Dark-adapted Rod Outer Segments Research Articles

Distribution and Translocation of Photoreceptor Gβγ-phosducin System in Medaka Retina‡

Unlike other vertebrates, teleosts have rod- and cone-specific phosducins (PD-R and PD-C) in the retina. To evaluate the teleost Gbetagamma-PD systems, we isolated cDNAs encoding medaka Gbeta1 and GbetaC, which selectively expressed rods and cones. Immunohistochemical studies showed that the strong reactivity of GbetaC but not PD-C was detected in cone outer segments. In rod outer segments (ROS), PD-R reactivity was stronger in light-adapted retina than in dark-adapted retina. Western blot analyses of fractions torn from the cryosections showed that the PD-R concentration was low in dark-adapted ROS. It is suggested that PD-R is translocated to ROS and effectively downregulates the phototransduction cascade in light-adapted rods.

Enhanced Shutoff of Phototransduction in Transgenic Mice Expressing Palmitoylation-deficient Rhodopsin*♦

Palmitoylation is a reversible, post-translational modification observed in a number of G-protein-coupled receptors. To gain a better understanding of its role in visual transduction, we produced transgenic knock-in mice that expressed a palmitoylation-deficient rhodopsin (Palm(-/-)). The mutant rhodopsin was expressed at wild-type levels and showed normal cellular localization to rod outer segments, indicating that neither rhodopsin stability nor its intracellular trafficking were compromised. But Palm(-/-) rods had briefer flash responses and reduced sensitivity to flashes and to steps of light. Upon exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in wild-type retinas. Since quench of rhodopsin begins with its phosphorylation, these results suggest that palmitoylation may modulate rod photoreceptor sensitivity by permitting rhodopsin to remain active for a longer period.

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors.

The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which approximately 40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

Calcium-dependent activation of guanylate cyclase by S100b.

Calcium concentration in the dark-adapted retinal rod outer segment is in the 200 to 600 nM range, and the guanylate cyclase of rod outer segments is thought to be activated in response to a fall in calcium concentration triggered by light. Calcium-binding proteins that mediate such activation, i.e., activation in the absence of or presence of low nanomolar calcium concentrations, have been identified and termed GCAPs (Guanaylate Cyclase Activating Proteins). In the course of our search for GCAP-like proteins in bovine retina, we isolated a protein fraction that stimulated rod outer segment cyclase activity at calcium concentrations higher than those in dark-adapted rod outer segments. We purified the protein responsible for this calcium-dependent stimulation of cyclase activity and found it to be of 6-7 kDa molecular weight as judged by electrophoresis under denaturing conditions and about 40 kDa by gel filtration analysis. Maximum stimulation of cyclase activity was observed at 3-4 micromolar concentration of the protein. It required about 1.5 micromolar free calcium concentration for half-maximal activation of the enzyme. Partial amino acid sequencing of peptide fragments of the activator suggested that the protein was identical with S100b, a previously described calcium-binding protein. Further characterization with antibody specific for S100b supported this possibility. However, the protein isolated in our laboratory and termed CD-GCAP (Calcium-Dependent Guanylate Cyclase Activator Protein) was found to differ significantly from commercially available S100b in the magnitude and calcium dependence of cyclase activation. It was also found to be inactivated by hydroxylamine while S100b was resistant. Investigation into these differences showed that purification methods had a significant influence on the properties of the activator, producing a less active (S100b) or more active (CD-GCAP) protein, but that it was, otherwise, one and the same protein. We conclude from this study that rod outer segment guanylate cyclase, unlike any cyclase known so far, is capable of activation by two different types of calcium-binding proteins, one that activates in response to a decrease in calcium concentration, and the other, described here, which activates in response to an increase in calcium-concentration. We hypothesize that this cyclase and others like it will be colocalized with one or the other type of activator depending upon the physiological requirement, i.e., activation in response to decreasing or increasing calcium concentration.

