Showed Saturation Kinetics Research Articles

Identification and characterization of nuclear location signal-binding proteins in nuclear envelopes

A radioiodinated, photoactivable synthetic nonapeptide corresponding to the nuclear location signal (NLS) of SV40 large T antigen has been used in photolabelling reactions with purified mouse liver nuclei, nuclear envelopes and other cellular fractions, to identify specific NLS-binding proteins which may be involved in selective transport of karyophilic proteins. SDS-polyacrylamide gel analysis of photolabelled products demonstrates that a 60 kDa nuclear protein and four nuclear envelope proteins (67, 60, 53 and 47 kDa) bind specifically to the native NLS and not to a mutant NLS or unrelated sequences. This binding shows saturation kinetics, with highest affinity of the NLS for the 60 and 67 kDa proteins. The nuclear 60 kDa NLS-binding proteins is identical to the nuclear envelope 60 kDa NLS-binding protein by two-dimensional gel analysis of labelled proteins. Biochemical fractionation of labelled nuclear envelopes suggests that the 53 and 47 kDa proteins are peripheral membrane proteins whereas the 67 and 60 kDa proteins can be localized to the pore complex. The NLS also binds to solubilized 67, 60, 53 and 47 kDa proteins but with decreased affinity. Our results suggest that one of the early steps in selective nuclear transport of proteins may be the recognition of the NLS by the 60 kDa and/or 67 kDa binding proteins present in the nuclear pore complex.

Effects of superoxide dismutase on the autoxidation of 1,4-hydroquinone

During autoxidation of 1,4-hydroquinone (H 2Q, <1 mM) at pH 7.4 and 37°C, stoichiometric amounts of 1,4-benzoquinone (Q) and hydrogen peroxide were formed during the initial reaction. The reaction kinetics showed a significant induction period which was abolished by minute amounts of Q. Hydrogen peroxide and catalase were without effect on the autoxidation process. Transition metals apparently were not involved, since chelators like EDTA, DETAPAC, and desferrioxamine or FeSO 4 had no influence on the autoxidation kinetics. Superoxide dismutase (SOD) did not abolish the induction period but dramatically enhanced the autoxidation rate by more than two orders of magnitude. The stimulatory effect was first-order in SOD concentration but showed saturation kinetics. The dependence of Q and hydrogen peroxide formation rates on H 2Q concentration shows a biphasic behaviour: dependence on the square at low H 2Q, but on the square root at high H 2Q concentration. As revealed by calculatory simulations the results can be adequately described by the known reaction rate constants. The reaction starts with the comproportionation of H 2Q and Q to yield two semiquinone molecules which autoxidize to give two superoxide radicals and two molecules of Q which enter into a new cycle of comproportionation. Because of unfavourable equilibria the autocatalytic reaction soon comes to steady state, and the further reaction is governed by the rate of superoxide removal. At excess SOD, the comproportionation reaction is rate-limiting, thus explaining the saturation effects of SOD. The experiments do not allow a decision between the two functions of SOD; the conventional action as a superoxide:superoxide oxidoreductase or as a semiquinone:superoxide oxidoreductase. In the latter reaction SOD is thought to be reduced by semiquinone with Q formation. In the second step the reduced enzyme would be re-oxidized by a superoxide radical which is formed during autoxidation of the second semiquinone molecule generated in the comproportionation reaction. From thermodynamic considerations, the latter function of SOD appears to be plausible.

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.

PH-stat experiments in proximal renal tubules

The pH-stat technique has been used to measure H+ fluxes in gastric mucosa and urinary bladder "in vitro" while keeping mucosal pH constant. We now report application of this method in renal tubules. We perfused proximal tubules with double-barreled micropipettes, blocked luminal fluid columns with oil and used a double-barreled Sb/reference microelectrode to measure pH, and Sb or 1 N HC1-filled microelectrodes to inject OH- or H+ ions into the tubule lumen. By varying current injection, pH was kept constant at adjustable levels by an electronic clamping circuit. We could thus obtain ratios of current (nA) to pH change (apparent H(+)-ion conductance). These ratios were reduced after luminal 10(-4) M acetazolamide, during injection of OH-, but they increased during injection of H+. The point-like injection source causes pH to fall off with distance from the injecting electrode tip even in oil-blocked segments. Therefore, a method analogous to cable analysis was used to obtain H+ fluxes per cm2 epithelium. The relation between JH+ and pH gradient showed saturation kinetics of H fluxes, both during OH- and H+ injection. This kinetic behavior is compatible with inhibition of JH by luminal H+. It is also compatible with dependence on Na+ and H+ gradients of a saturable Na/H exchanger. H(+)-ion back-flux into the tubule lumen also showed saturation kinetics. This suggests that H+ flow is mediated by a membrane component, most likely the Na(+)-H+ exchanger.

