Cation-activated ATPase activity of plasmalemma-enriched membrane preparations from maize coleoptiles

The ATPase activity present in plasmalemma-enriched preparations from maize coleoptiles shows an optimum at pH 6, a strong dependence on Mg²⁺, and is stimulated by K⁺ and other monovalent cations, both organic and inorganic. The activation of ATPase by K⁺ obeys Michaelis Menten kinetics, saturation being reached at 50 mM K⁺ concentration. K⁺, Mg²⁺-stimulated ATPase activity is strongly inhibited by N,N-dicyclohexylcarbodiimide and by diethylstilbestrol and, to a lesser extent, by octylguanidine.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DTE dithioerythritol - Ellmans r 5-5 dithiobis (2 nitrobenzoic) acid - FC fusicoccin - NPA naphthylphthalamic acid - OG octylguanidine - PCMBS p-chloromercuribenzensulphonate     

Solubilization of a divalent cation dependent ATPase from dog heart sarcolemma

Heart sarcolemma has been shown to contain an ATPase hydrolizing system which is activated by millimolar concentrations of divalent cations such as Ca²⁺ or Mg²⁺. Although Ca²⁺-dependent ATPase is released upon treating sarcolemma with trypsin, a considerable amount of the divalent cation dependent ATPase activity was retained in the membrane. This divalent cation dependent ATPase was solubilized by sonication of the trypsin-treated dog heart sarcolemma with 1% Triton X-100. The solubilized enzyme was subjected to column chromatography on a Sepharose-6B column, followed by ion-exchange chromatography on a DEAE cellulose column. The enzyme preparation was found to be rather labile and thus the purity of the sample could not be accurately assessed. The solubilized ATPase preparations did not show any cross-reactivity with dog heart myosin antiserum or with Na⁺ + K⁺ ATPase antiserum. The enzyme was found to be insensitive to inhibitors such as ouabain, verapamil, oligomycin and vanadate. The enzyme preparation did not exhibit any Ca²⁺-stimulated Mg²⁺ dependent ATPase activity. Furthermore, the low affinity of the enzyme for Ca^2– (Ka = 0.3 mM) rules out the possibility of its involvement in the Ca²⁺ pump mechanism located in the plasma membrane of the cardiac cell.     

Contribution of plasma membrane Ca ATPase to cerebellar synapse function

The cerebellum expresses one of the highest levels of the plasma membrane Ca(2+) ATPase, isoform 2 in the mammalian brain. This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex; i.e. the Purkinje neurons (PNs). Here we review recent evidence, including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2 (PMCA2) knockout mouse, to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour. These studies have also revealed that deletion of PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development, they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.     

The prime plasmalemma ATPase of the halophilic alga Dunaliella bioculata: purification and characterization

The prime plasmalemma ATPase of the halophilic green alga Dunaliella bioculata has been solubilized by Triton X-100 from a plasmalemma-rich membrane fraction and purified by anion-exchange chromatography. Vanadate-sensitive ATPase activity was totally enriched about 230-fold to a specific activity of approx. 250 nkat·mg protein^–1. The presence of Mg²⁺ or Mn²⁺ is essential for ATP hydrolysis by the enzyme. In addition to an equimolar requirement (11 Mg²⁺: ATP), there is further stimulation by Mg²⁺ (up to 20 mM) and by (100 mM) monovalent cations (K⁺ NH ₄ ⁺ >Rb⁺ -Na⁺ >Cs⁺ >Li⁺-choline⁺). Most anions have no or little effect. With a molecular mass of about 105 kDa for the single subunit, sensitivity to vanadate and N,N-dicyclohexylcarbodiimide (50% inhibition at about 1 M and 0.3 mM, respectively), strict ATP-specificity, and an acidic pH optimum, this enzyme shows the typical characteristics of the common type of H⁺-ATPase in the plasmalemma of higher plants and fungi. These results undermine the hypothesis of a wider distribution of a special (high salt) type of plasmalemma ATPase as found in the marine alga Acetabularia.Abbreviations BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - Mega-9 nonanoyl-N-methyl-glucamide - Mes N-morpholinoethanesulfonic acid - Mops N-morpholinopropanesulfonic acid - PAGE polyacrylamide-gel electrophoresis - PM plasmalemma-enriched membrane fraction - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft; we thank Drs. M. Ikeda and D. Oesterhelt (MPI für Biochemie, Martinsried, FRG) for generous and valuable information about their work prior to publication.     