Light-dependent Association of Src with Photoreceptor Rod Outer Segment Membrane Proteinsin Vivo

In vivo light exposure results in tyrosine phosphorylation of several rod outer segment (ROS) proteins (Ghalayini, A. J., Guo, X. X., Koutz, C. A, and Anderson, R. E. (1998) Exp. Eye Res. 66, 817-821). We now report the presence of Src in ROS and its increased association with bleached ROS membranes. Immunoprecipitation with anti-phosphotyrosine revealed that tyrosine kinase activity recovered from light-adapted ROS membranes was twice that recovered from dark-adapted ROS. Other experiments revealed the presence of both bleached rhodopsin and arrestin in immunoprecipitates of LROS, suggesting the formation of a multimeric complex containing Src, arrestin, and bleached rhodopsin. Additionally, when immobilized Src homology domains 2 and 3 (SH2 and SH3, respectively) were used to study the association of Src with ROS membranes, only bleached opsin and arrestin were found to associate with the SH2 domain of Src. These data strongly suggest that Src through its SH2 domain interacts with bleached rhodopsin and arrestin either directly or indirectly. Similar results were also obtained when dark-adapted and light-adapted retinas were used instead of ROS membranes. Our data strongly suggest that light exposure in vivo activates Src and promotes its association through its SH2 domain with a complex containing bleached rhodopsin and arrestin. A hypothesis for the functional significance of this phenomenon is presented.

Effect of Light on Phosphatidate Phosphohydrolase Activity of Retina Rod Outer Segments: The Role of Transducin

The aim of the present paper is to evaluate the modulation of phosphatidate phosphohydrolase (PAPase) and diacylglyceride lipase (DGL) activities in bovine rod outer segment (ROS) under dark and light conditions and to evaluate the role of transducin (T) in this phenomenon. In dark-adapted ROS membranes exposed to light, PAPase activity is inhibited by 20% with respect to the activity found under dark conditions. To determine whether the retinal G protein, T, participates in the regulation of PAPase activity in these membranes, the effects of GTPγS and GDPβS on enzyme activity were examined. Under dark conditions in the presence of GTPγS, which stabilizes T in its active form (Tα + Tβγ), enzyme activity was inhibited and approached control values under light conditions. GDPβS, on the other hand, which stabilizes the inactive state of T (Tαβγ), stimulated PAPase activity by 36% with respect to control light conditions. ADP-ribosylation by cholera and pertussis toxin was also studied. In ADP-rybosilated ROS membranes with pertussis toxin under dark conditions, PAPase activity was 36% higher than the activity found under control light conditions. ADP-ribosylation by CTx, on the other hand, inhibited PAPase activity by 22%, with respect to dark control conditions, mimicking light effect. The effects of GTPγS and GDPβS and conditions of ADP-ribosylation by PTx and CTx on DGL activity were similar to those of PAPase activities. Based on NEM sensitivity we have also demonstrated that the PAPase present in ROS is the PAP 2 isoform. Our findings therefore suggest that light inhibition of PAP 2 in ROS is a transducin-mediated mechanism.

Light Activation of Phosphatidylethanolamine N-Methyltransferase in Rod Outer Segments and its Modulation by Association States of Transducin

Phosphatidylethanolamine N-Methyltransferase (PE N-MTase) is the enzyme responsible for the synthesis of phosphatidylcholine from phosphatidylethanolamine by successive transfer of methyl groups. This enzyme is present in bovine rod outer segments (ROS) and it is the only pathway for the synthesis of phosphatidylcholine in the outer segment of rod photoreceptor cells. In dark-adapted ROS membranes PE N-MTase activity is stimulated by 100% when ROS membranes are incubated under light condition. To determine whether the retinal G protein, transducin (Gt), intervenes in the regulation of PE N-MTase in these membranes, the effects of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and guanosine 5′-O-(2-thiodiphosphate (GDPβS) on the enzyme activity were examined. In dark, GTPγS which induces dissociation of Gt, stimulates the enzyme activity mimicking the stimulation by light. On the contrary, GDPβS stabilizes the inactive state of Gt, inhibiting the stimulation by light of PE N-MTase without affecting basal activities. In addition, adenosine 5′-diphosphate (ADP)-ribosylation by cholera and pertussis toxin was studied. ADP-ribosylation of ROS membrane with pertussis toxin, which stabilizes transducin in its inactive state, prevents the light-induced increase in PE N-MTase activity. On the contrary ADP-ribosylation with cholera toxin stimulates the enzyme activity. Our findings therefore suggest that light-stimulated effect of PE N-MTase activity is transducin-mediated.

