We have recently characterized the major hydroperoxide-reducing enzyme of human plasma as a glutathione peroxidase (Maddipati, K. R., Gasparski, C., and Marnett, L. J. (1987) Arch. Biochem. Biophys. 254, 9-17). We now report the purification and kinetic characterization of this enzyme. The purification steps involved ammonium sulfate precipitation, hydrophobic interaction chromatography on phenyl-Sepharose, anion exchange chromatography, and gel filtration. The purified peroxidase has a specific activity of 26-29 mumol/min/mg with hydrogen peroxide as substrate. The human plasma glutathione peroxidase is a tetramer of identical subunits of 21.5 kDa molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is different from human erythrocyte glutathione peroxidase. The plasma peroxidase is a selenoprotein containing one selenium per subunit. Unlike several other glutathione peroxidases this enzyme exhibits saturation kinetics with respect to glutathione (Km for glutathione = 4.3 mM). The peroxidase exhibits high affinity for hydroperoxides with Km values ranging from 2.3 microM for 13-hydroperoxy-9,11-octadecadienoic acid to 13.3 microM for hydrogen peroxide at saturating glutathione concentration. These kinetic parameters are suggestive of the potential of human plasma glutathione peroxidase as an important regulator of plasma hydroperoxide levels.     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

Phorbol esters and calcium ionophores inhibit internalization and accelerate recycling of receptors in macrophages

Exposure of macrophages to phorbol esters or the calcium ionophore A23187 increases the number of several surface receptors due to recruitment of receptors from internal pools (Buys, S. S., Keogh, E. A., and Kaplan, J. (1984) Cell 38, 569-576). We have examined the mechanism by which these agents increase surface receptor number. Cells which were preloaded with either fluid phase or receptor-mediated ligands did not lose ligand following exposure to ionophore or phorbol ester. The rate of movement of ligands to the lysosome was also unaffected. These results suggest that A23187 does not induce the fusion of ligand-containing compartments with the cell surface. Ionophore treatment did, however, produce a severalfold increase in the rate at which unoccupied receptors reappear on the cell surface. These results suggest that the compartment of receptors affected by the ionophore formed subsequent to the dissociation of ligand from receptor. The altered rate of receptor reappearance was transitory (90 s), and the increase in receptor number was subsequently maintained by a decrease in the rate of internalization. Changes in the rate of receptor internalization did not correlate with changes in the rate of fluid phase pinocytosis, suggesting that the effect on receptor internalization was selective.     

Cell surface dynamics of neutrophils stimulated with phorbol esters or retinoids

Neutrophils undergo rapid morphological changes as well as metabolic perturbations when stimulated with certain phorbol esters. Stimulated cells initially exhibit pronounced projections emanating from the cell bodies, followed by rounding of the cells, reduction in granule number, and the appearance of intracellular vesicles. We show these vesicles to be derived, at least in part, from the plasmalemma. The experimental approach involved labeling stimulated and unstimulated cells with native ferritin and cationized ferritin, along with the cytochemical localization of ecto-5'-nucleotidase. The labeling patterns of the vesicles indicate that these structures are involved in both phorbol ester-stimulated adsorptive and fluid-phase endocytosis. Neutrophils stimulated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibit two distinct rates of superoxide release in which the second, prolonged level is approximately 50% of the initial rate. All-trans-retinal, which we have recently shown to stimulate O2- release but not granule exocytosis or cell vesiculation, induces a single prolonged rate of maximal O2- release. Neutrophils treated with both all-trans-retinal and TPA exhibit only a single sustained rate of maximal O2- release similar to that observed with all-trans-retinal alone. Moreover, treatment of cells with all-trans-retinal blocks the vesiculation of neutrophils induced by TPA in a dose-dependent manner. This observation provides a possible explanation for the differences in the kinetics of superoxide release.     

