Disruption of insect photoreceptor membrane by divalent ions: Dissimilar sensitivity of light- and dark-adapted mosquito rhabdomeres

Summary The conditions that lead to the formation of myelin figures in rhabdomere microvilli were studied in the larval ocelli of the mosquito Aedes aegypti. These artifacts can result from the addition of divalent ions, such as Ca²⁺, to primary-aldehyde fixatives, but they form subsequently during postfixation with OsO₄. In light-adapted ocelli, myelin figures are concentrated at the proximal ends of the microvilli along the cytoplasmic margin of the rhabdomere. The severity of the artifact is proportional to the ion concentration: scattered myelin whorls are induced by Ca²⁺ concentrations as low as 5 mM; they become abundant at 15 mM to 25 mM, and displace much of the rhabdomere margin at 50 mM. In contrast, even at high concentrations of Ca²⁺ few membrane whorls form in dark-adapted rhabdomeres, and these are mostly located at the distal ends of the microvilli. The differential response of the rhabdomere microvilli in light and darkness does not result from a direct action of light during fixation; it reflects an underlying difference between light- and dark-adapted photoreceptor membranes. We suggest that this differential sensitivity to divalent ions is associated with the shedding of membranes from the rhabdomere, a process that is enhanced by light and reduced in darkness.This work was supported by a grant (BNS 76-18623) from the National Science Foundation     

Production and properties of ∝-mannosidase from aspergillus sp.

Summary Aspergillus sp NCIM 508 produced 22 U/L of extracellular -mannosidase activity in a medium containing 8 % brewer's yeast cells. The optimum period and pH range for maximum production of the enzyme were 7 days and 4.0–6.0, respectively. The optimum pH and temperature for enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable for 24 h at 28°C, in the pH range 6.0–7.0. The enzyme retained 100 and 65 % of its original activity after heating for 15 min at 45 and 55°C, respectively. The Km and V_max for p-nitrophenyl--D- mannoside (PNPM) were 71M and 7.5 × 10^–2 moles/min/mg, respectively. The enzyme was strongly inhibited by 1 mM Hg⁺⁺ and Cu⁺⁺ and partially by Co.⁺⁺ (NCL Communication No.; 5780)     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

Ca++ fluxes in isolated cells of rat pancreas. Effect of secretagogues and different Ca++ concentrations

Summary Secretagogues of pancreatic enzyme secretion, the hormones pancreozymin, carbamylcholine, gastrin I, the octapeptide of pancreozymin, and caerulein as well as the Ca⁺⁺-ionophore A 23187 stimulate⁴⁵Ca efflux from isolated pancreatic cells. The nonsecretagogic hormones adrenaline, isoproterenol, secretin, as well as dibutyryl cyclic adenosine 3,5-monophosphate and dibutyryl cyclic guanosine 3,5-monophosphate have no effect on⁴⁵Ca efflux. Atropine blocks the stimulatory effect of carbamylcholine on⁴⁵Ca efflux completely, but not that of pancreozymin. A graphical analysis of the Ca⁺⁺ efflux curves reveals at least three phases: a first phase, probably derived from Ca⁺⁺ bound to the plasma membrane; a second phase, possibly representing Ca⁺⁺ efflux from cytosol of the cells; and a third phase, probably from mitochondria or other cellular particles. The Ca⁺⁺ efflux of all phases is stimulated by pancreozymin and carbamylcholine. Ca⁺⁺ efflux is not significantly effected by the presence or absence of Ca⁺⁺ in the incubation medium. Metabolic inhibitors of ATP production, Antimycin A and dinitrophenol, which inhibit Ca⁺⁺ uptake into mitochondria, stimulate Ca⁺⁺ efflux from the isolated cells remarkably, but inhibit the slow phase of Ca⁺⁺ influx, indicating the role of mitochondria as an intracellular Ca⁺⁺ compartment. Measurements of the⁴⁵Ca⁺⁺ influx at different Ca⁺⁺ concentrations in the medium reveal saturation type kinetics, which are compatible with a carrier or channel model. The hormones mentioned above stimulate the rate of Ca⁺⁺ translocation.The data suggest that secretagogues of pancreatic enzyme secretion act by increasing the rate of Ca⁺⁺ transport most likely at the level of the cell membrane and that Ca⁺⁺ exchange diffusion does not contribute to the⁴⁵Ca⁺⁺ fluxes.With the technical assistance of C. Hornung.     

