Iodination (125I) of the apical plasma membrane of toad bladder epithelium: Electron-microscopic autoradiography and physiological effects

Summary The apical plasma membrane of toad bladder epithelial cells has been enzymatically iodinated, using lactoperoxidase, H₂O₂ (generated by a glucose-glucose oxidase system) and NaI. The site of labeling was demonstrated by electron-microscopic autoradiography; the silver grains (¹²⁵I) were found exclusively overlying the luminal plasma membranes of the epithelium. The iodination reaction reached completion in less than 5 min. The dependence of the degree of iodination on NaI concentrations (range=6.3×10^–8 to 6.3×10^–2 m) in the mucosal medium was determined. The results suggest that three classes of sites are iodinated within this concentration range. At concentrations of NaI of 6.3×10^–6 m or less, iodination of the apical membrane had no significant effect on either the fine structure of the epithelium or on electrophysiological properties. The baseline short-circuit current (SCC) remained steady and the response to vasopressin was unimpaired. At concentrations of 6.3×10^–5 m NaI and greater, the baseline SCC was depressed and the response to vasopressin was partially inhibited. The results indicate that¹²⁵I may serve as a covalent marker (specific for tyrosine and histidine residues) of the apical plasma membrane of epithelia.     

Comparative studies of fructose 1,6-diphosphate aldolase fromEscherichia coli 518 andLactobacillus casei var.rhamnosus ATCC 7469

A comparative study has been carried out with FDP aldolases fromEscherichia coli 518 andLactobacillus casei ATCC 7469, which had been purified 17.6- and 65-fold, respectively. The aldolase ofL.casei was stable only in the presence of mercaptoethanol, whereas that ofE.coli was strongly inhibited at low (1.0×10^–4 m) and activated at high concentrations (2.0×10^–1 m) of the same compound.p-Chloromercuric benzoic acid inhibited both aldolases, with 40% inhibition at 2×10^–5 m withE.coli aldolase against at 2×10^–4 m withL.casei aldolase. Significant differences were also observed in pH optima and Km values.E.coli aldolase exhibited a maximal activity at pH 9.0 and gave a Km value of 1.76×10^–3 m FDP with strong substrate inhibition above 7×10^–3 m, against pH 6.8–7.0 and a Km of 7.04×10^–3 m FDP forL.casei aldolase. Strong resistance ofL.casei aldolase against inhibition by EDTA, Ca²⁺ and Mn²⁺ was observed compared with complete inhibition at concentrations of 20mm, 40mm and 20mm, respectively, withE. coli aldolase. Polyacrylamide gel electrophoresis did not reveal any differences between the two enzyme preparations.The differences of the properties of FDP aldolases from different bacterial genera are discussed in relation to other Class II aldolases.     

Kinetic studies on 3-hydroxykynureninase from rat liver

Summary 3-Hydroxykynureninase was purified from rat liver. The Michaelis constants for L-kynurenine and L-3-hydroxykynurenine were determined to be 2.33 × 10^–4 m and 6.85 × 10^–5 m, respectively, at pH 8.41 and 37°. With L-kynurenine as substrate, the enzyme was competitively inhibited by L-alanine, 3-hydroxyanthranilic acid, and several other compounds which contained structural features of either amino acid or aryl portions of the substrate. The effect of pH on the initial velocity, maximal velocity, and Michaelis constant, using L-kynurenine as substrate, was studied. Maximal velocity was strongly pH-dependent, with a maximum at pH 8.4. The Michaelis constant decreased from 11.4 × 10^–4 m at pH 7.1 to 1.30 × 10^–4 m at pH 9.0. Logarithmic plots of these data showed pKa's for functional groups ionizing in the enzyme-substrate complex and free enzyme active center of 7.6 and 8.5, respectively. Possible groups responsible for these ionizations were discussed.Supported in part by a Faculty Creative Endeavor Grant from Central Michigan University, Mount Pleasant, Michigan.     