Erythroid anion transporter assembly is mediated by a developmentally regulated recruitment onto a preassembled membrane cytoskeleton

Analysis of the expression and assembly of the anion transporter by metabolic pulse-chase and steady-state protein and RNA measurements reveals that the extent of association of band 3 with the membrane cytoskeleton varies during chicken embryonic development. Pulse-chase studies have indicated that band 3 polypeptides do not associate with the membrane cytoskeleton until they have been transported to the plasma membrane. At this time, band 3 polypeptides are slowly recruited, over a period of hours, onto a preassembled membrane cytoskeletal network and the extent of this cytoskeletal assembly is developmentally regulated. Only 3% of the band 3 polypeptides are cytoskeletal-associated in 4-d erythroid cells vs. 93% in 10-d erythroid cells and 36% in 15-d erythroid cells. This observed variation appears to be regulated primarily at the level of recruitment onto the membrane cytoskeleton rather than by different transport kinetics to the membrane or differential turnover of the soluble and insoluble polypeptides and is not dependent upon the lineage or stage of differentiation of the erythroid cells. Steady-state protein and RNA analyses indicate that the low levels of cytoskeletal band 3 very early in development most likely result from limiting amounts of ankyrin and protein 4.1, the membrane cytoskeletal binding sites for band 3. As embryonic development proceeds, ankyrin and protein 4.1 levels increase with a concurrent rise in the level of cytoskeletal band 3 until, on day 10 of development, virtually all of the band 3 polypeptides are cytoskeletal bound. After day 10, the levels of total and cytoskeletal band 3 decline, whereas ankyrin and protein 4.1 continue to accumulate until day 18, indicating that the cytoskeletal association of band 3 is not regulated solely by the availability of membrane cytoskeletal binding sites at later stages of development. Thus, multiple mechanisms appear to regulate the recruitment of band 3 onto the erythroid membrane cytoskeleton during chicken embryonic development.     

Small-angle neutron-scattering and electron microscope studies of the chicken liver fatty acid synthase

A structural model for the chicken liver fatty acid synthase is proposed based on electron microscope and small-angle neutron-scattering studies of the enzyme. The model has the overall appearance of two side by side cylinders with dimensions of 160 X 146 X 73 A, with each subunit 160 A in length and 73 A in diameter. The model was constructed by dividing each cylinder into three domains having lengths of 32, 82, and 46 A, with the domain structures in the two subunits being related to each other by a dyad axis. The model is consistent with chemical cross-linking studies which indicated that the subunits are arranged in a head to tail fashion. The cross-linking studies further showed that the beta-ketoacyl synthase active site contains a cysteine and a pantetheine residue from adjacent subunits. It is proposed that the domains which catalyze the addition of C2 units from malonate to the growing fatty acid chain lie in the crevice between the two subunits and that the two independent sets of fatty acid-synthesizing centers lie on the major axis of the model on opposite ends of the molecular dyad.     

Control of the adipogenic differentiation of 3T3-F442A cells by retinoic acid, dexamethasone, and insulin: a topographic analysis

Differentiation of 3T3-F442A adipocytes, monitored by accumulation of neutral lipid and by using the sensitive marker glycerophosphate dehydrogenase, is inhibited by incubation of confluent 3T3-F442A fibroblasts in medium containing retinoic acid or dexamethasone. When added together, dexamethasone (0.25 microM) potentiates about 50-fold the inhibitory effect of retinoic acid (10 microM). Insulin cannot counteract the retinoic acid blockade; however, it can overcome the inhibition of differentiation elicited by dexamethasone. These differential effects of insulin are used for characterizing the adipose conversion cycle. We describe cell culture conditions where terminal differentiation of 3T3-F442A preadipocytes is achieved by low, physiological levels of insulin. They include the switch from a high-serum medium containing isobutyl methyl xanthine and dexamethasone to a serum-free, hormone-supplemented medium. The data reported establish the existence of two successive states for commitment to adipogenic differentiation: a first commitment point (CA) to differentiation which requires serum adipogenic factors, and a second commitment point (CH) controlled by lipogenic hormones, namely insulin, after which terminal maturation can resume. We demonstrate that retinoic acid can prevent and interrupt differentiation by blocking the cells within the early differentiation phase.     