Electrical properties of Paramecium caudatum: all-or-none electrogenesis

Summary Specimens of Paramecium immersed in solutions of CaCl₂ show graded electrogenesis in response to imposed transmembrane current. However, when BaCl₂ in a final concentration of 0.25 mM is added to a 1 mM CaCl₂ solution, an outward current pulse of 10^-10 amp or greater elicits an all-or-none transient reversal in membrane potential having a duration of about 40 msec. An increase of [Ba⁺⁺] results in (a) lower resting potential, (b) positive shift in critical firing level, (c) increased overshoot of the action potential, (d) decreased hyperpolarizing afterpotential, and (e) increased duration of the action potential (a.p.). If [Ca⁺⁺] is increased along with [Ba⁺⁺] so as to keep the ratio [Ba⁺⁺]/[Ca⁺⁺] constant, the same results are obtained except that the duration of the a. p. remains unaltered. Thus, effects a-d appear to be related to [Ba⁺⁺] and not to [Ca⁺⁺] or [Cl^-]. The degree of overshoot in 1 mM Ca is linearly related to log [Ba⁺⁺] with a slope of approximately 22 mv. With the ratio [Ba⁺⁺]/[Ca⁺⁺] constant, the slope closely approaches the ideal value of 29 mv. The evidence indicates that prolongation of the action potential is due to a delayed onset of Ba inactivation, and that this in turn is a function of surface-bound Ba. Other features of the action potential are absolute refractoriness during its rising and plateau phases, relative refractoriness lasting several seconds, and repetitive firing in response to steady current depolarization. The response is unaffected by TTX and TEA. Mn prolongs the action potential. Sr has an action similar to Ba, whereas the addition of K, Na, Rb, or Mg to the basic calcium medium is unaccompanied by all-or-none electrogenesis.On leave of absence from the Zoological Institute, Faculty of Science, University of Tokyo.Support came from National Science Foundation grant GB-5752x, U.S.P.H.S. grant NB-03664, and in part from Office of Naval Research grant Nonr 4785(00) administered by the Marine Biological Laboratory, Woods Hole.     

Consequences of skin color and fur properties for solar heat gain and ultraviolet irradiance in two mammals

Summary In animals with fur or feather coats, heat gain from solar radiation is a function of coat optical, structural, and insulative characteristics, as well as skin color and the optical properties of individual hairs or feathers. In this analysis, I explore the roles of these factors in determining solar heat gain in two desert rodents (the Harris antelope squirrel,Ammospermophilus harrisi, and the round-tailed ground squirrel,Spermophilus tereticaudus). Both species are characterized by black dorsal skin, though they contrast markedly in their general coat thickness and structure. Results demonstrate that changes in coat structure and hair optics can produce differences of up to 40% in solar heat gain between animals of similar color. This analysis also confirms that the model of Walsberg et al. (1978) accurately predicts radiative heat loads within about 5% in most cases. Simulations using this model indicate that dark skin coloration increases solar heat gain by 5%. However, dark skin significantly reduces ultraviolet transmission to levels about one-sixth of those of the lighter ventral skin.Symbols and abbreviations: (unless noted, all radiation relations refer to total solar radiation) absorptivity of individual hairs - _C absorptivity of the coat - backward scattering coefficient [reflectivity] of individual hairs - _C reflectivity of coat - _S reflectivity of skin - forward scattering coefficient [transmissivity] of individual hairs - _C transmissivity of coat - _S transmissivity of the skin - transmissivity of the coat and skin - transmissivity of the coat to ultraviolet radiation - _S transmissivity of the skin to ultraviolet radiation - [(1 – )² – ²] - h _C coat thermal conductance [W/m²-°C] - h _E coat surface-to-environment thermal conductance [W/m²-°C] - I probability per unit coat depth that a ray will be intercepted by a hair [m^–1] - K volumetric specific heat of air at 20°C [1200 J/m³-°C] - l _C coat thickness [m] - l _H hair length [m] - d hair diameter [m] - n hair density per unit skin area (m^–2] - Q _ABS heat load on animal's skin from solar radiation [W/m²] - Q _I solar irradiance at coat surface [W/m²] - r _E external resistance to convective and radiative heat transfer [s/m] - r _C coat thermal resistance [s/m]     