Regulation of bovine rod outer segment membrane guanylate cyclase by ATP, phosphodiesterase and metal ions.

In vertebrate retina, rod outer segment is the site of visual transduction. The inward cationic current in the dark-adapted outer segment is regulated by cyclic GMP. A light flash on the outer segment activates a cyclic GMP phosphodiesterase resulting in rapid hydrolysis of the cyclic nucleotide which in turn causes a decrease in the dark current. Restoration of the dark current requires inactivation of the phosphodiesterase and synthesis of cyclic GMP. The latter is accomplished by the enzyme guanylate cyclase which catalyzes the formation of cyclic GMP from GTP. Therefore, factors regulating the cyclase activity play a critical role in visual transduction. But regulation of the cyclase by some of these factors--phosphodiesterase, ATP, the soluble proteins and metal cofactors (Mg and Mn)--is controversial. The availability of different types of cyclase preparations, dark-adapted rod outer segments with fully inhibited phosphodiesterase activity, partially purified cyclase without PDE contamination, cloned rod outer segment cyclase free of other rod outer segment proteins, permitted us to address these controversial issues. The results show that ATP inhibits the basal cyclase activity but enhances the stimulation of the enzyme by soluble activator, that cyclase can be activated in the dark at low calcium concentrations under conditions where phosphodiesterase activity is fully suppressed, and that greater activity is observed with manganese as cofactor than magnesium. These results provide a better understanding of the controls on cyclase activity in rod outer segments and suggest how regulation of this cyclase by ATP differs from that of other known membrane guanylate cyclases.

Intracellular cGMP concentration in rod photoreceptors is regulated by binding to high and moderate affinity cGMP binding sites

cGMP is the second messenger for visual excitation in vertebrate rod photoreceptors. However, no direct correlation has been observed between the measured total cGMP concentration in the rod outer segment and the electrical response of these cells to photic stimulation. To address this discrepancy, we have quantitated the number and affinities of cGMP binding sites in the rod outer segment to determine the cytoplasmic free cGMP concentration that is involved in visual transduction. We identified two distinct classes of cGMP binding sites in amphibian rod outer segments: 1) high affinity binding sites with a KD1 = 60 nM and a site density of 30 microM, and 2) moderate affinity binding sites with a KD2 = 6.6 microM and a site density of 78 microM. These two classes of binding sites are calculated to bind 94% of the total cellular cGMP, thereby lowering the cytoplasmic cGMP concentration to 3.5 microM in dark-adapted rod outer segments. This value is consistent with predictions of the cytoplasmic cGMP concentration based on activation of the cGMP-gated ion channel of rod photoreceptors. The kinetics of cGMP dissociation from high affinity binding sites indicate that this class of sites would dissociate its bound cGMP too slowly to participate in visual excitation and recovery to flash illumination. This binding of cGMP to intracellular binding sites provides a non-enzymatic mechanism by which photoreceptor cells regulate the concentration and restrict the diffusion of this second messenger during visual transduction.

Effects of lithium on basal and modulated activities of the particulate and soluble guanylate cyclases in retinal rod outer segments.

