Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures

The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl--difluoromethylarginine or dl--difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl--difluoromethylornithine.Abbreviations ADC arginine decarboxylase - DFMA dl--dif-luoromethylarginine - DFMO dl--difluoromethylornithine - MPO N-methylputrescine oxidase - ODC ornithine decarboxylase - PMT putrescine N-methyltransferase We are indebted to Dr. E.W.H. Bohme of Merrell Dow Research Laboratories (Cincinnati, Ohio, USA) for kind gifts of DFMO and DFMA and to Dr. M.J.C. Rhodes for helpful advice and discussion.     

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain ofSerratia marcescens

Summary Proline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD ^–) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate--semialdehyde were produced as by-products.     

Metabolic links between the biosynthesis of pyrrolizidine alkaloids and polyamines in root cultures of Senecio vulgaris

Isotope feeding and inhibitor experiments were performed in order to elucidate the pathway common to polyamine and alkaloid biosynthesis in root cultures of Senecio vulgaris L. -Difluoromethylarginine, a specific inhibitor of arginine decarboxylase, prevented completely the incorporation of radioactivity from [¹⁴C]arginine and [¹⁴C]ornithine into spermidine and the pyrrolizidine alkaloid senecionine N-oxide. In contrast, -difluoromethylornithine, a specific ornithine-decarboxylase inhibitor, had no effect on the flow of radioactivity from labelled ornithine and arginine into polyamines and alkaloids. Thus, putrescine, the common precursor of polyamines and pyrrolizidine alkaloids, is exclusively derived via the arginine-agmatine route. Ornithine is rapidly transformed into arginine. Recycling of the guanido moiety of agmatine back to ornithine can be excluded. Putrescine and spermidine were found to be reversibly interconvertable and to excist in a highly dynamic state. In contrast, senecionine N-oxide did not show any turnover but accumulated as a stable metabolic product. In-vivo evidence is presented that the carbon flow from arginine into the polyamine/alkaloid pathway may be controlled by spermidine. The possible importance of the metabolic coupling of pyrrolizidine-alkaloid biosynthesis to polyamine metabolism is discussed.Abbreviations DFMA D,l--difluoromethylarginine - DFMO D,l--difluoromethylornithine - FW fresh weight     

Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna

The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine -Nmethyltransferase and -N-methylornithine decafboxylase were undetectable, indicating that -N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-¹⁴C]ornithine, l-[U-¹⁴C]arginine, [U-¹⁴C]agmaine and [1,4-¹⁴C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl--difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl--difluoromethylornithine. Together with the demonstration that label was incorporated from [U-¹⁴C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-¹⁴C] ornithine and l-[U-¹⁴C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.Abbreviations DFMA dl--difluoromethylarginine - DFMO dl--difluoromethylornithine We thank Miss E. Bent for valuable technical assistance and J. Eagles, K. Parsley and Dr. F. Mellon for mass-spectrometric analysis. We are grateful to Dr. A.J. Parr and Dr. M.J.C. Rhodes for helpful discussions. We are indebted to the Merrell Dow Research Institute, Cincinnati, Ohio, USA for supplying DFMA and DFMO.     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

Fluorescence methods for the histochemical demonstration of monoamines

Summary The histochemical fluorescence method of Falck and Hillarp for the demonstration of catecholamines and certain tryptamines, e.g. 5-hydroxytryptamine is based on the principle that these amines can be condensed with formaldehyde to yield strongly fluorescent 6,7-dihydroxy-3,4-dihydroisoquinolines and 6-hydroxy-3,4-dihydro--carbolines respectively. The investigation here reported presents the fluorescence characteristics and relative fluorescence yields for formaldehyde treated biogenic monoamines and certain related compounds enclosed in a dried protein layer. The fluorescence properties of some synthetic 6,7-substituted-3,4-dihydroisoquinolines and 3,4-dihydro--carbolines are given, and the fluorescence characteristics in relation to the molecular structure are discussed.Abbreviations used A adrenaline - DA dopamine - DOPA 3,4-dihydroxyphenylalanine - DOPS 3,4-dihydroxyphenyl-serine - 5-HT 5-hydroxytryptamine - 5-HTP 5-hydroxytryptophan - 5-MT 5-methoxytryptamine - -m-DA -methyl-dopamine - -m-DOPA -methyl-3,4-dihydroxyphenylalanine - -m-NA -methyl-noradrenahne - MTA 3-methoxy-tyramine - NA noradrenaline - NM normetanephrine - T Tryptamine - Try Tryptophan     

Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy

Summary MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450–500 nm excitation/>510 nm emission) or rhodamine (514 nm excitation/>530 emission) type dyes. A second 5- to 10-fold brighter emission for 450–500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled domes and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 m riboflavin resulted in accumulation in domes of 565±80 m. The transport rate was calculated to be 189±30 pmol/min-cm². Onemm probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10mm nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatable with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.     

Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

The activity of powdery-mildew haustoria after feeding the host cells with different sugars,as measured with a potentiometric cyanine dye

The biotrophic parasite Erysiphe graminis f. sp. hordei produces haustoria within the cells of its host Hordeum vulgare. To determine the physiological activity of these haustoria, the electric potential across the membranes in the mitochondria of the haustorium was studied. The membrane potential was estimated with the fluorescent potentiometric cyanine dye 3,3-dibutyloxacarbocyanine iodide. The addition of depolarizing agents (carbonylcyanide m-chlorophenylhydrazone, 2,4-dinitrophenol or KCN) to infected cells resulted in an increase of fluorescence after the addition of low concentrations or a decrease of fluorescence after the addition of higher concentrations. When the infected host cell was fed with increasing concentrations of d-glucose (25, 50, 75 mM), corresponding decreases of fluorescence were measured immediately in the mitochondria of the fungal haustoria. Sucrose induced a similar reduction of fluorescence about 20 min late. d-Galactose and d-fructose induced a somewhat smaller reduction of fluorescence, l-glucose and d-glucitol had no effect. The results indicate that haustoria take up glucose from the host cells immediately. Sucrose, d-galactose and d-fructose seem to require time to be metabolized before their products reach the fungal haustorium or mitochondria.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DiOC₄(3) 3,3-dibutyloxacarbocyanine iodide - DNP 2,4-dinitrophenol     

Effects of chemical modification of amino and sulfhydryl groups on KATP channel function and sulfonylurea binding in CRI-G1 insulin-secreting cells

The effects of several group-specific chemical reagents were examined upon the activity of the ATP-sensitive potassium (K_ATP) channel in the CRI-G1 insulin-secreting cell line. Agents which interact with the sulfhydryl moiety (including 1 mM N-ethylmaleimide (NEM), 1 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DNTB) and 1 mm o-iodobenzoate) produced an irreversible inhibition of K_ATP channel activity when applied to the intracellular surface of excised inside-out patches. This inhibition was substantially reduced when attempts were made to eliminate Mg²⁺ from the intracellular compartment. ATP 50 m and 100 m tolbutamide were each shown to protect against the effects of these reagents. The membrane impermeable DNTB was significantly less effective when applied to the external surface of outside-out patches. Agents which interact with peptide terminal amine groups and amino groups of lysine [1 mm methyl acetimidate and 1 mm trinitrobenzene sulfonic acid (TNBS)] and also the guanido group of arginine (1 mm methyl glyoxal) produced a Mg²⁺-dependent irreversible inhibition of K_ATP channel activity which could be prevented by ATP but not tolbutamide. The irreversible activation of the K_ATP channel produced by the proteolytic enzyme trypsin was prevented only when methyl glyoxal and methyl acetimidate were used in combination to inhibit channel activity. Radioligand binding studies showed that the binding of ³H glibenclamide was unaffected by any of the above agents with the exception of TNBS which completely inhibited binding with a EC₅₀ of 307 ±6 m.These results provide evidence for the presence of essential sulfhydryl (possibly cysteine), and basic amino acid (possibly lysine and arginine) residues associated with the normal functioning of the K_ATP channel. Furthermore, we believe that the sulfhydryl group in question is situated at the internal surface of the membrane, possibly near to the channel pore.K.L. is a Wellcome Prize Student. This work was supported by the Wellcome Trust, MRC and BDA.     