Adrenergic regulation of chloride secretion across the opercular epithelium: The role of cyclic AMP

Summary Activation of the -adrenergic receptors of the opercular epithelium ofFundulus heteroclitus stimulates Cl^– secretion, while activation of the -adrenergic receptors inhibits Cl^– secretion (Degnan and Zadunaisky, 1979). The possible involvement of adenosine 3, 5-monophosphate (cAMP) in these adrenergic responses was investigated. Isolated opercular epithelia incubated in Ringer, containing 10 mM theophylline, had cAMP levels ranging between 5.3 and 19.3 pmoles·mg protein^–1 (mean=9.5±1.0 pmoles·mg protein^–1). Activation of the -receptors by 10^–5 M isoproterenol increased the mean cAMP level 430% (P<0.001). Blockage of the -receptors with propranolol greatly reduced the increase in cAMP in response to isoproterenol. Activation of the -receptors by 10^–5 M arterenol stimulated the mean cAMP level 270% (P<0.01). However, when the -receptors were blocked with propranolol, arterenol had no effect on the cAMP level. The possible involvement of Ca⁺⁺ in these adrenergic responses was investigated. Neither the stimulatory effect of isoproterenol, nor the inhibitory effect of arterenol on the Cl^– secretion were diminished in the absence of extracellular Ca⁺⁺. The Ca⁺⁺ ionophore, A23187, and the calmodulin inhibitor, trifluoperazine, had no effects on the Cl^– secretion. The Ca⁺⁺-channel blocker, D600, had a significant inhibitory effect (P<0.005). Guanosine 3,5-monophosphate (cGMP) had no effect on the Cl^– secretion.The results indicate that -adrenergic stimulation of Cl^– secretion across the opercular epithelium is accompanied by an elevation in tissue cAMP levels. -adrenergic inhibition of Cl^– secretion does not involve changes in the tissue cAMP. Neither of these responses appear to require Ca⁺⁺.     

Glutaraldehyde-fixation of yeast cells: Kinetics of a model system for enzyme cytochemistry

The kinetics of glutaraldehyde inactivation of a protoplasmic (-fructofuranosidase) and an extracytoplasmic (acid phosphatase) enzyme inSaccharomyces rouxii cells were studied at pH 5.5 and 30°C. The effects of glutaraldehyde concentration (0.5–3%), pH value, and temperature were surveyed by varying the fixation conditions. Cells from 1- to 10-day cultures retained 50–75% of their acid phosphatase activity and 15–24% of their -fructofuranosidase activity after 1-h exposures to 0.5% glutaraldehyde. The surviving -fructofuranosidase activity remained physically cryptic and was revealed only after further membrane perturbation with ethyl acetate. This crypticity barrier disappeared after overnight incubation of the treated cells at 4°C, with or without added glutaraldehyde, during which time the enzyme was resistant to further inactivation. The velocity ratio for raffinose versus sucrose, as substrate, decreased in treated cells, and changes inV _max andK _m were indicative of frank destruction of some enzyme molecules as well as modification of survivors. A comparable set of changes was also generated by treating cell-free extract with glutaraldehyde. Glutaraldehyde (0.5%) killed all yeast cells at 30°C within 5 min; at 4°C survival rates were quite high—81% after 15 min and 65% after 1 h. The bearing of these examples of enzyme inactivation, permeability barrier abolition, and structural stabilization on the general problems of yeast cytochemistry is discussed.     