Cadmium accumulation and metallothionein-like proteins in the sea starAsterias rubens

Sea stars were exposed for four months to cadmium at 50 μg Cd/L. The cadmium content of stomachs, pyloric caeca, gonads and body wall was determined after 2, 4, 6, 8, and 16 weeks of exposure. The highest accumulation rates were found for body wall and pyloric caeca. The lowest accumulation rate was found for the gonads. In all tissues cadmium accumulation showed saturation kinetics. The body wall contained the largest part of the accumulated cadmium.

Uptake of palmitic acid by rabbit alveolar type II cells

Alveolar type II cells require a source of palmitic acid for synthesis of dipalmitoyl phosphatidylcholine (DPPC), a major constituent of pulmonary surfactant. Previous studies indicated that maximal rates of DPPC synthesis are achieved only if exogenous palmitate is available to the type II cell. Little is known of the mechanisms by which fatty acids enter type II cells. To determine if uptake is mediated by a membrane carrier system, as described in other cell types, we examined the kinetics of palmitate uptake. Using freshly isolated rabbit type II cells, we demonstrated that radiolabeled palmitate uptake was maximal and linear for 45 s; after 1 min the apparent rate of uptake declined. The initial uptake phase was taken as a measure of cellular fatty acid influx because intracellular radiolabeled palmitate remained 80% nonesterified at this time but was 55% esterified by 2 min. Cellular influx of palmitate showed saturation kinetics with increasing concentration of nonalbumin bound palmitate. Michaelis constant was 52.6 nM, and maximum velocity was 152 pmol.10(6) cells-1.min-1. The hypothesis that saturable cellular influx of palmitate is likely linked to the previously identified membrane fatty acid binding protein (MFABP) was supported by Western-blot analysis of rat lung tissue with an antibody to MFABP that demonstrated the presence of this carrier protein in lung tissue. These data suggest that palmitate uptake by type II cells is saturable and may be mediated by a membrane-associated carrier as described in other cell types.

Concentration of neutral lipids in the phospholipid surface of substrate particles determines lipid transfer protein activity

To better understand the mechanism of lipid transfer protein (LTP) action and the effects of altered lipoprotein composition on its activity, we evaluated the dependence of LTP activity on the concentrations of cholesteryl ester (CE) and/or triglyceride (TG) in the phospholipid bilayer of substrate particles. Phosphatidylcholine (PC)-cholesterol liposomes containing up to 2 mole% TG and/or CE were prepared by cholate dialysis and used as either the donor of lipids to, or the acceptor of lipids from, low density lipoproteins (LDL). CE or TG transfer from liposomes of varying neutral lipid content to LDL showed saturation kinetics with an apparent Km of less than or equal to 0.2 mole%. Throughout this concentration-dependent response. PC transfer, which depended on the same LTP-donor particle binding interactions as those required for neutral lipid transfer, was essentially unchanged. Lipid transfer in the reverse direction (from LDL to liposomes of varying neutral lipid content) followed the same kinetics showing that transfer between the two particles is tightly coupled and bidirectional. When liposomes contained both TG and CE, these lipids competed for transfer in a manner analogous to that previously noted with lipoprotein substrates. In conclusion, CE and TG transfer activities are determined by the concentration of these lipids in the phospholipid surface of donor and acceptor particles. At low TG and CE concentrations, LTP bound to the liposome surface as indicated by PC transfer, but only a portion of these interactions actually facilitated a neutral lipid transfer event. Thus, the overall rate of neutral lipid transfer, and the competition between TG and CE for transfer, depend on the concentrations of these lipids in the phospholipid layer.

Antibody-Catalyzed Porphyrin Metallation

An antibody elicited to a distorted N-methyl porphyrin catalyzed metal ion chelation by the planar porphyrin. At fixed Zn2+ and Cu2+ concentrations, the antibody-catalyzed reaction showed saturation kinetics with respect to the substrate mesoporphyrin IX (2) and was inhibited by the hapten, N-methylmesoporphyrin IX (1). The turnover number of 80 hour-1 for antibody-catalyzed metallation of 2 with Zn2+ compares with an estimated value of 800 hour-1 for ferrochelatase. The antibody also catalyzed the insertion of Co2+ and Mn2+ into 2, but it did not catalyze the metallation of protoporphyrin IX (3) or deuteroporphyrin IX (4). The antibody has high affinity for several metalloporphyrins, suggesting an approach to developing antibody-heme catalysts for redox or electron transfer reactions.