Forskolin increases osmotic water permeability of rabbit cortical collecting tubule

Summary Forskolin is a unique diterpene that may directly activate the catalytic subunit of adenylate cyclase. We therefore examined the effect of 50 m forskohn on osmotic water permeability in rabbit cortical collecting tubules perfusedin vitro. Forskolin increased net volume flux (J _v , from 0.30 to 1.22 nl/mm/min,P<0.02) in all tubules. The hydro-osmotic effect of forskolin was similar with respect to magnitude and time course to that produced by a maximal dose (250 U/ml) of arginine vasopressin. An additive effect onJ _v andL _p was not observed when maximal concentrations of forskolin and arginine vasopressin were given simultaneously. The compound d(CH₂)₅Tyr(Et) VAVP, which noncompetitively inhibits the vasopressin receptor, significantly reduced collecting tubular hydro-osmotic response to arginine vasopressin. In contrast, the hydro-osmotic response to forskolin was maintained in the presence of d(CH₂)₅ Tyr(Et)VAVP. However, the hydro-osmotic response to forskolin could be inhibited by 1.0 m guanine 5-(,-imido) triphosphate (GppNHp) and by the calmodulin inhibitor N-(6-amenohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). These results demonstrate that forskolin exerts an hydro-osmotic effect in the mammalian nephron which occurs independent of the vasopressin receptor. Guanine nucleotide regulatory proteins may modulate the osmotic water permeability effect of forskolin. Finally, calmodulin is required for full expression of the effect of forskolin to increase osmotic water flux.     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

The use of gel electrophoresis to study the reactions of activated amino acids with oligonucleotides

We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonucleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5-phosphates and ethylenediamine. We find that arginine and amino acids that can interact with oligonucleotides through stacking interactions react most efficiently. D-and L-amino acids give indistinguishable families of products.Correspondence to: L.E. Orgel 1444     

Use of Congo red as a microscopic fluorescence indicator of hyphal growth

Congo red was found to be feasible as a microscopic fluorescence indicator of hyphal growth at the single-hypha level. When 1 m Congo red was applied to mold of Aspergillus niger, the dye was found to a specific cell-wall component, chitin, without causing any inhibitory effect on hyphal growth. The bound Congo red emitted fluorescence at 614 nm. This binding reaction, however, proceeded more slowly than the growing speed of hypha. Consequently the fluorescence intensity was low at the apex where the surface area of the hypha was expanding rapidly. In contrast, as an apex where the growth was retarded, the fluorescence intensity became remarkably high. Therefore growing hyphae could be distinguished from non-growing hyphae by using Congo red.     

Impurity controlled phase transitions of phospholipid monolayers

The phase diagram of monolayers of l--dimyristoyl phosphatidic acid has been studied by fluorescence microscopy. For pressures corresponding to the nearly horizontal slope in the pressure area diagram the growth of crystalline platelets can be observed. They are of dendritic nature; their sizes can be controlled via pressure, compression speed, temperature and pH, and increased up to 100 m. Due to repulsive interaction a hexagonal arrangement of crystalline platelets can be established.It is shown that the textures do not depend on the dye probe for concentrations below 3 mol%. On the other hand via incorporation of impurities in concentrations of about 1 mol% the coexistence of lipid and solid phases can be controlled. Since, for a constant surface pressure, this coexistence can be maintained, these monolayers are suitable model systems to study the interactions of proteins and vesicles with coexisting fluid and solid membrane areas.Abbreviations DMPA l--dimyristoyl phosphatidic acid - DPPC l--dipalmitoylphosphatidylchline - DP-NBD-PE l--dipalmitoyl-nitrobenzoxadiazol-phosphatidylethanolamin - diO-C₁₈ (3) 3,3-dioctadecyl-oxocarbocyanin     

Evidence for essential arginine residues at the active sites of maize branching enzymes