Allelic expression in intergeneric fox hybrids (Alopex lagopus × Vulpes vulpes). II. Erythrocyte glucose-6-phosphate dehydrogenase in arctic and silver foxes: Purification and properties

The functional properties of purified glucose-6-phosphate dehydrogenase (G6PD) from the erythrocytes of Arctic foxes (Alopex lagopus) and silver foxes (Vulpes vulpes) were investigated. It was found that pH optima for G6PD range from 8.15 to 8.25 in Arctic foxes and from 10.2 to 10.4 in silver foxes. For G6P, the estimated K _m values were 74×10^–6 m (at pH 8.2) and 166×10^–6 m (at pH 10.2) in Arctic foxes and 58×10^–6 m (at pH 10.2) and 40×10^–6 m (at pH 8.2) in silver foxes. The K _m values for NADP were estimated as 62×10^–6 m (at pH 8.2) and 86×10^–6 m (at pH 10.2) in the Arctic foxes and 15×10^–6 m (at pH 10.2) and 12×10^–6 m (at pH 8.2) in the silver foxes. It was found that Mg²⁺ ions exert a significant activating effect on G6PD in the Arctic fox and do not affect appreciably its activity in the silver fox. The experimental data indicate that slight differences in the electrophoretic mobility of G6PD are associated with considerable functional differences in this enzyme between the two fox species.     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringer's solution at 10^–4, 10^–3 and 5×10^–3 m concentration. Resistance was reduced by 10^–4 m Cd⁺⁺ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10^–4 and 10^–3 m Cd⁺⁺ produced additive stimulation which was reversible by washing with Cd⁺⁺-free Ringer's. If SCC was first stimulated by Cd⁺⁺, further stimulation by vasopressin was additive with 10^–4 m Cd⁺⁺ but completely inhibited by 10^–3 m Cd⁺⁺. Elevating the calcium ion (Ca⁺⁺) concentration of the outer Ringer's from 10^–3 m to 5×10^–3 m or 10^–2 m prior to Cd⁺⁺ treatment did not reduce the magnitude of SCC stimulation by Cd⁺⁺. Removal of Ca⁺⁺ from the outside Ringer's with 2×10^–3 m EDTA increased SCC as predicted. Subsequent addition of 5×10^–3 m Cd⁺⁺ drastically reduced SCC below control levels while equimolar concentrations of Cd⁺⁺ and EDTA reduced SCC only to control levels. These results suggest that Cd⁺⁺ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca⁺⁺ removal and may be a useful probe for elucidating these components.     

Probing the role of active site histidine residues in the catalytic activity of lacrimal gland peroxidase

The role of active site histidine residues in SCN^– oxidation by lacrimal gland peroxidase (LGP) has been probed after modification with diethylpyrocarbonate (DEPC). The enzyme is irreversibly inactivated following pseudo-first order kinetics with a second order rate constant of 0.26 M^–1 sec^–1 at 25°C. The pH dependent rate of inactivation shows an inflection point at 6.6 indicating histidine derivatization. The UV difference spectrum of the modified versus native enzyme shows a peak at 242 nm indicating formation of N-carbethoxyhistidine. Carbethoxyhistidine formation and associated inactivation are reversed by hydroxylamine indicating histidine modification. The stoichiometry of histidine modification and the extent of inactivation show that out of five histidine residues modified, modification of two residues inactivates the enzyme. Substrate protection with SCN^– during modification indicates that although one histidine is protected, it does not prevent inactivation. The spectroscopically detectable compound II formation is lost due to modification and is not evident after SCN^– protection. The data indicate that out of two histidines, one regulates compound I formation while the other one controls SCN^– binding. SCN^– protected enzyme is inactive due to loss of compound I formation. SCN^– binding studies by optical difference spectroscopy indicate that while the native enzyme binds SCN^– with the K_d of 15 mM, the modified enzyme shows very weak binding with the K_d of 660 mM. From the pH dependent binding of SCN^–, a plot of log K_d vs. pH shows a sigmoidal curve from which the involvement of an enzyme ionizable group of pKa 6.6 is ascertained and attributed to the histidine residue controlling SCN^– binding. LGP has thus two distinctly different essential histidine residues – one regulates compound I formation while the other one controls SCN^– binding.     