Interaction of ruthenium red with isolated sarcolemma

Summary The stain ruthenium red binds very strongly to isolated sarcolemma and the maximal binding is about 125 nmoles/mg protein, decreasing slightly in lipid-extracted membranes. The binding is half maximal when the free stain concentration is about 1.0 M, for both intact and lipid-depleted material. The nucleotide, ATP, reduces markedly the binding and the apparent affinity of the membranes for the stain. Minute concentrations of ruthenium red (10 M) inhibit by 80 to 90% the Ca⁺⁺-binding by sarcolemma. The inhibition does not depend on the Ca⁺⁺ concentration and is similar in both intact and lipid-extracted preparations. Ruthenium red inhibits the ATPase activity of sarcolemma. The inhibition is decreased by increasing the ATP concentration in the medium.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia

Summary Pure lateral (L) cilia may be separated from the remaining (R) cilia types ofMytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with³²P-labeled ATP at low Ca⁺⁺ (10^–7 M), where L cilia beat, or in high Ca⁺⁺ (2–20 M), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5–15% gels, and autoradio-graphed. Neither cilia type undergoes Ca⁺⁺-dependent phosphorylation of specific proteins, suggesting that neither Ca⁺⁺-induced arrest in L cilia nor the Ca⁺⁺ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca⁺⁺-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca⁺⁺-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP₂). PIP₂ increases at least 3-fold under Ca⁺⁺-arrest conditions.Aequipecten gill lateral cilia, which require higher Ca⁺⁺ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca⁺⁺ levels. Phosphorylation of other lipids is Ca⁺⁺-independent. In the Ca⁺⁺-insensitive or activated R cilia, PIP₂ levels are intermediate, increasing only marginally with increased [Ca⁺⁺]. The formation of PIP₂ in response to Ca⁺⁺, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca⁺⁺ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca⁺⁺ ions, acting directly on the axoneme. After Ca⁺⁺-induced arrest, the formation of PIP₂ may be involved in sequestering Ca⁺⁺ or in augmenting Ca⁺⁺ pump activity, thus reducing Ca⁺⁺ levels so that motility may resume quickly.     

Resistance adaptation of the freshwater crayfish and thermal inactivation of membrane-bound enzymes

Summary Heat death and resistance adaptation of freshwater crayfish are thought to be properties of its muscle membranes. The inactivation at high temperatures of a membrane-bound enzyme, the Ca⁺⁺-stimulated ATPase of crayfish abdominal muscle sarcoplasmic reticulum, and the effect of thermal acclimation of crayfish upon the inactivation kinetics have been investigated. In the absence of KCl, the Ca⁺⁺-stimulated ATPase is irreversibly inactivated with pseudo-first order kinetics at temperatures that cause heat death in the whole animal. 0.1–10.0 mM KCl resulted in slower inactivation, while 100 mM KCl activated the enzyme to 120–180% of its original activity. Enzyme activation by KCl and heat involved a shift in the enzyme concentration/activity curve. Thermal acclimation of crayfish had no significant effect upon the kinetics or Arrhenius activation energy for enzyme inactivation (100.6±10.5 and 92.3±14.6 kcal/mole for preparations from 4°C and 25°C acclimated crayfish).Ca⁺⁺-stimulated ATPase isolated from heat dead crayfish exhibited normal in vitro activity due presumably to the high intracellular K⁺ concentration. Nevertheless, the close correspondence between heat death temperatures and inactivation temperatures for several membrane-bound enzymes of muscle is thought to reflect some perturbation of muscle structure that occurs during heat death.Abbreviations ATP Ademosine 5-Triphosphate - EGTA Ethyleneglycol-bis [-amino-ethyl ether] - N N-tetraacetic acid - Hepes N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic acid - FSR Fragmented sarcoplasmic reticulum - Tris Tris (hydroxymethyl)aminomethane     