Presence of 5-hydroxytryptamine (serotonin) transport coupled with vacuolar-type H(+)-ATPase in neurosecretory granules from bovine posterior pituitary.

Electrogenic H(+)-ATPase was found in neurosecretory granules from bovine posterior pituitary. This enzyme was sensitive to bafilomycin, a specific inhibitor of vacuolar H(+)-ATPase, and was inactivated completely by cold treatment in the presence of MgATP and NaNO3. Immunoblot analysis showed the presence of immunologically identical polypeptides (72, 57, and 34 kDa) in the ATPases of the neurosecretory granules and chromaffin granules. The granules showed MgATP-dependent activity for 5-hydroxytryptamine (serotonin) uptake. This uptake was temperature-dependent and showed saturation kinetics (apparent Km for 5-hydroxytryptamine, 2 microM) and counter-flow. Reserpine and tetrabenazine at 1 microM inhibited the uptake, whereas imipramine at 2 microM had no effect. Dopamine, epinephrine and norepinephrine were also inhibitory. The uptake was abolished by various treatments that dissipated the electrochemical H+ gradient or inhibited the H(+)-ATPase. These results indicate that a vacuolar type H(+)-ATPase in the neurosecretory granules forms an electrochemical H+ gradient that drives 5-hydroxytryptamine uptake by a specific transport system. A similar granular fraction from the anterior pituitary had no ATP-dependent activity for 5-hydroxytryptamine uptake.

The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3.radical.

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3.radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H. H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R. M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4. Although EDTA (100 microM) decreased the CCl4 inhibition, the metal-complexing agents deferoxamine (100 microM) and diethyldithiocarbamate (100 microM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 micrograms/ml), superoxide dismutase (15 micrograms/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCl4 for the Ca2+ pump results from the metabolism of CCl4 to the CCl3. radical. This radical then directly oxidizes the Ca2+ pump, leading to decreased Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of fasting on D-3-hydroxybutyrate metabolism in the perfused rat heart

The effects of fasting for 24 h and 48 h on D-3-hydroxybutyrate utilization and acetoacetate, L-lactate and pyruvate production by the isolated non-working perfused rat heart were investigated over a wide range of DL-3-HB concentrations. D-3-HB utilization is concentration dependent and shows saturation kinetics, D-3-HB oxidation is correlated with D-3-HB concentration. Acetoacetate production is proportional to DL-3-HB concentration. L-lactate production is proportional to DL-3-HB concentration up to 5 mM following a 24 h fast and up to 10 mM after a 48 h fast, but further increase in DL-3-HB concentration decreases the rate of lactate production. Fasting enhances D-3-HB utilization at 16 mM DL-3-HB by 16% and 25% in 24 h and 48 h fast respectively, but has no significant effect at lower concentration. Fasting has no effect on acetoacetate production. Fasting for 48 h doubled the half-saturation concentration (Ku) without significant change in the maximum rate of utilization (Vu) of D-3-HB.

Uptake of aspartate aminotransferase into mitochondria in vitro causes efflux of malate dehydrogenase and vice versa

Incubation of intact mitochondria with aspartate aminotransferase results in efflux of malate dehydrogenase and vice versa. The export process is specific and rapid. It shows saturation kinetics with respect to the effector enzyme consistent with involvement of a receptor for the effector in the mitochondrial membrane system. Export is inhibited by both β-mercaptoethanol and by the metal chelating agent bathophenanthroline; both substances inhibit release of malate dehydrogenase by aspartate aminotransferase competitively whereas for release of aspartate aminotransferase by malate dehydrogenase inhibition is non-competitive. The efflux processis dependent on a trans-membrane pH gradient. Exported enzymes differ from the native forms in their dependence of activity on pH. Export of both aspartate aminotransferase and malate dehydrogenase is effected by incubation of mitochondria with the newly-synthesised precursor of aspartate aminotransferase; this observation provides supporting evidence for the physiological significance of the other results reported here. It is speculated that exported enzymes are on a pathway to degradation, and that coupled uptake and export is involved in the co-ordination of synthesis and breakdown of mitochondrial proteins.

Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc.

Photoaffinity labeling of purified cellulose synthase with [beta-32P]5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of [beta-32P]5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.

Binding of staphylococci to mucus in vivo and in vitro.

