Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Effect of amino acids on glutathione production by Saccharomyces cerevisiae

Summary The constituent amino acids of the glutathione (GSH) tripeptide chain, glutamate, cysteine and glycine, were investigated for positive effects on GSH production in shake-flask cultures of Saccharomyces cerevisiae with glucose as the carbon source. Cysteine was confirmed as the key amino acid for increasing the specific GSH production rate, g, but showed some growth inhibition, especially in the second growth phase (ethanol-assimilation phase). An intracellular cysteine delivery agent, thiazolidine, showed a similar pattern of increased GSH production and growth inhibition, but to a slightly lesser degree, compared with free cysteine. The initial cysteine concentration affected both the specific growth rate, µ, and g, up to about 5 mm for µ and about 2–3 mm for g. Results of the [³⁵S]cysteine-labelling experiments suggest a complicated role of cysteine in increasing GSH production and further investigation may be necessary. Offprint requests to: S. Shioya     

Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

Production of ammonium dependent on basicL-amino acids byAnacystic nidulans

The incubation of the cyanobacteriumAnacystis nidulans withL-Arg,L-Lys orL-Orn, but neither with the correspondingD-isomers nor with other twentyL-amino acids, resulted in the production of large amounts of ammonium which accumulated in the outer medium. Relevant properties of thisin vivo ammonium production activity have been studied in cell suspensions treated with the glutamine synthetase inactivatorL-methionine-D,l-sulfoximine (MSX) to prevent assimilation by the cells of the resulting ammonium. In addition to its specificity for the basicL-amino acids, the system exhibited a set of properties (K _m value for substrates, requirement of oxygen which is taken up stoichiometrically with the production of ammonium, inhibition by o-phenanthroline and divalent cations) all of which are shared by a peculiarL-amino acid oxidase recently isolated fromA. nidulans. The data strongly suggest the participation of this enzyme in the production of ammonium from basic amino acids byA. nidulans, an activity that could account for the ability of this cyanobacterium to use arginine as a nitrogen source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanide p-trifluoromethoxy-phenylhydrazone - MSX L-methionine-D,l-sulfoximine     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Purification and characterization of anl-amino-acid oxidase fromChlamydomonas reinhardtii

Anl-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelvel-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity fromChlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55° C, respectively, with a Q₁₀ (45–55° C) of 1.7 and an activation energy of 45 kJ · mol^–1. Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated flavin content of 7.7 mol FAD per mol of native enzyme. ApparentK _m values of the twelvel-amino acids which can act as substrates ofl-amino-acid oxidase ranged between 31 M for phenylalanine and 176 M for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.This work was supported by Grant 780-CO2-01 from CICYT, Spain. The skillful secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.     

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers

We determined the extent of Na⁺-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na⁺-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na⁺-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, -alanine, -aminoisobutyric acid (AIB), -methylaminoisobutyric acid (MeAIB), -amino-n-butyric acid and l-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both d-serine and d-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and d-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2,7-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (I _sc) in voltage-clamped Caco-2 cell monolayers in Na⁺-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, d-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pH_i and inward I _sc. In conclusion, Caco-2 cells express a Na⁺-independent, H⁺-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.This study was supported by a Wellcome Trust Fellowship (to DTT). Charlotte Ward, Maureen Sinclair and Ken Elliott provided excellent technical assistance.     

Light and dark metabolism ofl- andd-amino acids in 5 strains ofChlorella vulgaris and 4 strains of the genusAnkistrodesmus

In the light, 2 out of the 4 newly testedChlorella vulgaris strains were found to use about as many amino acids as a source of nitrogen as the previously investigated strain Delft; the other 2C. vulgaris strains and 3 of theAnkistrodesmus strains used only a few. The 4thAnkistrodesmus strain used none.On the average,l-amino acids supported better growth thand-amino acids, butd-serine was preferred tol-serine by 3Ankistrodesmus strains.In the dark, growth was only obtained withC. vulgaris strain Delft, and only on a few of thel-amino acids,l-leucine in particular.The author is indebted to the Direction of the Academic Hospital Dijkzigt, Rotterdam and to Prof. Dr. H. Esseveld, Head of the Central Bacteriological Laboratory, Rotterdam, for providing facilities for the performance of this study.He thanks Mrs. Dr. H. J. Leijnse-Ybema for her help in making the chromatograms, and Mr. J. B. Lenstra, pharmacist, for advice in matters of organic chemistry.     