In-vitro auxin transport in membrane vesicles from maize coleoptiles

When membrane vesicles from maize (Zea mays L.) coleoptiles are extracted at high buffer strength, a pH-driven, saturable association of [¹⁴C] indole-3-acetic acid is found, similar to the in-vitro auxin-transport system previously described for Cucurbita hypocotyls. The phytotropins naphthylphthalamic acid and pyrenoylbenzoic acid increase net uptake, pressumably by inhibiting the auxin-efflux carrier.Abbreviations IAA indole-3-acetic acid - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA, 2-NAA 1-, 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid     

The outer epidermis of Avena and maize coleoptiles is not a unique target for auxin in elongation growth

A controversy exists as to whether or not the outer epidermis in coleoptiles is a unique target for auxin in elongation growth. The following evidence indicates that the outer epidermis is not the only auxin-responsive cell layer in either Avena sativa L. or Zea mays L. coleoptiles. Coleoptile sections from which the epidermis has been removed by peeling elongate in response to auxin. The magnitude of the response is similar to that of intact sections provided the incubation solution contains both auxin and sucrose. The amount of elongation is independent of the amount of epidermis removed. Sections of oat coleoptiles from which the epidermis has been removed from one side are nearly straight after 22 h in auxin and sucrose, despite extensive growth of the sections. These data indicate that the outer epidermis is not a unique target for auxin in elongation growth, at least in Avena and maize coleoptiles.Abbreviations IAA indole-3-acetic acid - PCIB p-chlorophenoxyiso-butyric This research was supported by grants from the National Aeronautics and Space Administration and from the U.S. Department of Energy. The help of S. Ann Dreyer is gratefully acknowledged.     

Kinetics of amyloplast sedimentation in gravistimulated maize coleoptiles

Inner mesophyll cells from coleoptiles of Zea mays L. cv. Merit were fixed after varying periods of gravistimulation. A statistically significant amount (17–21%) of amyloplast sedimentation occurred in these cells after 30 s of gravistimulation. The presentation time is approx. 40 s or less. The accumulation of amyloplasts near the new lower wall shows a linear relationship to the logarithm of the gravistimulation time (r=0.92 or higher). The intercept of this line with the baseline value of amyloplasts in vertical coleoptiles indicates that the number of amyloplasts on the new lower wall begins increasing 11–15 s after the onset of gravistimulation. Direct observations of living cells confirm that many amyloplasts sediment within less than 15–30 s. These rapid kinetics are consistent with the classical statolith hypothesis of graviperception involving the sedimentation of amyloplasts to the vicinity of the new lower wall.     

Calcium transport by pea root membranes

Calcium transport has been studied using purified endomembrane vesicles from dark-grown roots of Pisum sativum L. Membranes from a mixed microsomal (non-mitochondrial) fraction showed ATP-dependent calcium uptake which was released by the ionophore A 23187, had a pH optimum of 7.2 and required Mg²⁺ for uptake. Membranes were further purified using a rapid sucrosedensity-gradient technique yielding vesicles suitable for transport studies, and were identified using marker enzymes. Uptake by plasma membrane, tonoplast, endoplasmic reticulum and Golgi apparatus was indicated. Uptake by membranes of low density (predominantly tonoplast) had a pH optimum of 7.2–7.4 and nucleotide specificity ATP> guanosine 5-triphosphate>inosine 5-triphosphate>ADP>, while that by high-density membranes had a pH optimum of 7.5–7.9 and less specificity for ATP. The importance of regulating sucrose concentrations in calcium transport studies was demonstrated.Abbreviations ER endoplasmic reticulum - GTP guanosine 5-triphosphate - IDPase inosine diphosphatase - IIP inosine 5-triphosphate     