Calcium entry in rabbit corneal epithelial cells: Evidence for a nonvoltage dependent pathway

We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca⁺⁺ channels, Na⁺/Ca⁺⁺ exchange, and nonvoltage-dependent Ca⁺⁺-permeable channels. Whole cell inward currents carrying either Ca⁺⁺ or Ba⁺⁺ were not detected using voltage clamp techniques. We also used imaging technology and the Ca⁺⁺-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca⁺⁺ concentration ([Ca]_i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 m), or Ni⁺⁺ (40 m), a blocker of many voltage-dependent calcium channels, did not affect [Ca⁺⁺]_i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca⁺⁺]_i. These results are inconsistent with the presence of voltage gated Ca⁺⁺ channels. Nonvoltage gated Ca⁺⁺ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K⁺ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn⁺⁺ and bath perfusion with 5 mm Ni⁺⁺ decreased [Ca⁺⁺]_i suggesting that the Ca⁺⁺ conductance was blocked. These results are most consistent with a nonvoltage gated Ca⁺⁺ influx pathway. Finally, replacing extracellular Na⁺ with Li⁺ resulted in an increase in [Ca⁺⁺]_i if the cells were first Na⁺-loaded using the Na⁺ ionophore monensin and ouabain, a Na⁺-K⁺-ATPase inhibitor. These results suggest that Na⁺/Ca⁺⁺ exchange may also regulate [Ca⁺⁺] in this cell type.The authors are grateful to Chris Bartling for expert technical assistance with the imaging experiments, Helen Hendrickson for cell preparation, and Jonathon Monck for helpful discussions regarding imaging technology. This work was supported by National Institutes of Health grants EYO3282, EYO6005, DK08677, and an unrestricted award from Research to Prevent Blindness.     

Calcium efflux from human erythrocyte ghosts

Summary The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with⁴⁵Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that⁴⁵Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1mm Ca in the external solution ([Ca⁺⁺] _o ), the rate constant of passive Ca efflux (k min^–1, fraction of⁴⁵Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min^–1. There was no further increase at higher [Ca⁺⁺] _o . The relation between the rate constant of Ca efflux and [Ca⁺⁺] _o is thus characterized by saturation kinetics. The passive transfer system for Ca could also be activated by Sr. The alkali metal ions Na, K and Li did not seem to have any significant influence on passive Ca transfer. The passive Ca efflux was slightly inhibited by Mg and strongly inhibited by Pb. Under most experimental conditions, a fraction of 15 to 50% of the intracellular Ca seemed to be inexchangeable. The inexchangeable fraction decreased with increasing [Ca⁺⁺] _o and increased with increasing [Ca⁺⁺] _i . It was not influenced by alkali metal ions, CN or Pb, but it could be completely removed from the cells by the addition of 0.1mm Mersalyl to the incubation medium or by hemolysis with addition of a detergent. The active ATP-dependent Ca transport differed characteristically from passive transfer; the rate constant decreased with increasing [Ca⁺⁺] _o , and the inexchangeable Ca fraction increased with increasing [Ca⁺⁺] _o . The experimental results suggest that there exists a carrier-mediated Ca–Ca exchange diffusion in the erythrocyte membrane and that only a fraction of the ghost cell population participates in the Ca exchange diffusion.     

Cytochemical localization of myrosinase (β-thioglucosidase) in root tips ofSinapis alba

Summary A method for electron microscopic cytochemical localization of a-thioglucosidase (myrosinase) has been developed. Since sulphate is one of the products of the hydrolysis of sinigrin by myrosinase, it was felt that if the incubation was carried out in the presence of Pb⁺⁺-ions an insoluble precipitate of electron-dense PbSO₄ would be formed at the reaction sites. Following formaldehyde fixation a few different cell organelles in the extreme root tip ofSinapis alba showed reaction specificity for myrosinase but following glutaraldehyde fixation the enzymatic activity was inhibited. Biochemical tests of the isolated enzyme showed complete inhibition of the myrosinase by glutaraldehyde. Variations in the substrate concentration and incubation time indicated that the enzyme was confined to the dilated cisternae of the endoplasmic reticulum and in a limited extent to the mitochondria.     