The association of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus with tissues of the upper respiratory tract were compared by using an in vivo ferret model. Ferrets were challenged intranasally with a 1-ml volume of radiolabeled staphylococci (3 mg [dry weight]), were allowed to clear the bacteria in vivo for 90 min, and were sacrificed. Tissues from the right nasal fossa were harvested and processed for radioassay or histology. Of the recoverable staphylococci, greater than or equal to 96% was associated with mucus gel overlaying mucosa of the turbinates. A quantitative radioassay was developed to study the binding of labeled staphylococci to immobilized crude ferret nasal mucin (FM) and bovine submaxillary gland mucin (BM). Binding showed saturation kinetics and was blocked specifically by BM but not by human Tamm-Horsfall glycoprotein nor orosomucoid. Binding to both FM and BM was significantly inhibited (P less than or equal to 0.01) when cocci were pretreated with trypsin but not when treated with beta-galactosidase or sodium metaperiodate (except for binding of S. saprophyticus to FM). These results suggest that mucin-binding receptors of the cocci may have protein components. The staphylococcus-binding receptors of both FM and BM appear to contain protein components, based on sensitivity to pretreatment with trypsin.

Transport of coenzyme M (2-mercaptoethanesulfonic acid) and methylcoenzyme M [(2-methylthio)ethanesulfonic acid] in Methanococcus voltae: identification of specific and general uptake systems

A transport system for coenzyme M (2-mercaptoethanesulfonic acid [HS-CoM]) and methylcoenzyme M [(2-(methylthio)ethanesulfonic acid (CH3-S-CoM)] in Methanococcus voltae required energy, showed saturation kinetics, and concentrated both forms of coenzyme M against a concentration gradient. Transport required hydrogen and carbon dioxide for maximal uptake. CH3-S-CoM uptake was inhibited by N-ethylmaleimide and monensin. Both HS-CoM and CH3-S-CoM uptake showed sodium dependence. In wild-type M. voltae, HS-CoM uptake was concentration dependent, with a Vmax of 960 pmol/min per mg of protein and an apparent Km of 61 microM. Uptake of CH3-S-CoM showed a Vmax of 88 pmol/min per mg of protein and a Km of 53 microM. A mutant of M. voltae resistant to the coenzyme M analog 2-bromoethanesulfonic acid (BES) showed no uptake of CH3-S-CoM but accumulated HS-CoM at the wild-type rate. While the higher-affinity uptake system was specific for HS-CoM, the lower-affinity system mediated uptake of HS-CoM, CH3-S-CoM, and BES. Analysis of the intracellular coenzyme M pools in metabolizing cells showed an intracellular HS-CoM concentration of 14.8 mM and CH3-S-CoM concentration of 0.21 mM.

Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport

Nitrate-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) contain a 45-kDa protein as a major protein in the cytoplasmic membrane but ammonium-grown cells lack it. A mutant (M45) was constructed by inactivating the gene encoding the 45-kDa protein. M45 did not grow under low concentrations of nitrate but high concentrations of nitrate could support its growth, with the optimal concentration being 40-70 mM. The growth rate of M45 was as high as that of the wild-type cells when ammonium was the nitrogen source. The 45-kDa protein was absent in M45 irrespective of the growth conditions. The activities of nitrate and nitrite reductases were higher in M45 than in wild type. The rate of nitrate-dependent O(2) evolution in wild type measured in the presence of L-methionine D,L-sulfoximine and D,L-glyceraldehyde showed saturation kinetics with respect to nitrate concentration in the external medium. The nitrate concentration required to produce half the maximal rate was 1 muM. In M45, the rate of nitrate-dependent O(2) evolution was nearly zero at nitrate concentrations <1 mM and was linearly increased as the concentration increased. The presumed absence of nitrate transport in M45 demonstrated by these results suggested that the 45-kDa protein is a nitrate transporter.

Characterization of 19-NOR-10-0X0-25-hydroxyvitamin D 3 production by solubilized chick kidney mitochondria and bovine serum albumin