Physiological evidence that the primary site of auxin action in maize coleoptiles is an intracellular site

To locate functionally the primary site of auxin action in growing cells, the pool of auxin relevant to induction of growth in maize (Zea mays L.) coleoptile sections was determined. A positive correlation was consistently noted between growth and intracellular levels of indole-3-acetic acid (IAA), i.e. growth appears to be relatively independent of the external level of IAA. N-1-Naphthylphthalamic acid (NPA), a potent inhibitor of auxin transport, was used to enhance accumulation of IAA in coleoptile cells. From the use of NPA, it is shown that: 1) increasing the accumulation of IAA in cells, while the external concentration is held constant, resulted in a concomitant increase in growth, and 2) blocking the exit of IAA from cells with NPA sustained an IAA-induced growth response in the absence of externally applied IAA. Furthermore, the absence of any alterations in auxin binding to microsomal fractions by NPA indicates that the action of NPA in causing enhancement of auxin-induced growth is based upon its inhibition of efflux of IAA from the cells. This research was supported by National Science Foundation grant No. DMB 8515925. The careful assistance of Laurie Brulport is gratefully acknowledged.     

Phototropism of maize coleoptiles Influences of light gradients

The lateral fluence-rate gradients in unilaterally irradiated maize (Zea mays L.) coleoptiles were calculated on the basis of the proportions of P _fr (far-red-absorbing form of phytochrome) measured spectroscopically in transverse slices of the coleoptiles (top 1 cm). The results showed the occurrence of significant gradients that are wavelength-dependent. The gradient at 449 nm was steeper than those measured at 516, 534 and 551 nm, which were steeper than that measured at 665 nm. The ratios between the sides proximal and distal to the light source were, for example, 1:0.12 (449 nm), 1:0.23 (534 nm), and 1:0.28 (665 nm). Fluence-response curves for coleoptile phototropism (first positive curvature produced by less than 100 s unilateral irradiation) were measured at 449, 516, 534 and 551 nm. Comparison of the threshold fluences indicated that the responsiveness to 551 nm is about 10^4.8 less than that to 449 nm. Increasing wavelengths led to a decrease in maximal curvature, which correlated with the decrease of the fluence-rate ratios between the proximal and distal sides. Phototropic fluence-response curves were also measured using bilateral irradiation (449 nm). In one set of experiments, the fluence ratio was kept constant (either 1:1/2, 1:1/4 or 1:1/16) and the total fluence was varied, and in the other set the fluence applied to one side was kept constant and the fluence ratio was varied. A simple model based on the assumption that only one photoreaction occurs, and that the response is a function of the difference between the proximal and distal sides in the local photoreceptor action was tested. A fluence-response curve for this local photoreceptor action was calculated based on the fluence-rate ratio and the phototropic fluence-response curve measured for 449 nm. This curve was used, in conjunction with the measured fluence-rate ratios, as a basis for calculating phototropic fluence-response curves for other wavelengths and those for 449 nm obtained with bilateral irradiation. The calculated fluence-response curves showed excellent agreement with the experimental data. It is concluded that the threshold for maize coleoptile phototropism reflects the apparent photoconversion cross-section of the blue-light receptor whereas the maximal curvature depends on the steepness of the light gradient across the coleoptile.Abbreviations and symbols I(x) fluence rate at the depth x - P _fr phytochrome (far-red absorbing) - P _r phytochrome (red absorbing) - P _tot total phytochrome (P _r+P _fr) - photoconversion cross-section     