A minimum mechanism for Na+−Ca++ exchange: Net and unidirectional Ca++ fluxes as functions of ion composition and membrane potential

Summary Both simultaneous and consecutive mechanisms for Na⁺–Ca⁺⁺ exchange are formulated and the associated systems of steady-state equations are solved numerically, and the net and unidirectional Ca⁺⁺ fluxes computed for a variety of ionic and electrical boundary conditions. A simultaneous mechanism is shown to be consistent with a broad range of experimental data from the squid giant axon, cardiac muscle and isolated sarcolemmal vesicles. In this mechanism, random binding of three Na⁺ ions and one Ca⁺⁺ on apposing sides of a membrane are required before a conformational change can occur, translocating the binding sites to the opposite sides of the membranes. A similar (return) translocation step is also permitted if all the sites are empty. None of the other states of binding can undergo such translocating conformational changes. The resulting reaction scheme has 22 reaction steps involving 16 ion-binding intermediates. The voltage dependence of the equilibrium constant for the overall reaction, required by the 31 Na⁺Ca⁺⁺ stoichiometry was obtained by multiplying and dividing, respectively, the forward and reverse rate constants of one of the translocational steps by exp(–FV/2RT). With reasonable values for the membrane density of the enzyme (120 sites m²) and an upper limit for the rate constants of both translocational steps of 10⁵·sec^–1, satisfactory behavior was obtainable with identical binding constants for Ca⁺⁺ on the two sides of the membrane (10⁶ m ^–1), similar symmetry also being assumed for the Na⁺ binding constant (12 to 60m ^–1). Introduction of order into the ion-binding process eliminates behavior that is consistent with experimental findings.     

Characteristics of tree bark as an indicator in high-immission areas

Summary In the immission area of Frankfurt a.M. measurements of pH value, sulfur content and concentrations of Ca⁺⁺, K⁺ and Na⁺ of tree bark have been made. In the case of the deciduous trees Fraxinus excelsior, Acer platanoides and Tilia spp. all parameters can be used as an indicator for the degree of immissions. Even in the desert of lichens a division into four different zones of load is possible. Because in the Rhein-Main area winds from the southwest predominate, the size of zones of greatest air pollution depends on the distribution of winds. Correlations between the sulfur content of bark and the arithmetical mean value of SO₂ in the air have been found. The sulfur values of bark range from 50 to 270 g/cm², the SO₂ values from 0.08 to 0.13 mg/m³. A correlation between electric conductivity and Ca⁺⁺ values in the bark suspension was also found. With distance from the immission center the Ca⁺⁺ content of bark is decreasing. In the immission area of Frankfurt a.M. pH value and sulfur content of bark of deciduous trees must be regarded as indicators for air quality and the Ca⁺⁺ values as indicators for dust immissions.This paper is part of a doctoral thesis presented by the junior author to the Fachbereich Biologie, J.W. Goethe-Universität, Frankfurt a.M.     

Further studies on the ultrastructural localization of a magnesium dependant neutral ATPase in arteries