Phytic acid. A natural antioxidant

The catalysis by iron of radical formation and subsequent oxidative damage has been well documented. Although many iron-chelating agents potentiate reactive oxygen formation and lipid peroxidation, phytic acid (abundant in edible legumes, cereals, and seeds) forms an iron chelate which greatly accelerates Fe2+-mediated oxygen reduction yet blocks iron-driven hydroxyl radical generation and suppresses lipid peroxidation. Furthermore, high concentrations of phytic acid prevent browning and putrefaction of various fruits and vegetables by inhibiting polyphenol oxidase. These observations indicate an important antioxidant function for phytate in seeds during dormancy and suggest that phytate may be a substitute for presently employed preservatives, many of which pose potential health hazards.     

Behavioural and physiological adaptations to a burrowing lifestyle in the snake blenny, Lumpenus lampretaeformis, and the red band-fish, Cepola rubescens

Observations have been made on the mode of burrow construction in the snake blenny, Lumpenus lampretaeformis , under laboratory conditions. It appears that head probing and lateral oscillations of the body are principally responsible for the excavation of the burrow which is completed within 24 h. The burrow structure has been analysed in detail, showing a mean depth of 7.2 cm with a maximum observed length of 73 cm, with most systems between 20 and 35 cm in length. Initially linear burrows with two openings are usually provided with a small side tunnel, giving the system a characteristic Y-shape.
Burrow irrigation was investigated for the first time in L. lampretaeformis. The mean duration of burrow irrigation, by flexions of the tail of the fish, was 21 s with over 13 min h⁻¹ spent in irrigating the burrow. The mean water displacement per irrigation period was 3.1 ml. The PO ₂ and PCO ₂ were measured in both surface water and within the burrow system of L. lampretaeformis. Surface water values for PO ₂ were high (> 150 Torr) and PCO ₂ low (<0.4 Torr). Hypoxic and hypercapnic conditions were measured in the burrow system itself, with PO ₂ values ranging between 57 and 129 Torr and PCO ₂ rising to > 1.3 Torr in some burrows.
A comparative study of Cepola rubescens burrows indicated similar surface water PO ₂ and PCO ₂ values as in L. lampretaeformis. Burrow water PO ₂ values ranged between 60 and 94 Torr, with PCO ₂ values as high as 1.5 Torr being recorded. These results are discussed in relation to the adaptation of both species to a burrowing lifestyle.     

Autostimulation of dihydrostreptomycin uptake in Bacillus subtilis

In Bacillus subtilis it was shown that the membrane potential (delta psi) has to reach a threshold value of -180 to -190 mV for efficient uptake of dihydrostreptomycin to occur. The magnitude of delta psi is raised above this threshold, and dihydrostreptomycin uptake greatly enhanced, not only by dihydrostreptomycin itself (autostimulation) and by other miscoding aminoglycoside antibiotics, but also by puromycin, bacitracin and N,N'-dicyclohexylcarbodiimide. Stimulation of uptake by dihydrostreptomycin or puromycin was dependent on a specific interference with ongoing protein synthesis. Thus, chloramphenicol prevented the stimulating effect of puromycin by lowering the magnitude of delta psi. Although normally severely antagonizing streptomycin accumulation, K+, as well as spermidine and putrescine, which are known to stabilize ribosomes, consequently enhanced autostimulation of dihydrostreptomycin uptake in a K+-retention mutant with impaired protein synthesis. It is suggested that miscoding aminoglycosides and puromycin both enhance dihydrostreptomycin uptake by increasing delta psi due to ion fluxes, which are themselves caused by a dramatic stimulation of intracellular proteolysis of faulty proteins.