Kinetic modelling of phototropism in maize coleoptiles

Blue-light-induced phototropism of maize (Zea mays L.) coleoptiles was studied with a view to kinetic models. Red-light-grown plants were used to eliminate complication arising from the activation by blue light of phytochrome-mediated phototropism. In the first part, mathematical models were developed to explain the phototropic fluence-response data, which were obtained for the responses induced by a single unilateral pulse (30 s) and those induced by a unilateral pulse (30 s) given immediately after a bilateral pulse (30 s, fixed fluences). These data showed bell-shaped fluence-response curves, characteristic of first positive curvature. Modelling began with the assumptions that the light gradient plays a fundamental role in phototropism and that the magnitude of the response is determined by the gradient, or the concentration difference, in a photoproduct between the irradiated and the shaded sides of the tissue. Minimal mathematical models were then derived, by defining chemical kinetics of the photoreaction and introducing the minimum of parameters needed to correlate the incident fluencerate to the functional fluence-rates within the tissue, the functional fluence-rate to the rate constant of the photoreaction, and the photoproduct concentration difference to the curvature response. The models were tested using a curve-fitting computer program. The model obtained by assigning first-order kinetics to the photoreaction failed to explain the fluence-response data, whereas application of second-order kinetics led to a successful fit of the model to the data. In the second part, temporal aspects of the photosystem were examined. Experimental results showed that a high-fluence bilateral pulse eliminated the bell-shaped fluence-response curve for an immediate unilateral pulse, and that the curve gradually reappeared as the time for unilateral stimulation elapsed after the bilateral pulse. The model based on a second-order photoreaction could be extended to explain the results, with assumed changes in two components: the concentration of the reactant for the photoproduct, and the light-sensitivity of the reaction. The reactant concentration, computed with the curvefitting program, showed a gradual increase from zero to a saturation level. This increase was then modelled in terms of regeneration of the reactant from the photoproduct, with an estimated first-order rate constant of about 0.001·s^-1. The computed value for the constant reflecting the light-sensitivity showed a sharp decline after the high-fluence pulse, followed by a gradual return to the initial level. From these analytical results, the appearance of second positive curvature was predicted.Abbreviations FPC first positive curvature - SPC second positive curvature CIW-DPB publication No. 884     

Isoform-specific up-regulation of plasma membrane Ca2+ATPase expression during colon and gastric cancer cell differentiation

In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.     

Auxin causes oscillations of cytosolic free calcium and pH inZea mays coleoptiles

In epidermal cells of maize (Zea mays L.) coleoptiles, cytosolic pH (pH_c), cytosolic free calcium, membrane potential and changes thereof were monitored continuously and simultaneously (pH_c/, _m, Ca²⁺/ _m) using double-barrelled ion-sensitive microelectrodes. In the resting cells the cytosolic pH was 7.3–7.5 and the concentration of free calcium was 119±24 nM. One-micromolar indole-3-acetic acid (IAA), added to the external medium at pH 6.0 triggered oscillations in _m, pH_c and free calcium with a period of 20 to 30 min. Acidification of the cytosolic pH increased the cytosolic free calcium. The _m oscillations are attributed to changes in activity of the H⁺-extrusion pump at the plasmalemma, triggered off by pH and controlled by pH regulation (pH oscillation). The origin of the pH_c and Ca²⁺ changes remains unclear, but is possibly caused by auxin-receptor-induced lipid breakdown and subsequent second-messenger formation. It is suggested that the observed cytosolic pH and Ca²⁺ changes are intrinsically interrelated, and it is concluded that this onset of regulatory processes through the phytohormone IAA is indicative of calcium and protons mediating early auxin action in maize coleoptiles. It is further concluded that the double-barrelled ion-sensitive microelectrode is an invaluable tool for investigating in-vivo hormone action in plant tissues.Abbreviations and symbols FC fusicoccin - IAA indole-3-acetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - pH_c cytosolic pH - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - _m membrane potential difference (mV)     

Factors affecting the binding between fusicoccin and plasma membranes from maize roots

Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at −20° C decreased both the number of the low-affinity sites and their dissociation constant (K_D); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [³H]dihydroFC and the competition between [³H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H. The authors are indebted to Dr. L.M. Krasnopolskaya (Institute of Agricultural Biotechnology, Moscow, Russia) for fusicoccins A, C, J, and H, and to Dr. A.V. Galkin (Institute of Agricultural Biotechnology, Moscow, Russia) for valuable comments and ren dering the paper into English.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Mutations in PMCA2 and hereditary deafness: a molecular analysis of the pump defect

The inner ear converts sound waves into hearing signals through the mechanoelectrical transduction (MET) process. Deflection of the stereocilia bundle of hair cells causes the opening of channels that allow the entry of endolymph K⁺ and Ca²⁺. Ca²⁺ that enters is crucial to the hearing process and is exported to the endolymph by the plasma membrane Ca²⁺ pump (isoform PMCA2w/a): disturbances of the balance between Ca²⁺ penetration and ejection, e.g. by pump mutations, generate deafness. Hearing loss caused by PMCA defects is frequently exacerbated by mutations in cadherin 23, a single pass stereociliar Ca²⁺ binding protein that forms the tip links which permit the deflection of the stereocilia bundle and thus the opening of the MET channels. The PMCA2w/a pump ejects Ca²⁺ to the endolymph even in the absence of the natural activator calmodulin. This satisfies the special Ca²⁺ homeostasis requirements of the stereocilia/endolymph system. Here we have analyzed a mice and a human previously described pump mutant. The human mutant only exacerbated the deafness produced by a cadherin 23 mutation. The murine mutant overexpressed in model cells displayed an evident defect both in the basal activity of the pump and in the long range ejection of Ca²⁺, the human mutant instead failed to impair the Ca²⁺ ejection by the pump.     

A Ca-stimulated ATPase activity in rabbit neutrophil membranes

An ATPase activity specifically stimulated by micromolar Ca²⁺ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca²⁺-ATPase activity with regard to neutrophil function.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Blue-light-induced phosphorylation of a plasma-membrane protein from phototropically sensitive tips of maize coleoptiles

Tips of maize coleoptiles, which function as esential light sensors for the phototropic growth reaction, exhibit a rapid blue-light-induced phosphorylation of a plasma-membrane-associated 100-kDa protein. Characteristics of this reaction are as follows: (i) The functional unit involved in the light-dependent phosphorylation consists of a photoreceptor, a protein kinase and the 100-kDa protein. This complex is only localized in the plasma membrane of tips but not in other parts of the seedling, (ii) The photoreceptor is a cryptochrome-like compound, (iii) The pH optimum of the light-dependent phosphorylation on isolated plasma membranes is around pH 7.8 whereas the light-independent phosphorylation of other membrane proteins occurs at lower values (pH 6.2). (iv) The light-induced in-vitro phosphorylation of the 100-kDa protein is strongly inhibited by the protein-kinase inhibitor staurosporine (IC₅₀=4 nM). (v) The ³²P-moiety of a ³²P-[100 kDa]-protein complex generated after a light pulse with the aid of a membrane-associated protein kinase in the presence of [γ-³²P]ATP cannot be removed by a 100-fold higher level of (unlabelled) ATP. This fact indicates that protein and phosphate are covalently connected and that the complex is not a short-lived intermediate. (vi) The 100-kDa protein is not identical to the plasma-membrane H⁺-ATPase, as shown by immunostaining on Western blots. (vii) Irradiation-dependent in vivo phosphorylation of the 100-kDa protein in tips is already saturated by a light pulse of 5 s. In contrast, the de-phosphorylation of the protein in the dark is a slow reaction lasting about 30 min. It is suggested that the blue-light-triggered phosphorylated status of the 100-kDa protein is an early step in phototropism of the coleoptile, affecting the transport of auxin primarily in the irradiated flank.