d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei

Summary The new enzyme d-2-hydroxyisocaproate dehydrogenase (NAD⁺-dependent) was detected in strains of the genus Lactobacillus and related genera. Straight and branched chain aliphatic as well as aromatic 2-ketocarboxylic acids are stereospecifically reduced to the corresponding d-2-hydroxycarboxylic acids according to the following equation:R-CO-COOH + NADH + H⁺ R-CHOH-COOH + NAD⁺ The enzyme is called d-hydroxyisocaproate dehydrogenase by us because 2-ketoisocaproate is the substrate with the lowest K_M-value. NAD(H) as a cofactor cannot be replaced by NADP(H). Because of its broad substrate specificity we chose the strain Lactobacillus casei ssp. pseudoplantarum (DSM 20 008) for enzyme production and characterization. d-2-hydroxyisocaproate dehydrogenase could be purified 180-fold starting with 500 g of wet cells.The purification procedure involved liquid-liquid extraction with aqueous two-phase systems and ion-exchange chromatography. At this stage the enzyme has a specific activity of 25 U/mg and can be used for technical applications. Further purification up to a homogeneous protein with a specific activity of 110 U/mg can be achieved by chromatography on Amberlite CG 50 at pH 3.5. Properties important for technical application of the d-HicDH were investigated, especially the substrate specificity and the optimum pH- and temperature ranges for activity and stability of the catalist.     

Production of carbon monoxide from aromatic amino acids by Morganella morganii

Morganella morganii produced CO when cultured in a medium containing casamino acids or peptone as the sole carbon source. Although the production of CO was distinctly enhanced by the addition of hemin to the medium, the amounts of CO produced in the absence of hemin were nearly proportional to the amounts of peptone added to the culture media. Examination of 20 amino acids for their ability to produce CO by resting cells revealed that phenylalanine, tyrosine, histidine and tryptophan were the sources of CO. Oxygen and hemin were necessary for CO production from the amino acids except tryptophan which produced CO in the absence of hemin. When cells were incubated for 4 h at 30° C in the mixture containing 40 mol tyrosine and 1 mol hemin, about 15 mol CO was produced; the activity of CO production was about 1.2 mol CO/h · mg cell nitrogen. Phenylpyruvic acid, p-hydroxyphenylpyruvic acid and imidazolepyruvic acid also produced CO in the presence of hemin, while indolepyruvic acid produced CO regardless of the presence or absence of hemin. The production of CO by the 2-oxo acids proceeded spontaneously and did not require the presence of M. morganii cells.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Secondary transport of amino acids in Nitrosomonas europaea

Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity ( molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake of l-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that l-alanine is transported by a H⁺/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed.     

Production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans containing aminotransferase activity

Summary To establish an efficient production method for l-phenylalanine, the production of l-phenylalanine from phenylpyruvate by Paracoccus denitrificans pFPr-1 containing aminotransferase activity was investigated. By using intact cells, 0.74M l-phenylalanine was produced from 0.8M phenylpyruvate (conversion yield, 92.5%). Moreover, by using immobilized cells with -carrageenan, when the space velocity was 0.1 h^-1 at 30°C, 0.135 M l-phenylalanine was produced from 0.15 M phenylpyruvate (conversion yield, 90%). The half-life of the l-phenylalanine-forming activity of the column was estimated to be about 30 days at 30°C.     

Purification and characterization of trypsin from an entomopathogen,Nomuraea rileyi NRRL 13755

Nomuraea rileyi isolate NRRL-13755 produced a large amount of trypsin enzyme when cultured on basal salt medium containing 1% (w/v) gelatin. The trypsin was purified nearly 60-fold, with a recovery of about 13% of the initial activity from the culture supernatant. This protease exhibited a remarkably high specific activity of nearly 370,000 IU/mg protein. The native molecular weight was estimated by gel permeation chromatography to be 30 kDa, and the subunit molecular weight was determined to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH and temperature optima were determined to be 8.5 and 35°C, respectively. With a relative trypsin activity of 100%, this purified preparation showed about 10% chymoelastase and nearly 50% chymotrypsin activity. Metal-chelating agents such as EDTA and EGTA at 2mm inhibited the enzyme activity by 40%, whereas N-carbobenzoxy-glycyl-l-phenylalaninamide (CBZ-gly-phe-NH₂) (2mm) and DTT (2mm) had no effect on activity. Trypsin inhibitor from turkey egg-white at 100 g/ml strongly inhibited the enzyme activity.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Occurrence and structure of lipoteichoic acids in the genus Staphylococcus