The vitamin D 3 metabolite obtained from the incubation of 3-((cholamidopropyl) dimethylammonio]-2-hydroxy-l-propanesulfonate (CHAPSO)-solubilized chick kidney mitochondria with 25-hydroxy vitamin D 3 (25-OH-D3) was identified to be 5( E)-19-nor-10-oxo-25-hydroxyvitamin 03 (5( E0)-19-nor). The production of 19-nor was dependent on time and on protein concentration, but was not dependent on the pH of the incubation. 19-Nor was not formed in the absence of protein or when protein had been heat-treated following detergent solubilization. 19-Nor was not further metabolized to any other product upon incubation with the CHAPSO-solubilized proteins. No 19-nor-10-oxo derivative of 1,25(OH) 2D 3 was formed when 1,25(OH) 2D 3 was used as substrate in the incubation. Kinetic analysis showed a substrate saturation with an apparent V max of about 4.1 pmol/nun β mg and S 0.5 of approximately 1.3 × 10 −6M. The production of 19-nor was not restricted to the CHAPSO-soluble protein fraction of kidney mitochondria but was also found in both the CHAPSO-soluble and -insoluble fractions of chick liver mitochondria and CHAPSO-treated bovine serum albumin (BSA). 19-Nor production by detergent-treated BSA also showed saturation kinetics with a similar So S 0.5 and an apparent V max which was about 5-fold higher than that obtained with CHAPSO-solubilized mitochondria. The evidence suggests that the formation of 19-nor is not mediated by a traditional enzyme, but does require protein. A mechanism for the conversion of 25-OH-E 3 to 19-nor is proposed, in which the naturally-occurring 5(Z)-25-OH-D 3 substrate binds to protein, isomerizes to 5( E)-25-OH-D 3 and is oxidized by hydrogen peroxide to 5( E)- 19-nor via a dioxetane intermediate.

Papillomavirus detection using the polymerase chain reaction

be 2.8k0.41 x lo-’ ( n = 6 ) and 1.5+0.12x lo-’ ( n = 6 ) mol/mg of protein for adult and neonate, respectively ( P = 0.1 14). Fe3+ and Fez+ uptake by BBM vesicles showed saturation kinetics (Fig. l a and b ) with similar results being obtained using vesicles prepared from neonates and adults. Values for Fe3+ uptake at the highest concentration used (256 PM) were similar to those obtained at 91 p~ (Debnam et al., 1987) confirming saturation of the uptake process. These results imply that the enhanced duodenal iron uptake by the neonatal guinea-pig noted previously (Srai et al., 1988) does not involve changes in iron binding or uptake at the BBM. This is in contrast to adaptation of iron uptake following iron deficiency, where the increased iron absorption is a consequence of enhanced uptake at the BBM (Muir & Hopfer, 1985). This suggests that iron uptake by enterocytes is subject to control at more than one level. Possible alternative explanations for increased iron absorption in the neonate include alteration in the profile of enterocyte binding proteins (Srai et af., 1987) and change in movement across the basolateral membrane. We wish to thank North East Thames Regional Health Authority for financial support.

Kinetics of ferric chelate reduction by roots of iron-deficient peanut (Arachis hypogea)

SUMMARY Experiments were conducted to evaluate the effect of Fe3+-chelate concentration and chemical properties on the rate of reduction by roots of Fe-deficient peanut plants (Arachis hypogea L. cv. Florigiant). Reduction studies with the Fe3+-chelates of ethylenediaminetetraacetate (EDTA), hydroxyethylethylenediaminetriacetate (HEDTA), N,N-ethylenediaminediacetate, and diethylenetriamine-pentaacetate (DTPA) each showed saturation kinetics characteristic of active processes by plants. Km values varied from 23 to 85 μM, and Vmax from 0·55 to 5·5 μmol h–1 (g fresh roots)–1. Vmax values declined as the Fe3+-chelate formation constant increased. The removal of plant shoots reduced initial (< 6 h) reduction rates by 22%. The Km′ for Fe3+-chelates was much higher than Fe3+-chelate concentrations in soil solutions of problem calcareous soils.

Adipogranular cells from the mantle tissue of Mytilus edulis L. I. Isolation, purification and biochemical characteristics of dispersed cells

High-density adipogranular (ADG) cells were isolated from the mantle tissue of female mussels Mytilus edulis L. by enzymic dispersion and centrifugation through Percoll of density 1.08 g/ml. The cells were viable as judged by microscopic examination, exclusion of Trypan Blue, nonleakage of intracellular glutamate-oxaloacetate transaminase (GOT) and malate dehydrogenase (MDH), and utilization of exogenous [2- 3H] and [U- 14C] glucose. In dual-labelled experiments, the levels of [ 3H]glucose and [ 14C]glucose in cellular glycogen were equivalent to the utilization of 80 nmol glucose/h/g wet wt and 120 nmol glucose/h/g wet wt, respectively, at an external glucose concentration of 0.5 mM. The rate of 14CO 2 production was very low (18 nmol glucose/h/g wet wt), representing 6% of the total glucose utilization as measured by detritiation of [2- 3H] glucose. Recovery of 3H 2O from [2- 3H] glucose was linear with time over a 3-h incubation and showed saturation kinetics for glucose utilization, with an apparent K m of 0.56 ± 0.09 mM and a V max of 632 ± 48 nmol glucose/h/g wet wt.