Stimulation of lymphocyte receptor capping by the ionophore monensin

Summary The carboxylic ionophore monensin has a biphasic effect on antibody-induced Thy-1 cap formation. At higher concentrations, 5×10^–6–5×10^–5 m monensin causes a significant inhibition of receptor capping similar to that previously found with the Ca²⁺ selective ionophore A23187. At lower concentrations, 5×10^–8–5×10^–7 m capping is stimulated. It is concluded that capping at lower ionophore concentrations is a secific response to the ability of monensin to induce a rise in intracellular Na⁺, which indirectly elevates intracellular Ca²⁺ activity. This in turn activates the contractile machinery required for the aggregation of surface receptors into capped structures. At higher concentrations monensin acts as a nonspecific detergent, which causes detrimental structural alterations in some of the membrane components involved in the capping process.     

Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

Pyrimidine biosynthesis in Serratia marcescens: Polypeptide interactions of three nonsequential enzymes

Orotidine-5-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3.). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the K _ms for PRPP and orotate were stoichiometric: 2.3×10^–6 m and 2.6×10^–6 m, respectively. Following separation, the K _ms were significantly different: 1.3 × 10^–6 m for PRPP and 4.1×10^–6 m for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of nonsequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.This work was supported by grants from the National Science Foundation (NSF GB 5811) and the Office of Naval Research (Nonr 4413). One of us (J.W.) was a National Science Foundation Graduate Fellow.     

Argininosuccinate synthetase activity in cultured human lymphocytes

The activity of argininosuccinate synthetase (E.C. 6.3.4.5), a urea cycle enzyme, was measured in cultured human lymphocytes using a new radioactive assay. Control cells had a maximum specific activity of 15.7±8.7 nmoles per hour per milligram of protein and an apparent K _m for citrulline of 2 × 10^–4 m, whereas cells derived from a patient with citrullinemia had no detectable activity. A nutritional variant, selected out of the citrullinemic lymphocyte population by ability to grow in citrulline, had a maximum specific activity of 10.7±3.8 nmoles/hr/mg and an apparent K _m for citrulline of 2 × 10^–2 m. These measurements confirm the observation that citrullinemia is associated with a defect in argininosuccinate synthetase activity and provide further evidence that citrullinemia is expressed in cultured lymphocytes. The emergence of a nutritional variant with a partial defect in argininosuccinate synthetase enzyme suggests that this citrullinemic patient has a heterogeneous population of cells, some totally defective and others only partially defective in argininosuccinate synthetase. The new activity assay is described in detail.This research was supported by a National Institutes of Health Training Grant (5-TO1-GM-0071) and NIH Program Project Grant (2-PO1-GM-15419).     

6-Phosphogluconate dehydrogenase in cell free extracts of Escherichia coli K-12

Summary Investigations into the properties of 6-PG dehydrogenase in cell free extracts of Escherichia coli revealed a pH optimum at pH 9.5 with a sharp decline on both sides of the optimum. The addition of 1.0×10^-2 m MgCl₂ produced maximal activity, whereas higher concentrations caused inhibition. The K _m values were 2.5×10^-4 m for 6-phosphogluconate and 2.5×10^-5 m for NADP⁺ as substrate. The enzyme was extremely stable for at least 5 hours if stored at 4°C in Tris–NaCl–MgCl₂ buffer at pH 7.5. 6-PG dehydrogenase activity was shown to be proportional to cell free extract concentration over the range 0–0.3 mg protein. An assay method based on the new optimal conditions has been established and has been shown to be 33% more sensitive than a number of commonly used methods.Meinem hochverehrten Lehrer Herrn Professor A. Rippel zum 80. Geburtstage.     