Lipoteichoic acids were isolated from eleven species of the genus Staphylococcus using phenol-water partition and hydrophobic chromatography on octyl-Sepharose CL-4B. The lipoteichoic acids purified could be visualized by SDS-PAGE. They were shown to be composed of a hydrophilic poly(glycerophosphate) chain covalently linked to gentiobiosyldiacylglycerol, the common lipid anchor of these molecules. Glycerophosphate units of the hydrophilic chain were found to be partly substituted with ester-linked d-alanine, except in the case of S. cohnii. The lipoteichoic acids isolated from S. cohnii, S. hominis, S. saprophyticus and S. simulans contain (1–2)-linked N-acetylglucosamine as an additional substituent of the poly(glycerophosphate) backbone.Abbreviations GLC gas-liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TLC thin-layer chromatography     

Expression of secondary alcohol dehydrogenase in methanogenic bacteria and purification of the F420-specific enzyme from Methanogenium thermophilum strain TCI

In four species of methanogens able to grow with secondary alcohols as hydrogen donors the expression and properties of secondary alcohol dehydrogenase (sec-ADH) were investigated. Cells grown with 2-propanol and CO₂ immediately started to oxidize secondary alcohols to ketones if transferred to new media. In the presence of H₂, such cells reduced ketones or aldehydes to alcohols. In the absence of H₂, aldehydes were dismutated (without growth) to primary alcohols and fatty acids. None of these reactions was catalyzed by cells grown with only H₂ and CO₂ at non-limiting concentration. This indicated an induction or derepression of sec-ADH by its substrate. Apparently, sec-ADH in all strains enabled not only the reduction of ketones or aldehydes, but also the dismutation of the latter. Sec-ADH was also expressed if strains were grown on H₂ and CO₂ in the presence of non-oxidizable, tertiary alcohols. Methanogenium thermophilum expressed sec-ADH even without added alcohol when H₂ became limiting. From this species, an F₄₂₀-specific sec-ADH was purified; the final gel filtration chromatography yielded a single protein peak that coincided with the activity. The enrichment was 12-fold, the activity recovery 26%. SDS polyacrylamide gel electrophoresis indicated that the enzyme was a homodimer with an apparent M _r of 79,000. At the pH optimum around 4.2, the specific activity for oxidation of 2-propanol (130 mM) and reduction of acetone (20 mM) was 176 and 110 mol/ min·mg, respectively (40°C). The apparent K _m for 2-propanol and acetone (with 15 M F₄₂₀) was 2.5 and 0.25 mM, respectively. Aldehydes also were reduced.Non-standard abbreviations ADH alcohol dehydrogenase - Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - F₄₂₀ N-(N-L-lactyl--L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mb. Methanobacterium - Mg. Methanogenium - Ms. Methanospirillum - OD₅₇₈ optical density at 578 nm - SDS sodium dodecyl sulfate     

Long-term kinetics of the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in plants with and without 2-carboxyarabinitol 1-phosphate

The light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in two species: Phaseolus vulgaris L., which has high levels of the inhibitor of Rubisco activity, carboxyarabinitol 1-phosphate (CA1P), in the dark, and Chenopodium album L., which has little CA1P. In both species, the ratio of initial to fully-activated Rubisco activity declined by 40–50% within 60 min of a reduction in light from high a photosynthetic photon flux density (PPFD; >700 mol · m^–2 · s^–1) to a low PPFD (65 ± 15 mol · m^–2 · s^–1) or to darkness, indicating that decarbamylation of Rubisco is substantially involved in the initial regulatory response of Rubisco to a reduction in PPFD, even in species with potentially extensive CA1P inhibition. Total Rubisco activity was unaffected by PPFD in C. album, and prolonged exposure (2–6 h) to low light or darkness was accompanied by a slow decline in the activity ratio of this species. This indicates that the carbamylation state of Rubisco from C. album gradually declines for hours after the large initial drop in the first 60 min following light reduction. In P. vulgaris, the total activity of Rubisco declined by 10–30% within 1 h after a reduction in PPFD to below 100 mol · m^–2 · s^–1, indicating CA1P-binding contributes significantly to the reduction of Rubisco capacity during this period, but to a lesser extent than decarbamylation. With continued exposure of P. vulgaris leaves to very low PPFDs (< 30 mol · m^–2 · s^–1), the total activity of Rubisco declined steadily so that after 6–6.5 h of exposure to very low light or darkness, it was only 10–20% of the high-light value. These results indicate that while decarbamylation is more prominent in the initial regulatory response of Rubisco to a reduction in PPFD in P. vulgaris, binding of CA1P increases over time and after a few hours dominates the regulation of Rubisco activity in darkness and at very low PPFDs.Abbreviations CA1P 2-carboxyarabinitol 1-phosphate - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat substrate-saturated turnover rate of fully carbamylated enzyme - PPFD photosynthetically active photon flux density (400–700 nm) - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate     

