ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning from Vicia faba mesophyll and epidermis: Characterization and influence of abscisic acid and fusicoccin

Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K⁺ -stimulated, mg²⁺-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, K_m for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). V_max -480 nmol (mg protein)¹ min¹ (whole leaf) and 510 nmol (mg protein)¹ min¹ (epidermis), I₅₀ (Na₃,VO₄) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO₃(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca²⁺ inhibited the ATPase [I₅₀, C²⁺: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca²⁺ inhibited the ATPase [I₅₀, C²⁺ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.     

Recognition of vesicular lipoproteins by the apolipoprotein B,E receptor of cultured fibroblasts

Vesicular lipoproteins (e.g., lipoprotein-X) are found in plasma in cholestasis or following infusion of Intralipid or phospholipid. To investigate the metabolism of vesicular lipoproteins, we isolated them from the plasma of subjects with cholestasis or following chronic or single Intralipid infusion. Cholestasis and chronic Intralipid therapy were found to be associated with elevated plasma concentrations of apoE, as determined by radioimmunoassay. Vesicular lipoproteins purified from each of the three types of plasma contained apoE, as well as other proteins. In cholestasis, in which levels of apoE were up to five times normal, a major portion of the plasma apoE was on vesicular lipoproteins. Normalized for apoE content, all preparations of vesicular lipoproteins displaced 125I-labeled LDL from apoB,E receptors of cultured fibroblasts identically. This displacement was inhibited by monoclonal antibodies that block receptor binding of apoE. Vesicular lipoproteins containing 125I-labeled apoE were internalized and degraded by fibroblasts. Different preparations caused small losses or gains of cellular cholesterol, with appropriate stimulation or suppression of apoB,E receptors. Thus, vesicular lipoproteins contain apoE, and apoE mediates their interaction with the apoB,E receptor. Our results suggest that the catabolism of cholesterol-rich vesicular lipoproteins, formed during cholestasis or following infusions of Intralipid or phospholipid, may be receptor-mediated.     

Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium

Summary Phage Mud1 cts (Ap^r lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.     

Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii

Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.     

Metabolism of viprostol — A synthetic vasolidator PGE2 analog

Metabolic studies were done with ¹⁴C-Viprostol (I) administered by various routes (I.V., oral and topical) to six animal species and to man. Total radioactivity and metabolic profiles were analyzed in plasma, tissues and excreta. The main metabolites were isolated and identified by capillary GC/MS.Plasma and urinary metabolic profiles were qualitatively similar across species, with two major metabolic reactions being predominant: rapid hydrolysis to the pharmacologically active free acid (II)_ and oxidation of the alpha-chain to dinor and tetranor acids (III, IV). In the monkey and man, reduction of the 9-keto group lead to PGF₂ type metabolites (VI–VIII). In the rat, omega oxidation of the beta-chain occurred as well, resulting in the formation of dicarboxylic acids (V).     

Effects of granuloma modulation induced by regulatory-T-lymphocyte activity on angiotensin II/III production by granuloma macrophages in murine schistosomiasis mansoni

Angiotensins are produced by granuloma macrophages in murine Schistosoma mansoni. During the course of infection, granuloma undergo a T-cell-dependent process called modulation in which their maximal size decreases. This study was undertaken to establish whether angiotensin production by granuloma macrophages is altered by immunoregulatory lymphocytes. Granuloma macrophages from modulated lesions released and contained more angiotensin II/III (AII/III) and less angiotensin I (AI) than those from the acute infection. Captopril, a specific angiotensin-converting-enzyme (ACE) inhibitor, appreciably decreased AII/III produced by macrophages from modulated granulomas. Adoptive transfer of splenic T lymphocytes from chronically infected donors into acutely infected recipients altered angiotensin production by the granuloma macrophages in a manner similar to that seen in modulated lesions. However, no difference was detected in the capacity of granuloma macrophages from acutely or chronically infected mice to metabolize 125I-AI or -AII added to cell cultures. Similarly, captopril did not alter the metabolism of exogenously administrated angiotensins. These findings suggest that regulatory T lymphocytes influence the metabolism by granuloma macrophages of endogenously produced angiotensins at least in part by induction of macrophage ACE activity. However, the degradation of extracellular AI and AII may result from the activity of enzymes other than ACE which are not inducible by modulation.     

Induction of granuloma modulation in murine schistosomiasis mansoni by enteric exposure to schistosome eggs

Enteric administration of antigen can induce systemic tolerance. In murine schistosomiasis mansoni, blood flukes produce eggs which enter the intestine. An immunologic phenomenon associated with this disease is a spontaneous diminution in the intensity of the granulomatous response in the liver, lungs, and colonic mucosa with chronic infection, which is termed modulation. It was determined whether modulation of liver granulomas could be induced by enteric immunization with schistosome eggs. Mice infected for 4 wk were immunized by injection of 25,000 eggs into cecal pouches. This induced modulation of liver granulomas by the eighth week of infection. Neither cecal injection of normal saline nor i.p. or subcutaneous injection of eggs could induce the modulatory process. Modulation could be adoptively transferred from enterically immunized donors by injection of spleen cells into infected recipients or into uninfected recipients with synchronous liver granulomas induced by the hepatic embolization of schistosome eggs. Spleen cells treated with anti-Thy-1.2 or anti-Lyt-1.1 and complement could no longer adoptively transfer modulation. These data show that enteric immunization with schistosome eggs can induce modulation of the liver granuloma by a cellular mechanism similar to that described for the natural infection.     

Quantitative analysis of intermediary metabolism in hepatocytes incubated in the presence and absence of glucagon with a substrate mixture containing glucose, ribose, fructose, alanine and acetate.

Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II; and 'futile' cycling at each of the five substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed about 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.