Alignment of 23 branching enzyme (BE) amino acid sequences from various species showed conservation of two arginine residues. Phenylglyoxal (PGO) was used to investigate the involvement of arginine residues of maize BEI and BEII in catalysis. BE was significantly inactivated by PGO in triethanolamine buffer at pH 8.5. The inactivation followed a time- and concentration-dependent manner and showed pseudo first-order kinetics. Slopes of 0.73 (BEI) and 1.05 (BEII) were obtained from double log plots of the observed rates of inactivation against the concentrations of PGO, suggesting that loss of BE activity results from as few as one arginine residue modified by PGO. BE inactivation was positively correlated with [¹⁴C]PGO incorporation into BE protein and was considerably protected by amylose and/or amylopectin, suggesting that the modified arginine residue may be involved in substrate binding or located near the substrate-binding sites of maize branching enzymes I and II.Abbreviations BE branching enzyme - BCA bicinchoninic acid - BSA bovine serum albumin - Glc-1-P glucose-1-phosphate - IPTG isopropyl-d-thiogalactoside - PGO phenylglyoxal - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium docecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - TEA triethanolamine     

Use of a fluorescence proble for hydrophobic groups, anilinonaphthalene sulphonic acid, in the supravital study of unusual slug oocyte nucleoli

Synopsis Oocytes of the slug,Limax flavus, display cytoplasmic and nucleolar fluorescence in the presence of 8-anilino-1-naphthalene sulphonic acid, a fluorescent probe for hydrophobic groups. When nuclei are isolated in a balanced salt solution, or in 0.25 m sucrose, chromosomes also become fluorescent, but chromosomes never display fluorescence in intact cells. In the complex multiform nucleolus characteristic of slug oocyte nuclei, the nucleolar cap displays conspicuously high levels of fluorescence and patterns of differential fluorescence are obvious. It would appear that this and similar reagents could be of significant value in the investigation of nucleoli in supravital and unfixed preparations of isolated nuclei.     

Monoaminhaltige Strukturen im Zentralnervensystem der Trichoptera (Insecta)

Zusammenfassung Mit der fluoreszenzmikroskopischen Methode nach Falck und Hillarp wurden biogene Monoamine im Ganglion cerebrale und -suboesophageale bei adulten Trichopteren lokalisiert. Es konnten nur spezifisch grün fluoreszierende Strukturen festgestellt werden. Den überwiegenden Teil dieser Strukturen bilden terminale Varikositäten. Bestimmte Zentren, darunter die primären Assoziationszentren, weisen eine besonders hohe Konzentration und regelmäßige Anordnung fluoreszierender Bereiche auf. Hierzu gehören die Medulla externa und-interna, das Corpus centrale mit seinen Knollen, der -Lobus und -Lobus, das Corpus ventrale und das Neuropil des Unterschlundganglions. Im Deutocerebrum nimmt die Anzahl der fluoreszierenden Fasern stark ab, im Tritocerebrum sind sie kaum noch vorhanden. Die Lamina ganglionaris, der Pedunculus und die Glomeruli fluoreszieren nicht. Fluorophorhaltige Zellkörper lassen sich nur vereinzelt darstellen. Die meisten dieser Zellen (im Lobus opticus, Protocerebrum lateral der -Loben, in der caudalen Pars intercerebralis, im Suboesophageal-ganglion) konnten unter konstanten technischen Bedingungen regelmäßig nachgewiesen werden. Selten waren dagegen Zellen in der Ganglienzellschicht zwischen Proto- und Deutocerebrum sowie eine Zelle im Tritocerebrum darstellbar.

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Monoamine-containing structures in the central nervous system in Trichoptera (Insecta), part I

Summary The distribution of biogenic monoamines in the cerebral ganglion and suboesophageal ganglion of adult Trichoptera (Insecta) was studied by means of the histochemical fluorescence method of Falck and Hillarp. All structures that showed a specific fluorescence emitted a green light. Most of these structures were varicose fibres, spread over almost all the neuropile of the ganglia. In many areas (Medulla externa and interna, -lobe, -lobe, Corpora ventralia, Corpus centrale, and in the Ganglion suboesophageale) the catecholamine-containing varicosities appeared densely packed. In the deutocerebrum, the fluorescent structures were less numerous, the lowest amount of adrenergic neurons being found in the tritocerebrum. No specific fluorescence could be detected in the lamina ganglionaris, the pedunculus, and the glomeruli of the mushroom bodies.Perykarya displaying a specific fluorescence could be divided into two groups: those that could be constantly seen under standardized technical conditions and those found only occasionally. The former were situated in the cell layer of the optical lobe, the protocerebrum, close to the tops of the -lobes, in the caudal pars intercerebralis, and in the suboesophageal ganglion. In the last-mentioned ganglion, there appeared dorsally and ventrally in the caudal part two pairs of cell bodies which exhibited a green fluorescence also in their processes. Only occasionally catecholamine-containing cell bodies could be demonstrated in the cell layer between protocerebrum and deutocerebrum as well as in the tritocerebrum.