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: II. Effects of La3+, EGTA,and calmodulin antagonists on the current-voltage relation

Summary The steady N shapeI/V curves were obtained by applying slow ramp hyper- and depolarization pulses toChara cells under the voltage-clamp condition. Application of calcium channel blocker, 20 m La³⁺, to theChara membrane caused, in about 30 min, a marked reduction of the transient inward current and later almost complete blocking of the pump current, while the steady outward current remained almost unaffected. Removal of external Ca²⁺ with 0.5mm EGTA caused similar results. Application of calmodulin antagonists, 10 m TFP or 20 m W-7, also gave very similar results, i.e., the decrease of the transient inward current and of H⁺-pump activity. These results suggest that not only the excitatory mechanisms but also the H⁺-pump activity ofChara membrane are regulated by calmodulin within a comparatively narrow range of internal Ca²⁺ level.     

N-(indol-3-ylacetyl)amino acids as sources of auxin in plant tissue culture

N-(Indol-3-ylacetyl) derivatives (IAA conjugates) of aliphatic amino acids with a two- to six-carbon backbone including -l-amino acids, (-amino acids, and the ,-diamino acids ornithine and lysine were prepared, chemically characterized, and tested as sources of auxin in plant tissue culture. Stimulation of unorganized growth in Solanum nigrum L. callus and callus induction and developmental effects in tomato (Lycopersicon esculentum Mill. cv. Marglobe) hypocotyl explants were studied systematically. Relative auxin activities were estimated by comparing physiologically equivalent concentrations, in the optimal and suboptimal range, of the individual IAA conjugates. While the growth-promoting properties of some of the conjugates were species-dependent, those containing straight-chain two- to four-carbon -l-amino acid moieties were generally up to 100 times more active than those of their five- to six-carbon homologues. Branching of the amino acid backbone at C- (norvaline vs. valine and norleucine vs. isoleucine) and C- (norleucine vs. leucine) had a minor effect, but substitution of H- by a methyl group (-amino-l-butyric vs. -aminoisobutyric acids) almost completely blocked growth-promoting activity. IAA conjugates of -amino acids were, in most cases, nearly as active as those of their -amino-l-isomers. Among the conjugates of ,-diamino acids N -(IAA) ornithine was less active than N -(IAA)lysine. The activity of N -(IAA)lysine was less than for the -(IAA) isomer, and that of N ,N -(IAA)₂-lysine was different in tomato and Solanum nigrum. The l-alanine and -lysine conjugates were also found to be useful for induction and development of Oenothera leaf callus and in tomato cell-suspension culture, two systems which require highly active sources of auxin.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indol-3-ylacetic acid the abbreviations for N-(indol-3-ylacetyl)amino acids are listed in Table 1.     

Mitochondrial permeability for alcohols,aldoses, and amino acids

Summary Mitochondria isolated from potato tubers were placed in solutions containing various alcohols, aldoses, or neutral amino acids. Based on the osmotic responses in the different media, the reflection coefficients and hence the relative permeabilities of the nonelectrolytes could be determined. The reflection coefficients ( _j 'S) of the potato tuber mitochondria for alcohols became progressively larger as hydroxymethyl groups were added to the molecule,viz. methanol ( _j =0.07), ethylene glycol (0.25), glycerol (0.44),meso-erythritol (0.71) and adonitol (0.98). This increase in _j (decrease in permeativity) with increasing chain length parallels the decreasing lipid-water partition coefficients of the solutes. The reflection coefficients ofd-sorbitol (1.02) and ofd-mannitol (0.99) indicate that these six-carbon polyhydroxy alcohols are relatively impermeant and hence they would be suitable osmotica in which to suspend mitochondria. The _j 'S varied from 0.96 to 1.02 forD-ribose,D-xylose,D-lyxose,D-arabinose, -D-glucose, -D-glucose,D-galactose,D-mannose, glycine,L-alanine,L-threonine,L-phenylalanine,L-methionine andL-cysteine, indicating that these sugars and amino acids do not readily diffuse across the pair of membranes surrounding potato mitochondria. By contrast, the _j 'S of liver mitochondria for glycine and of pea chloroplasts for most of the same aldopentoses and amino acids are close to zero. Thus, different organelles can vary widely in their permeability properties for nonelectrolytes.