A large amount of information regarding the kinetics of biochemical reactions involved in visual transduction was derived from electrophysiological studies on dark-adapted rod outer segments. Hodgkin et al. [(1985) J. Physiol. 358, 447-468] observed that when Na was replaced with Li in the perfusion solution bathing the rod outer segment, the dark current slowly declined to zero. This decline was thought to result from a rise in intracellular calcium which was hypothesized to inhibit guanylate cyclase activity and reduce the cyclic GMP concentration. Rod outer segments contain membrane and soluble guanylate cyclase activities, and we show here that Li directly inhibits both types of activities very strongly. Both the basal (at high calcium) and the stimulated (at low calcium) activities of the membrane enzyme were inhibited by Li. Half-maximal inhibition of the stimulated enzyme was at 30 mM Li while for the basal activity it was at 100 mM. Over 80% of the activated enzyme was inhibited at 110 mM Li. The soluble guanylate cyclase activity was stimulated by nitroprusside. One hundred millimolar Li inhibited the basal activity by 20-30%, but the inhibition of the nitroprusside-stimulated (soluble) enzyme was much stronger, resembling that of the activated membrane enzyme. Half-maximal inhibition occurred at 30 mM, and about 80% inhibition was found at 100 mM Li. Stimulation of the soluble enzyme by nitroprusside was independent of calcium in the physiological range. The inhibition of the stimulated enzyme by Li was similarly independent of calcium, except at unphysiologically high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of arrestin (S-antigen) in rods of pearl mutant and wild-type mice

The pearl mutant mouse is hypopigmented and exhibits a significantly elevated dark-adapted (DA) threshold in comparison to the congenic wild-type mouse. The primary cause of the elevated DA threshold is not known. The subcellular immunolocalization of arrestin/S-antigen reflects the state of adaptation of rod photoreceptors. In this study, quantitative immunoelectron microscopy was used to examine the subcellular distribution of arrestin in wild-type and pearl rods as a function of light exposure. The goal was to determine whether arrestin distribution within rods of pearl and wild-type mice responds to background luminance in a comparable manner. The level of arrestin immunolabeling in DA (unilluminated) rods of pearl retinas was indistinguishable from that measured in wild-type rods. By contrast, arrestin immunolabeling in light-adapted (LA) pearl rod outer segments (ROS) was significantly greater than in wild-type LA ROS. Relative to DA ROS, arrestin labeling density increased 1.6 fold in wild-type ROS following light adaptation, as compared to a 4 fold increase in pearl ROS. These data suggest that although arrestin levels in DA pearl rods are indistinguishable from that of DA wild-type rods, net changes in arrestin immunolocalization in response to light exposure reflect the effects of the pearl mutation at the level of the rod outer segment. The possible implication of this finding is discussed in view of the proposed role of arrestin in the down-regulation of the enzymatic cascade of phototransduction.

Interaction of recombinant rho A GTP-binding proteins with photoexcited rhodopsin

The small molecular mass GTP-binding proteins rho A, B and C are targets for ADP-ribosyltransferase activity of the botulinum exoenzyme C3. The possible interaction of recombinant rho A proteins expressed in E. coli with photoexcited rhodopsin was studied by reconstitution with bovine rod outer segment (ROS) membranes depleted of endogenous GTP-binding proteins by treatment with urea. As reported for C3 substrates present in untreated ROS membranes, ADP-ribosylation of recombinant rho A proteins, both normal and Val-14 mutant, by C3 was inhibited when reconstituted with illuminated compared to dark-adapted ROS membranes pretreated with urea. GDP reduced the light-induced inhibition, while GTP[S] and light inhibited ADP-ribosylation of rho A proteins in a synergistic manner.

Light activation of phospholipase A2 in rod outer segments of bovine retina and its modulation by GTP-binding proteins.

Light stimulates phospholipase A2 activity in rod outer segments (ROS) of bovine retina as measured by the liberation of arachidonate from phosphatidylcholine, in in vitro assays of dark-adapted ROS. A role for GTP-binding proteins (G or N proteins) in the light activation of phospholipase A2 is suggested by the capacity for guanosine 5'-O-(thiotriphosphate) (GTP gamma S) to activate phospholipase A2 in dark-adapted ROS. In contrast, addition of GTP gamma S coincident with light exposure inhibited the light activation of phospholipase A2, suggesting that phospholipase A2 activity in the ROS is under dual regulation by G proteins. Transducin, the major G protein of the ROS, mediates the activation of cGMP phosphodiesterase by light and is a substrate for both cholera and pertussis toxin. Treatment of dark-adapted ROS with either toxin inhibits both basal and light-activated phospholipase A2, mimicking the action of the toxins on the light-induced cGMP phosphodiesterase activity of ROS. There is a loss of light-sensitive phospholipase A2 activity coincident with extraction of transducin from ROS membranes. In addition, the light-sensitive phospholipase A2 activity can be partially restored by the addition of purified transducin to the extracted ROS membranes. Light activation of phospholipase A2 in ROS membranes thus occurs by a transducin-dependent mechanism.