Summary A further analysis of the ultrastructural localization of a Mg⁺⁺ dependant neutral ATPase in arteries (thoracic aorta and basilar artery) has been performed in light of recent findings concerning the use of differential fixation and pitfalls in the standard Wachstein-Meisel (W-M) technique. The localization of reaction product was documented following fixation in 5% and 10% formaldehyde and 5% glutaraldehyde, and following incubation in the standard W-M media with ATP, AMP, -glycerophosphate as substrates. These results were compared to the localization using a modified W-M medium with ATP in which the lead ion concentration was reduced to 1.8 mM. Using the standard W-M procedure, formalin fixation gave a more intense but also a more diffuse (both intra- and extracellular) precipitate of reaction product than glutaraldehyde. The localization to cell structure remained the same in both cases, namely to the outer cell membrane, within its invaginations and in pinocytotic vesicles of both endothelial and smooth muscle cells. Following incubation in a medium with lower lead ion concentration, less extracellular precipitate was found and following glutaraldehyde fixation, very sparse precipitate of reaction product was localized to the cell membrane and its invaginations, often on the cytoplasmic side. The reduction of extracellular precipitate following pre-incubation in 5 mM cystein was believed to be due to inhibition of an unspecific alkaline phosphatase and phosphomonoesterase which had diffused out of the cell following fixation. Cysteine had no effect on the ATPase of the vascular wall. The significance of these results was discussed in light of previous studies on blood vessels and newer insights into this technique.     

The effects of varying key steps in the non-radioactive in situ hybridization protocol: a quantitative study

Summary In situ hybridization represents a major advance in the study of gene expression and, thus, in the evaluation of cellular function in histological sections. The availability of oligonucleotide probes labelled with biotin and sensitive immunohistochemical detection systems makes the study of different types of mRNA by in situ hybridization easier. However, a large number of protocols have been reported, which is sometimes confusing. The present study analyses quantitatively the influence of each important step of in situ hybridization on the staining intensity of rat proinsulin mRNA. The aim was to optimize technical conditions, to make the method sensitive and to evaluate its reproducibility for proinsulin mRNA detection and measurements. The duration of fixation and the digestion have an important impact on the results. The optimal digestion time depends on the fixation. With a digestion of 30 g ml^–1 proteinase K for 12 min at 37°C, the optimal fixation time was 24 h. Section thickness also influences the staining intensity. The intensity of the staining increases as the section thickness increases from 3 to 5 m before slowly decreasing. A weak paraformaldehyde post-fixation (0.4% for 20 min) gives best results in comparison to a stronger post-fixation (4% for 10 min). An increase of probe concentration leads to a higher specific labelling, reaching a plateau at 800 ng ml^–1. Hybridization temperature (37–42°C) exerts little influence. However, the temperature of the washes and the immunodetection system have a major effect on the intensity of labelling.Quantification has permitted the evaluation of the influence of each key for optimizing and standardizing the non-radioactive in situ hybridization protocol. In these well-defined conditions, the intra and inter-assay coefficient of variation remains lower than 6% and thus the method can be used to quantify the content of proinsulin mRNA or other specific mRNAs in experimental and pathological conditions.     

Calcium binding to the subunit c ofE. coli ATP-synthase and possible functional implications in energy coupling

The 8-kDa subunit c of theE. coli F₀ ATP-synthase proton channel was tested for Ca⁺⁺ binding activity using a⁴⁵Ca⁺⁺ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds⁴⁵Ca⁺⁺ strongly with Ca⁺⁺ binding properties very similar to those of the 8-kDa CF₀ subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca⁺⁺ binding capacity, shown by loss of Ca⁺⁺ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca⁺⁺ binding, shown by Ca⁺⁺ binding being retained in twoE. coli mutants, Asp61Asn and Asp61Gly. The Ca⁺⁺ binding is pH dependent in both theE. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca⁺⁺ binding affinity to its F₀ binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca⁺⁺ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca⁺⁺ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80–100 nM was obtained using an EGTA-Ca⁺⁺ buffer system to control free Ca⁺⁺ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca⁺⁺ bound to the periplasimic side of theE. coli F₀ proton channel can block H⁺ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca⁺⁺ binding site is on the lumen side of the thylakoid, where Ca⁺⁺ binding can modulate acid-base jump ATP formation. The Ca⁺⁺ binding to the F₀ and CF₀ complexes is consistent with a pH-dependent gating mechanism for control of H⁺ ion flux across the opening of the H⁺ channel.This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia.