Rapid calcium release and proton uptake at the disk membrane of isolated cattle rod outer segments. 2. Kinetics of light-stimulated calcium release and proton uptake.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
W Junge
Publications and source records attributed to W Junge.
Interfacial potentials at the disk membranes of isolated intact cattle rod outer segments as a function of the occupation state of the intradiskal cation-exchange binding sites.
Two different methods have been used to determine the interfacial potential at the disk membranes of intact isolated bovine rod outer segments: (1) The photolysis products of rhodopsin are known to be dependent on pH. We have used this property in order to probe the interfacial potential at disk membranes which is considered to change the surface pH at the disk membrane seen by rhodopsin. (2) The pK value of the amphiphilic pH-indicating dye neutral red (uncharged basic form) in water is 6.6, but adsorbed to disk membranes at least 7.8. This makes the distribution of neutral red between disk membranes and bulk water dependent on the interfacial potential at the disk membrane if the pH in the bulk solution is less than 7.8. Both methods yielded comparable results on the influence of ions and ion carriers on the interfacial potential at disk membranes. In particular, we have studied the effect of different occupation states on the internal binding capacity (of rod outer segments) for divalent cations. In the presence of the ionophore A23187, addition of EDTA to a suspension of intact rod outer segments removed all endogenous divalent cations (Schnetkamp, P.P.M. (1979) Biochim. Biophys. Acta 554, 441--459) and resulted in an interfacial pH at the disk membrane surface of about 6.4, whereas the bulk pH was 7.4. Subsequent addition of 2 mM Mn2+ saturated the internal binding capacity and resulted in an apparent shift towards alkaline pH of the surface pH at the disk membrane by 1.0--1.1 pH units. This could indicate a change of the interfacial potential by 60--65 mV. The same change of ionic conditions resulted in a change of the interfacial potential by 72 mV as determined from the partitioning behaviour of neutral red. These results were independent of the presence of H+ ionophores such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and gramicidin. We conclude that the above results can be explained by the presence of fixed net negative charges (charge density: 0.5--1.5 electronic charges/rhodopsin molecule) at the intradiskal membrane surface. That the above charge density can be attributed to the intradiskal membrane surface is inferred from the observation that the presence of A23187 was required for access of divalent cations to the membrane interface involved in both rod outer segments with an intact as well as with a leaky plasma membrane.
Adsorption↗
Metarhodopsin I/metarhodopsin II transition triggers light-induced change in calcium binding at rod disk membranes.
The hypothesis of Yoshikami and Hagins that calcium ions act as diffusible transmitter molecules between the photochemistry of rhodopsin and the subsequent electrical events at the outer plasma membrane of rods initiated many investigations on light-stimulated calcium release in vertebrate photoreceptor cells (see refs 2, 3). Although it not seems firmly established that light has some effect on the redistribution of calcium in various disk preparations, reconstituted systems and intact rod outer segments, the physiological significance remained unclear. We previously reported a rapid, light-triggered calcium release from binding sites at the disk membrane in the presence of calcium ionophore A23187 (refs 3, 8). However, there is no evidence for rapid calcium release into the cytosol in the absence of ionophore. On fragmentation of intact rod outer segments, calcium release due to a light-requlated change of calcium binding appeared almost completely abolished. We describe here experiments with sonicated rod outer segments in which the previously observed loss of the calcium release capacity has been prevented. Calcium release in sonicated disks in the presence of A23187 kinetically follows the metarhodopsin I/metarhodopsin II transition (tau 1/2 = 10 ms, activation energy EA = 34 kcal mol-1), suggesting that calcium release is triggered by this photochemical transition.
Animals↗
[Experiences with ciliary block ("malignant") glaucoma (author's transl)].
Following a brief review of various interpretations of the disease and its treatment, the author's observations in 13 cases (18 eyes) treated during a period of ten years are reported. The data (Tables 2 and 3) are discussed with reference to the different forms of clinical manifestation of the disease (Table 4). Therapeutic aspects are outlined on the basis of the personal experience of the authors. If conservative measures (mydriasis, hyperosmotics, carboanhydrase inhibitors) fail to control ciliary block glaucoma, surgical procedures directed at the anterior hyaloid membrane are indicated. This structure is approached either via the pars plana or via the transpupillary route after lens extraction. In some cases lens extraction alone was sufficient.
Adult↗
Identification of a non-specific carboxylesterase in human pancreas using vinyl 8-phenyloctanoate as a substrate.
Human pancreatic tissue, pancreatic juice and sera of patients suffering from acute pancreatitis contain a vinyl 8-phenyloctanoate hydrolysing activity which was separated from true pancreatic lipase (EC 3.1.1.3). The enzyme, preliminary called "non-specific pancreatic carboxylesterase, was partially purified from human pancreatic tissue by DEAE-cellulose chromatography. Its molecular weight was found to be 54 000 by gel filtration on Sephadex G-100. The isoelectric point was estimated as 4.65 by isoelectric focusing. The results explain the poor correlation obtained when determinations of "serum lipase activity" using triolein and vinyl 8-phenyloctanoate as substrates are compared. However, since non-specific pancreatic carboxylesterase is liberated into the serum, determination of this new enzyme provides additional information in the diagnosis of pancreatic diseases.
Caprylates↗
Light-induced calcium release in isolated intact cattle rod outer segments upon photoexcitation of rhodopsin.
By applying flash-spectrophotometry with the calcium-indicating dye arsenazo III rapid light-triggered calcium release in various cattle rod outer segment preparations was studied. It is shown that light-induced calcium signals can be unambiguously discriminated from underlying absorption changes due to photolysis of rhodopsin and apparent absorption changes resulting from lightscattering transients. The following results have been obtained: 1. Calcium-induced arsenazo III responses can be quantitatively and kinetically resolved within the time domain of the visual transduction process. 2. Photoexcitation of rhodopsin results in calcium release from intradiscal binding sites. 3. Calcium released does not appear in the cytoplasmic space unless the disc membrane is made permeable to calcium ions by an ionophore. 4. The shortest observed half-rise time of calcium release (300 ms) is possibly limited by the ionophore. 5. The stoichiometric ratio of calcium released/rhodopsin bleached is 0.5 at a free calcium concentration of 2 microM. The amount of calcium released is proportional to the precentage of rhodopsin bleaching (from 1--10%). 6. Upon disruption of the disc stack by lysis of intact rod outer segments the light-induced calcium release is greatly altered. The results are discussed in relation to previous reports on a light-induced calcium release from retinal discs and in terms of the proposed role of calcium as an intracellular transmitter in vertebrate photoreceptors.
Animals↗
The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light.
The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent Km = 4 microM and satruation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is arrpox. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.
Chloroplasts↗
Characterization of the isoenzymes of pig-liver esterase. 1. Chemical Studies.
Three different subunits of highly purified pig liver esterase (EC 3.1.1.1) can be separated by analytical dodecyl sulfate electrophoresis, though their relative mobilities are very similar. The same subunit bands are obtained with microsomes, in which the esterases have been labeled with the specific active-site-directed inhibitor bis(4-nitro-[14C]phenyl)phosphate. The heterogeneity of the native trimeric enzyme is much more complex, as is demonstrated by isoelectric focussing and polyacrylamide gel electrophoresis. Fractions of esterase which were partially separated by preparative isoelectric focussing show differences in their subunit composition, their amino acid analyses, their tryptic peptide maps, and their C-terminal amino acids. From these experiments various features of the differing esterase subunits can be deduced. Based on the chemical results and on various experiments which did not indicate any secondary modification of the protein side-chains, the molecular basis of the esterase heterogeneity is discussed. We conclude that the native trimeric esterase is a mixture of numerous hybrids of at least three protein subunits with differing but closely related primary sequences. A comparison of the relative specificity of various preparations of pig liver microsomes indicates that genetic differences concerning the composition of liver esterase exist between individuals.
Amino Acids↗
Characterization of the isoenzymes of pig-liver esterase. 2. Kinetic studies.
Explore the source record for details and available documents.
Animals↗
[Haptoglobin phenotypes and liver cirrhosis. I (author's transl)].
The distribution of haptoglobin phenotypes (Hp) 1--1, 2--1 and 2--2 in 174 patients suffering from liver cirrhosis was determined and compared with a reference group consisting of 194 healthy subjects. The study revealed a high frequency of the Hp 1--1 phenotype (32%) in the patients as compared with the control group (14%). This difference is statistically highly significant (p less than 0.00025). It was calculated that in individuals of type Hp 1--1, the risk of liver cirrhosis is 4.3-fold higher than in persons with the phenotype Hp 2--2.
Adolescent↗
[Fluorescent antibody technique in identification of Neisseria gonorrhoeae-microcolonies grown on membrane filters (author's transl)].
Filter membranes (Nucleopore, No. N060, CPR 01300, pore size 0.6 mu) are inoculated with a pure suspension of N. gonorrhoeae or a Urethral smear from patients suffering from gonococcal urethritis. The membranes are placed on chocolate agar and incubated for three hours. After fixation in formaldehyd, grown microcolonies of N. gonorrhoea are detected microscopically after incubation of the membranes with a fluorescein-isothiocyanate labeled gonococcal antiserum (Difco, charge no. 605907) (Fig. 3--5). The high specificity of the antiserum was shown by lack of reaction with various other neisseriae (except for N. meningitidis, which showed a weak positive reaction) or other bacteria, respectively (table 1). To investigate the reliability of the new method urethral smears of 23 patients suffering from acute or chronic gonorrhoea were examined by culturing for gonococci in the conventional way and by means of the new technique. In all cases gonocococci were detected with both methods. This suggests that the fluorescent antibody technique has at least the same sensibility as the conventional one, and has the advantage that the result is obtained within one day.
Acute Disease↗
Gated proton conduction via the coupling factor of photophosphorylation modified by N,-N-orthophenyldimaleimide.
The membrane bound coupling factor of photophosphorylation is studied after pretreatment of broken chloroplasts with the bifunctional N,N-orthophenyldimaleimide under energization of the thylakoid membrane by mild flashing light. The proton conduction of the membrane is monitored both via the electrochromic absorption changes and via selective pH-indicating dyes. It is found that the coupling factor, after interaction with N,N-orthophenyldimaleimide during the preillumination period, shortcircuits one of the two protons pumped inside after excitation of chloroplasts with one short flash of light. In contrast to the low proton conductivity of the unperturbed thylakoid membrane (relaxation time for a proton gradient greater than 5s), this extra proton channel leads to a partial relaxation of a proton gradient within a few ms. Although limited to only one proton per electron, this extra proton conducting pathway is not otherwise specific. It operates with protons resulting from both Photosystem I and Photosystem II activity. In addition it operates with protons already present in the internal phase before firing of the exciting light flash. These effects are prevented by the presence of ATP (but not GTP) during the preillumination period. It is suggested that the modified coupling factor is gated open by the light induced electric field across the thylakoid membrane while self closing after passage of one proton per activated coupling factor.
Chemical Phenomena↗
On the mutual orientation of pigments in photosystem I particles from green plants.
Explore the source record for details and available documents.
Carotenoids↗
Rapid calcium release by passively loaded retinal discs on photoexcitation.
Explore the source record for details and available documents.
Animals↗
Ellipticity of cytochrome alpha3 and rotational mobility of cytochrome c-oxidase in the cristae membrane of mitochondria.
Explore the source record for details and available documents.
Animals↗
Proton release into the internal phase of thylakoids due to photosynthetic water oxidation. On the periodicity under flashing light.
Explore the source record for details and available documents.
Cell Membrane↗
The coupling factor of photophosphorylation and the electric properties of the thylakoid membrane.
The rate of ATP synthesis of illuminated chloroplasts is correlated with the electric conductance of their inner membranes. In agreement with previous studies it is shown that ATP synthesis is paralleled by an increased conductance of the thylakoid membrane. This conductance together with the ability to form ATP is abolished if chloroplasts are treated with an antibody against the coupling factor CF1. It is not influenced by the fragmented monovalent antibody. This parallels the lack of influence of the fragmented antibody on ATP synthesis in contrast to its influence on hydrolysis and exchange reactions. We conclude that there are different sites for the interaction of the coupling factor with adenine nucleotides. Extraction of the coupling factor is shown to increase the membrane conductance by more than two orders of magnitude. Reincorporation of the crude coupling factor partially restores the net conductance of the membrane (increase in resistance by a factor of 2.5), while a higher degree of restoration was observed for ATP synthesis and the proton conductivity of the membrane. We conclude that the extraction procedure opens different conductive channels in the membrane; a proton specific one, possibly associated with the binding protein for the coupling factor, plus other channels for "non-protons" which in contrast to the proton channel cannot be plugged by reincorporation of the coupling factor.
Adenosine Triphosphate↗
Symmetry, orientation and rotational mobility in the a3 heme of cytochrome c oxidase in the inner membrane of mitochondria.
The photoinduced linear dichroism of absorption changes resulting from photolysis of the complex between heme a3 of the cytochrome oxidase and CO is studied. The experiments started from isotropic solutions or suspensions of the enzyme both in its isolated form and in mitochondria. The anisotropy responsible for the linear dichroism was induced by excitation with a flash of linearly polarized light. The dichroic ratios observed with various systems; polymerized enzyme in solution, enzyme in mitochondria and in submitochondrial particles (at 20 degrees C as well as at liquid N2-temperature) all approached a value of 4/3 which characterizes a chromophore which is circularly degenerate. Therefrom we conclude that the interaction of heme a3 with its microenvironment within the protein does not break its four-fold symmetry. The experiments with mitochondria and submitochondrial particles suspended in aqueous buffer revealed similarly high dichoric ratios without any dichroic relaxation other than a rather slow one which could be attributed to the rotation of the whole organelle in the suspending medium. Therefrom we conclude that the cytochrome oxidase either is totally immobilized in the membrane, or that it carries out only limited rotational diffusion around a single axis coinciding with the symmetry axis of heme a3. In the light of independent evidence for a transmembrane arrangement of the oxidase and for the general fluidity of the inner mitochondrial membrane we consider anisotropic mobility of the cytochrome oxidase around an axis normal to the plane of the membrane as the most likely interpretation. Then our experimental results imply that the plane of heme a3 is coplanar to the membrane.
Animals↗