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Die vorliegende Arbeit wurde am Histologischen Institut der Universität Lund/Schweden durchgeführt. Ich danke Herrn Prof. K. Gösswald (Institut für Angewandte Zoologie, Würzburg), Herrn Prof. B. Falck, Herrn cand. med. A. Björklund, Herrn Dr. B. Ehinger (Histologisches Institut, Lund) sowie Herrn Dr. R. Elofsson und Herrn Dr. B. Tjeder (Zoologisches Institut, Lund) für die fachliche und technische Hilfe.Die Untersuchung wurde unterstützt durch The Swedish Natural Science Research Council, no. 99-35.     

Initial steps of αand β-d-glucose binding to intact red cell membrane

Summary The kinetics of the initial phases of d-glucose binding to the glucose transport protein (GLUT1) of the human red cell can be followed by stopped-flow measurements of the time course of tryptophan (trp) fluorescence enhancement. A number of control experiments have shown that the trp fluorescence kinetics are the result of conformational changes in GLUT1. One shows that nontransportable l-glucose has no kinetic response, in contrast to d-glucose kinetics. Other controls show that d-glucose binding is inhibited by cytochalasin B and by extracellular d-maltose. A typical time course for a transportable sugar, such as d-glucose, consists of a zero-time displacement, too fast for us to measure, followed by three rapid reactions whose exponential time courses have rate constants of0.5–100 sec^+–1 at 20°C. It is suggested that the zero-time displacement represents the initial bimolecular ligand/GLUT1 association. Exponential 1 appears to be located at, or near, the external membrane face where it is involved in discriminating among the sugars. Exponential 3 is apparently controlled by events at the cytosolic face. Trp kinetics distinguish the K _d of the epimer, d-galactose, from the K _dfor d-glucose, with results in agreement with determinations by other methods. Trp kinetics distinguish between the binding of the - and -d-glucose anomers. The exponential 1 activation energy of the -anomer, 13.6 ± 1.4 kcal mol^+–1, is less than that of -d-glucose, 18.4 ± 0.8 kcal mol^+–1, and the two Arrhenius lines cross at 23.5°C. The temperature dependence of the kinetic response following -d-glucose binding illustrates the interplay among the exponentials and the increasing dominance of exponential 2 as the temperature increases from 22.3 to 36.6°C. The existence of these interrelations means that previously acceptable approximations in simplified reaction schemes for sugar transport will now have to be justified on a point-to-point basis.We should like to express our thanks to Michael R. Toon for his important contributions. This work was supported in part by a grant-in-aid from the American Heart Association, by the Squibb Institute for Medical Research and by The Council for Tobacco Research.     

Cation effects on the conformations of muscle and non-muscle α-actinins

We examined the effects of changing KCl concentration on the secondary structures of -actinins using circular dichroism (CD), 1,1-bis(4-anilino) naphthalene-5,5-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mm KH₂PO₄, pH 7.5, increasing KCl from 0 to 120 mm led to decreases in -helix conformation for brain, platelet and heart -actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mm KCl, 0.65 mm calcium produced small changes in the CD spectra of both brain and platelet -actinin, but had no effect on heart -actinin. bisANS fluorescence of all three -actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mm KCl increasing concentrations of Ca²⁺ or Mg²⁺ did not have significant effects on the bisANS fluorescence of any -actinin. Digestion of brain, platelet and heart -actinins with -chymotrypsin showed an increase of proteolytic susceptibility in 120 mm KCl. These experiments also showed that increasing the concentration of Ca²⁺ or Mg²⁺ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mm KCl. The results suggest that -actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different -actinins.