Deaggregation and proteolytic modification of a galactosyltransferase of Poterioochromonas malhamensis

We isolated hybridomas that produced monoclonal antibodies specific for the UDP-galactose: sn -glycerol-3-phosphate α-D-galactosyltransferase (IFP synthase, EC 2.4.1.96), an enzyme involved in the volume regulation of Poterioochromonas malhamensis Peterfi. Western blotting of native gradient gels with the most reactive antibody S 162 revealed several immunoreactive proteins in crude homogenates suggesting the occurrence of multiple molecular mass species of the galactosyltransferase. The amount of the presumed enzyme monomer (64 kDa under native conditions) was strongly increased by a pH shift of crude homogenates from pH 8 to 6. During activation of the galactosyltransferase in the cell homogenate and also by shrinking the cells, the presumed enzyme monomer appeared to be proteolytically degraded generating stepwise products of 52 and 40 kDa. We assume that the proteolytically processed enzyme becomes highly active, but is very susceptible to further proteolytic degradation.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3−C4 intermediate species

Immunogold labelling has been used to determine the cellular distribution of glycine decarboxylase in leaves of C₃, C₃–C₄ intermediate and C₄ species in the genera Moricandia, Panicum, Flaveria and Mollugo. In the C₃ species Moricandia foleyi and Panicum laxum, glycine decarboxylase was present in the mitochondria of both mesophyll and bundle-sheath cells. However, in all the C₃–C₄ intermediate (M. arvensis var. garamatum, M. nitens, M. sinaica, M. spinosa, M. suffruticosa, P. milioides, Flaveria floridana, F. linearis, Mollugo verticillata) and C₄ (P. prionitis, F. trinervia) species studied glycine decarboxylase was present in the mitochondria of only the bundle-sheath cells. The bundle-sheath cells of all the C₃–C₄ intermediate species have on their centripetal faces numerous mitochondria which are larger in profile area than those in mesophyll cells and are in close association with chloroplasts and peroxisomes. Confinement of glycine decarboxylase to the bundle-sheath cells is likely to improve the potential for recapture of photorespired CO₂ via the Calvin cycle and could account for the low rate of photorespiration in all C₃–C₄ intermediate species.Abbreviation and symbol kDa kilodaltons - CO₂ compensation point     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

(2'-5')An-dependent endoribonuclease: enzyme levels are regulated by IFN beta, IFN gamma, and cell culture conditions

The levels of a (2'-5')An-dependent endonuclease (RNase L) were determined in extracts prepared from murine L cells and Ehrlich ascites tumor (EAT) cells by measuring specific binding of protein to a labeled derivative of (2'-5')An, (2'-5')A3[32P]pCp. RNase L levels were found to depend both on interferon (IFN) treatment and on cell growth conditions. Treatment of murine L cells and EAT cells with 100-2,000 IRU IFN beta or IFN gamma resulted in a similar 2-4-fold increase in the levels of RNase L when cells were present at low density. The levels of RNase L were also shown to increase 2-3-fold as cells approached saturation density. Serum-starved cells also displayed relatively high levels of RNase L. RNase L levels in cells maintained at high cell density did not change appreciably following treatment with IFN beta or IFN gamma. Regulation of RNase L levels by cell growth conditions as well as by IFN beta or IFN gamma treatment suggests that RNase L may play an important role in regulating the levels of cellular mRNAs as well as acting to degrade viral RNAs.     

The cytotoxicity of chrysotile asbestos fibers to pulmonary alveolar macrophages I. Effects of inhibitors of ADP-ribosyl transferase

Pulmonary alveoler macrophages exposedto very short chrysotile asbestos fibers present a typical cytotoxic response: extracellular releases of lactate dehydrogenase and -galactosidase, and a decrease in cellular ATP content. The objective of this study was to determine if nicotinamide and 3-aminobenzamide, two inhibitors of the ADP-ribosyl transferase, could modify the in vitro toxicity of chrysotilee fibers. After 30 min of pre-exposure with each of the two inihibitors, pulmonary alveolar macrophage monolayers were concominantly exposed for 18 hours to 50g of fibers. It was observed that, in a dose-effect relationship (5 to 30 mM), nicotinamide was very effective in reducing the extracellular liberation of the marker enzymes. At 30 mM, the enzyme releases in the medium had returned to control values; the restoration of cell viability was confirmed by ATP levels. Up to 5 mM 3-aminobenzamide did not provide any protection against chrysotile cytotoxicity. Nicotinic acid, a structural analogue of nicotinamide, but not an inhibitor of the ADP-ribosyl transferase, also showed no protective effect. Nicotinamide and 3-aminobenzamide increased the intracellular NAD⁺ pools, respectively by 350% and 250%. However, with or without additives, the chrysotile fibers caused a constant and significant decrease in NAD⁺ levels (40–55 pmoles). These results suggest that the inhibition of the nuclear ADP-ribosyl transferase is not the major mechanism by which nicotinamide protects pulmonary alveolar macrophages against the chrysotile asbestos fibers.Abbreviations 3-AB 3-aminobenzamide - ADPRT ADP-ribosyl transferase - -GAL -galactosidase - DTT dithiothreitol - FBS fetal bovine serum - FMN flavin mononucleotide - HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid - LDH lactate dehydrogenase - NAD⁺ nicotinamide adenine dinucleotide (oxidized form) - NADH nicotimide adenine dinucleotide (reduced forms) - NADPH nicotimide adenine dinucleotide phosphate (reduced form) - NAM nicotinamide - NIC nicotinic acid - ORS oxygen radical species - PAM pulmonary alveolar macrophages - S.E. standard error of the mean - TAPS tris (hydroxymethyl) methylamino-propane sulfonic acid - TRIS tris (hydroxymethyl) aminomethane - VSF very short chrysotile fibers     

电离辐射后大鼠离体胰腺腺泡淀粉酶释放及M受体数量变化

我们曾观察到大鼠经γ-射线照射后胰淀粉酶活性降低和分泌减少[1],为进一步探讨照射后胰酶分泌减少的机制,本研究制备出分散的大鼠胰腺腺泡悬液并以不同浓度的~3H-二苯羟乙酸-3-喹咛环酯(~3Hquinuclidinyll benzilatc,简称~3H-QNB)进行M受体结合测定,同时观察胆碱能介质氨甲酰胆碱刺激腺泡所引起的淀粉酶释放反应。结果表明,γ-射线10Gy照射后3天,大鼠分散的胰腺腺泡在氨甲酰胆碱刺激时淀粉酶释放量减少到对照的50％,腺泡M受体与~3H-QNB最大结合量(Bmax)减少到对照的38％,伋M受体与~3H-QNB结合的解离常数(K_D)无改变,说明胰腺腺泡细胞M受体数量的减少可能是照射后胰腺腺泡分泌淀粉酶减少的原因之一。     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.