Cyclic AMP stimulation of calcium efflux from kidney,liver, and heart mitochondria

Summary The effect of cyclic AMP on subcellular calcium turnover was studied in isolated kidney, liver and heart mitochondria. The calcium concentration of the incubating medium was determined by fluorometric methods after its separation by millipore filtration. Liver and kidney mitochondria take up calcium in exchange for H⁺ and lower the medium calcium to 1 to 40×10^–6 m in less than 2 min. Cyclic AMP produces an instantaneous release of calcium from mitochondria and a rise in the steady-state calcium concentration of the medium. A new medium calcium level of 0.7 to 3×10^–4 m is achieved in less than 3 sec and is proportional to cyclic AMP concentrations between 10^–7 and 3×10^–6 m. Cyclic AMP is inactive above 5×10^–6 m and below 10^–7 m. Cyclic IMP, 5 AMP, dibutyryl cAMP are inactive at any concentration. Cyclic GMP is active at 10^–5 m and competitively inhibits cyclic AMP action. The same staedy-state calcium level is reached from higher or, lower calcium concentrations, i.e. whether cyclic AMP is added before or after the addition of calcium to the mitochondrial suspension. At low calcium or phosphate concentrations, the calcium released by cyclic AMP is immediately reaccumulated by the mitochondria is less than 2 min with a further release of H⁺. This pulse can be repeated by sequential additions of cyclic AMP. The transient or sustained response to cyclic AMP depends on the medium calcium x phosphate product and presumably on the presence or absence of calcium phosphate precipitate inside the mitochondria. These results support the hypothesis that cyclic AMP regulates cytoplasmic calcium by controlling the mitochondrial calcium efflux rate. This mechanism may be involved in the regulation of calcium transport and in some hormonal effects mediated by cyclic AMP.     

Interactions of lanthanum with purified and intact cell acetylcholinesterase of electrophorus electricus

Summary The effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ ofE. electricus and on the activity of AChE in intact electro-plaques from the same species were studied. 0.1mm LaCl₃ produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl₃, initial activity was markedly increased. LaCl₃ exerted a marked, concentration-dependent inhibition of intact cell AChE.La³⁺ and Ca²⁺ appear to interact competitively. In the presence of both 10mm CaCl₂ and 0.1mm LaCl₃, the initial activity of purified AChE was increased at lower ACh concentrations and inhibited at ACh concentrations greater than 3 × 10^–4 m. Inhibition of intact cell enzyme by 0.1mm LaCl₃ was relieved by increasing the CaCl₂ concentration to 10mm at ACh concentrations less than 2 × 10^–4 m.The data were analyzed assuming Michaelis-Menten kinetics and interpreted with reference to the differential binding of divalent and trivalent cations to regulatory anionic sites which are separate and distinct from the anionic site of the active center of the enzyme.     

Na−K−2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues

Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC₅₀ values) and order of potency of these derivatives were benzmetanide, 5×10^–8 m> bumetanide 3×10^–7 m>piretanide 3×10^–6 m>furosemide 7×10^–6 m> amino piretanide 1×10^–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [³H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK _d and number of binding sites (n) were: bovine kidney,K _d =1.6×10^–7,n=10.5 pmol/mg protein; winter flounder intestine,K _d 1.2×10^–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K _d =5.3×10^–7,n=20.4 pmol/mg protein. The estimatedK _d for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10^–7 m. The similarity in magnitudes of IC₅₀ andK _d for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK _d andn were lower at the higher Cl concentration (5mm Cl:K _d =5.29×10^–7 m,n=20.4 pmol/mg protein; 100mm Cl:K _d =2.3×10^–7 m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.     

Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO₂. Membrane potential and coupling ratio (V ₂/V₁) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO₂, control cells consistently uncouple even if the CO₂ treatments are repeated every 15 min for 2.5 hr. CDZ (5×10^–8–1×10^–7 m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10^–7, 7×10^–8 and 5×10^–8 m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO₂, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.     

The effect of anions on K+-binding in aHalobacterium species

Summary Trinitrocresolate (TNC) at a concentration of 2×10^–3 m brings about rapid loss of K from starvingHalobacterium cells. Higher concentrations of other anions such as salicylate, thiocyanate, and perchlorate produce a similar effect. The TNC-induced K loss is not significantly reversed when TNC is removed from the ambient medium. The rate of K loss in the presence of 2×10^–3 m TNC is only slightly increased by the temperature in the ranges of 30 to 40°C and 0 to 20°C; between 20 and 30°C, however, the rate increases 10-fold. The K loss was partly replaced by Na⁺. These data are interpreted in terms of the hypothesis that K is retained in starvingHalobacterium sp. not by active transport, but rather by selective binding on loci which are modified by TNC.     

Receptor capping in mouse T-lymphoma cells: A Ca2+ and calmodulin-stimulated ATP-dependent process

Summary The roles that Ca²⁺, calmodulin, and ATP play in the redistribution of conconavalin A (Con A) binding sites on the surface of mouse T-lymphoma cells were examined. Membranes of cells labeled with fluorescein-conjugated Con A (Fl-Con A) were made permeable (skinned) to ions and proteins by incubation in a solution containing no added Ca²⁺, 7mm EGTA, and ATP. The intracellular ionic and protein concentrations could then be varied, and the degree of Con A receptor capping monitored simultaneously. A graded increase (9.0 to 30%) was found in the number of capped cells with increasing Ca²⁺ concentration from 10^–6–10^–4.9 m. Increasing concentrations of trifluoperazine, chlorpromazine, and promethazine (1.5×10^–6 to 1.0×10^–4 m) in cell suspensions containing 10^–4 m Ca²⁺ produced graded inhibition of capping in the same order that the drugs bind to calmodulin. Removal of extracellular Ca²⁺ dissociated (reversed) some of the caps into patches, thus reducing their number (12%). ATP was required for either capping or cap dissociation to occur. Addition of calmodulin (3.9×10^–8–6.3×10^–7 m) to the cell suspension increased the Ca²⁺ sensitivity. These results provide direct evidence that capping of Con A receptors is a reversible process (i) regulated by intracellular Ca²⁺ concentration, (ii) requiring ATP as an energy source, and (iii) susceptible to the influence of calmodulin. These findings are consistent with the hypothesis that the collection of surface receptor patches into cap structures is controlled by the interaction of actomyosin filaments, which in turn is regulated by a Ca²⁺-calmodulin-activated control system.     

Adaptation of thymidine utilization to changing rates of DNA synthesis in the cell cycle

Summary In synchronous cultures of P-815 murine mastocytoma and of Chinese hamster ovary (CHO) cells, the relative contribution of exogenous thymidine to DNA synthesis was studied by comparing rates of (³H)thymidine incorporation with the rate of DNA synthesis as derived from incorporation of (³H)thymidine (10^–5 m) in the presence of amethopterin. In synchronous P-815 cultures, time-dependent variations of DNA synthesis rates were in close agreement with those of (³H)thymidine incorporation rates at concentrations of the precursor ranging from 5 × 10^–8 to 10^–5 m. Similarly, in synchronous CHO cell cultures prepared by two different methods, time-dependent changes in DNA synthesis rate were almost identical with those of the rate of incorporation of (³H)thymidine supplied at 5 × 10^–8 m. Thus, at a given thymidine concentration in the medium, the proportion of thymine residues in DNA that were derived from exogenous thymidine remained nearly constant, even though rates of cellular DNA synthesis underwent pronounced changes. This indicates that in the synchronous culture systems used, utilization of exogenous thymidine is efficiently adapted to changing rates of DNA synthesis.In partial fulfillment of the requirements for the degree of Ph.D. by G.G.M.     

Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles

Summary A⁵⁹Fe assay was designed to detect an Fe(III) binding capacity in NP-40 solubilized proteins from rabbit reticulocyte endocytic vesicles. The iron binding capacity had an apparent molecular weight as determined by gel exclusion chromatography of 450,000 daltons. The iron binding moiety coincided with the major nontransferrin iron-containing material of endocytic vesicles labeled in vivo by incubation of cells with⁵⁹Fe,¹²⁵I-labeled transferrin. The material solubilized from vesicles with NP-40 exhibited two classes of saturable binding sites, one with an association constant for⁵⁹Fe-citrate of 3.63×10⁹ m ^–1 and with 6.6×10^–12 moles of iron bound per mg protein and the other with a constant of 3.96×10⁸ m ^–1 and 1.0×10^–12 moles of iron bound per mg protein. These affinities are sufficient to satisfy the sobulility characteristics of Fe(III) at pH 5.0. Most of the⁵⁹Fe bound both in vivo and in vitro to the iron binding moiety could be displaced with⁵⁶Fe and an equivalent amount of⁵⁹Fe could subsequently be rebound in vitro. The iron binding assay was adopted to vesicle proteins separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to nitrocellulose and revealed an iron binding activity of molecular weight approximately 95,000 daltons.     

Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum

Zusammenfassung Es wird die 25–30 fache Anreicherung einer löslichen NADH-Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.) aus R. rubrum durch Gelfiltration an Sephadex G 200 und Chromatographie an DEAE-Cellulose beschrieben. Das Enzym ist bei DEAE-Cellulosechromatographie nur in Gegenwart von FMN oder NADH stabil. Menadion, Ferricyanid, DCPIP, p-Benzochinon und Cytochrom c sind als Elektronenacceptoren wirksam. Cytochrom c ist ein schlechter Acceptor. Das pH-Optimum der Reaktion liegt bei 8,4. Km für NADH ist 3,0 · 10^-5 m. NADPH wird nur mit etwa 3–5% des Wertes von NADH umgesetzt. Die prosthetische Gruppe des Enzyms ist FMN, Km für FMN ist 0,3 · 10^-7 m. Das Enzymprotein wird bei Verdünnung in 0,05 m Puffer inaktiviert; FMN und FAD sowie NADH und NADPH haben einen stabilisierenden Effekt. Durch höhere Pufferkonzentrationen wird das Enzym zunehmend inaktiviert. Die Inaktivierung besteht in einer Labilisierung oder Abspaltung der prosthetischen Gruppe vom Enzymmolekül. Verschiedene Metalle inaktivieren das Enzym ebenfalls.

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Oxidation of reduced nicotinamide adenine dinucleotide in Rhodospirillum rubrum I. Characterization of a soluble NADH dehydrogenase

Summary A soluble NADH^* Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.] has been purified 25–30 fold from R. rubrum by gelfiltration with Sephadex G 200 and ionexchange chromatography on DEAE-Cellulose. During the second purification step the enzyme is stable only in the presence of either FMN or NADH. Electronacceptors which were found to be effective include menadione, ferricyanide, DCPIP, p-benzoquinone and cytochrome c, the latter substance being a poor acceptor. The optimum pH of the reaction is at about 8.4. Km for NADH is 3.0×10^-5 m. NADPH is oxidized at only about 3–5% the rate of NADH. The active prosthetic group of the enzyme is FMN with an appearant Km of 0.3×10^-7 m. When diluted in 0.05 m buffer the enzyme becomes rapidly inactivated. FMN, FAD and also NADH and NADPH exhibit a stabilizing protective effect. Higher concentrations of buffer cause increasing inactivation. The mechanism of the inactivation is thought to be a labilization or detachment of the prosthetic group from the enzyme molecule. Several metals also inactivate the enzyme.