Phagocytosis of light- and dark-adapted rod outer segments by cultured RPE cells: a reassessment

Rod outer segments (ROS) isolated from adult rat retinas are phagocytized by cultured rat retinal pigment epithelial (RPE) cells. Using a double immunofluorescent labeling procedure, we have compared the binding and ingestion of ROS isolated at different times of the day. After 2 hr of incubation, approximately 98% of the ROS are ingested, while 2% are still attached to the RPE cell surface, irrespective of the time of day or lighting conditions under which the ROS are isolated. These findings differ from those reported earlier, using a radioactive method for quantitating ROS phagocytosis ( Hall, 1978).

Effect of fluoride on the phosphodiesterase of bovine photoreceptors

In the absence of the specific hormone, fluoride is able to activate the adenylate cyclase because it interacts with the GTP-binding protein. It has been reported that fluoride activates also the phosphodiesterase of the light-sensitive enzymatic cascade in dark-adapted retinal rod outer segments, but there is no indication that the GTP-binding protein is involved in this process or not. We show here that also in the photoreceptor system fluoride does interact with the GTP-binding protein in order to activate the phosphodiesterase in the dark. Further, we show evidences that fluoride solubilizes the GTP-binding protein in the dark and that the resulting complex activates the phosphodiesterase in dark-adapted rod outer segment membranes.

Cyclic nucleotide-dependent phosphorylation of proteins in rod outer segments in frog retina. Characteristics of the phosphorylated proteins and their dephosphorylation.

To clarify the function of cyclic nucleotides in rod outer segments (ROS) of frog retinas, we studied cyclic nucleotide-dependent phosphorylation and dephosphorylation of protein. cGMP or cAMP with [gamma-32P]ATP in the dark enhanced the phosphorylation of two ROS proteins with Mr = 10,500 (Band 1) and 8,500 (Band 2) according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The phosphorylation was maximally enhanced at 2.0 mM cGMP and cAMP in the presence of Mg2+. The cGMP-activated protein kinase showed near-optimal activity between pH 6.5 and 8.0. GMP, GDP, GTP, AMP, and ADP did not enhance the phosphorylation. The stoichiometry of the phosphate incorporated into Bands 1 and 2 could not be calculated because the amount of Bands 1 and 2 was too small to measure. Both 32P-phosphorylated Bands 1 and 2 (32P-Bands 1 and 2) were solubilized during preparation and the molecular weight of each, in the native preparation, was 19,000. Their isoelectric point was 5.2. The sites of phosphorylation were the serine residue(s). DEAE-Sephadex A-50 column chromatography gave a good separation of Bands 1 and 2 from other 32P-phosphoproteins at 60 mM NaCl. Dephosphorylation of 32P-Bands 1 and 2 in dark-adapted ROS suspension required Mn2+ or Mg2+; the former was more effective than the latter at concentrations below 0.5 mM. Both phosphorylation and dephosphorylation were inhibited by Zn2+.

Photoreceptor GTP binding protein mediates fluoride activation of phosphodiesterase.

In this report, we show that fluoride activates dark-adapted rod outer segment phosphodiesterase, and that this activation is mediated, in analogy with adenylate cyclase, through a GTP binding protein. The GTP binding protein is released from dark-adapted rod outer segment membranes by exposure to fluoride and subsequent centrifugation. The 39-kilodalton subunit of the GTP binding protein, released from the membrane by this procedure, exhibits altered susceptibility to limited trypsin proteolysis, identical to that seen when hydrolysis-resistant GTP analogs are bound to that subunit. Repeated exposure of dark-adapted rod outer segment membranes to fluoride and subsequent centrifugation results in maximal activation of the membrane-bound phosphodiesterase. Thus, activation of phosphodiesterase by fluoride in the dark appears similar to fluoride activation of adenylate cyclase.

Detergent-induced specificity of an antirhodopsin serum for opsin micro-complement fixation studies

An antiserum elicited in rabbit against dark-adapted rod outer segment membranes has been characterized by means of the micro-complement fixation technique. Both particulate rhodopsin and opsin, either biochemically intact or denatured and either membrane-bound or in the absence of lipids, are able to interact with the antiserum. Solubilization of the antigens in increasing concentrations of Emulphogene BC-720 leads to complete loss of complement fixation with both rhodopsin and opsin, but in the case of opsin this requires almost 10-times more detergent. In the case of opsin this masking phenomenon is preceded by a drastic exposure of antigenic sites which in the membrane vesicles are not accessible to the antibodies. Absorption experiments show that the antigenic sites on membrane-bound rhodopsin and opsin, as well as on Emulphogene BC-720-solubilized opsin, are of the same nature. Competition experiments show that the masking effect of the detergent is due to an inhibition of the primary antigen- antibody interaction and not to the inhibition of lattice formation. The use of detergents other than Emulphogene BC-720 further demonstrates that detergents more efficiently mask the antigenicity of conformationally intact than of denatured rhodopsinoids. The balance between the masking and the denaturing efficiency of a particular detergent determines whether a detergent-induced immunological discrimination can be observed between rhodopsin and opsin. The detergent-induced masking effects described are typical for the present antiserum and are probably dependent on methodological details of the immunization procedure.

Autophosphorylation of rhodopsin kinase from retinal rod outer segments.

Rhodopsin kinase has been identified as a 68K protein that is more readily extracted from dark-adapted rod outer segments (dark-extract) than from illuminated rod outer segments (light-extract). We observed that a 68K protein is phosphorylated by endogenous protein kinase of dark- or light-extract of bovine rod outer segments and that the amount of incorporated radioactivity (32P) was greater in the dark-than in the light-extract. Phosphorylation of the 68K protein is neither stimulated by cyclic nucleotides nor affected by the light or dark conditions of the phosphorylation reaction. Light-and dark-extracts were centrifuged simultaneously on individual sucrose density gradients revealing that the 68K phosphoprotein cosediments with endogenous rhodopsin kinase activity and that both greater 32P incorporation and higher rhodopsin kinase activity are found in dark-extract as compared to light-extract. These findings suggest strongly that the 68K phosphoprotein and rhodopsin kinase are identical and that rhodopsin kinase undergoes autophosphorylation.

Complex formation between metarhodopsin II and GTP-binding protein in bovine protoreceptor membranes leads to a shift of the photoproduct equilibrium

FEBS LettersVolume 143, Issue 1 p. 29-34 Full-length articleFree Access Complex formation between metarhodopsin II and GTP-binding protein in bovine protoreceptor membranes leads to a shift of the photoproduct equilibrium D. Emeis, D. Emeis Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this authorH. Kühn, H. Kühn Institut für Neurobiologie der Kernforschungsanlage, Jülich GmbH, PO Box 1913, 5170 Jülich, FRGSearch for more papers by this authorJ. Reichert, J. Reichert Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this authorK.P. Hofmann, K.P. Hofmann Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this author D. Emeis, D. Emeis Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this authorH. Kühn, H. Kühn Institut für Neurobiologie der Kernforschungsanlage, Jülich GmbH, PO Box 1913, 5170 Jülich, FRGSearch for more papers by this authorJ. Reichert, J. Reichert Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this authorK.P. Hofmann, K.P. Hofmann Institut für Biophysik und Strahlenbiologie der Universität Freiburg in Breisgau, Albertstrasse 23, 7800 Freiburg i. Br. FRGSearch for more papers by this author First published: June 21, 1982 https://doi.org/10.1016/0014-5793(82)80266-4Citations: 214AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume143, Issue1June 21, 1982Pages 29-34 ReferencesRelatedInformation