Human Ribosomal Protein S26 Inhibits Splicing of Its Own Pre-mRNA

In vitro splicing was studied for a human ribosomal protein (rp) S26 pre-mRNA fragment containing the first exon, first intron, and a part of the second exon. Splicing yielded two products, one corresponding to a fragment of the mature rpS26 mRNA and the other retaining the 19 3-terminal nucleotides of the first intron between the first and second exons. Recombinant rpS26 inhibited generation of both splicing products in vitro. The inhibition was specific, because another recombinant human rp, S19, had no effect on the splicing of the pre-mRNA fragment. Toe-printing was used to map the rpS26-binding sites of the pre-mRNA in the regions of the conventional and alternative 3 splicing sites of the first intron. On the strength of the results, rpS26 was assumed to regulate the expression of its own gene at the level of pre-mRNA splicing via a feedback mechanism.     

Ribosomal Protein Binding with the First Intron of the Human rpS26 Pre-mRNA Stimulates Its Interaction with Proteins Extracted from HeLa Cells

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, proteins of the 40S ribosome subunit bind to the first intron of the rpS26 pre-mRNA. The binding involved mostly S23, S26 and, to a lesser extent, S13/16. Negligible binding was observed for S2/3a, S6, S8, S10, S11, and S20. Small-subunit proteins did not affect the efficiency of in vitro splicing of a pre-mRNA fragment corresponding to the first intron, second exon, second intron, and a part of the third exon of the rpS26 gene. However, ribosomal proteins substantially increased UV-induced adduction of the pre-mRNA fragments with nuclear extract proteins of HeLa cells. The same set of HeLa proteins was observed with each pre-mRNA fragment. Ribosomal proteins formed adducts only in the absence of HeLa proteins.     

Water chemistry changes in the gill micro-environment of rainbow trout: experimental observations and theory

Summary Soft water of low buffer capacity was drawn from near the branchial surface of rainbow trout (Salmo gairdneri) at 15°C, using opercular catheters, to determine pH changes in water passing over the gills. Latex masks allowed measurement of ventilation volume, and concentrations of carbon dioxide, oxygen, ammonia, and titratable base in expired water were compared to concentrations in inspired water. Water passing over the gills was more basic than inspired water if the inspired water was pH 4–6 (maximum increase: +0.7 pH units near pH 5). Expired water was more acidic than inspired water if the inspired water was pH 6–10 (maximum decrease: –1.7 pH units near pH 9). Ventilation volume (0.37 l·kg^–1·min^–1) and oxygen consumption (1.7 mmol·kg^–1·h^–1) were constant in the pH range 4.6–10.1, but both increased by 1.6–2.4× near pH 4. Carbon dioxide transfer near the gills was about 100 M, ammonia transfer about 15 M, and titratable base added at the gills was about 30 M. A theoretical model using CO₂, titratable base, and ammonia added at the gills, the titration characteristics of the defined soft water medium, and aquatic equilibria for CO₂ and ammonia, adequately explained the experimentally observed changes in pH near trout gills. Our observations and predictive model indicate that any gill contaminant whose toxicity varies with pH may be more or less toxic at the gills than predicted from bulk water chemistry alone.Abbreviations pH _ex expired pH - pH _in inspired pH     

Human ribosomal protein S13 inhibits splicing of its own pre-mRNA

Recombinant human ribosomal protein (rp) S13 was shown to specifically bind with its own pre-mRNA fragment containing the first exon, first intron, second exon, and a part of the second intron and to inhibit its splicing in vitro. The binding of rpS13 was specific: recombinant human rpS10 and rpS16 bound with the fragment to a lower extent. Moreover, rpS13 binding with the rpS13 pre-mRNA fragment was inhibited by non-labeled poly(AU) and an adenovirus pre-mRNA fragment to a lower extent than by the nonlabeled rpS13 pre-mRNA fragment. The specificity of splicing inhibition was inferred from the finding that, in contrast to rpS13, recombinant rpS10 and rpS16 did not affect the efficiency of first intron excision from the rpS13 pre-mRNA fragment. Enzymatic footprinting was used to determine the rpS13 pre-mRNA nucleotides whose accessibility to RNases T1, T2, and V1 changed in the presence of rpS13. Such nucleotides were detected close to the 5′ and 3′ splicing sites of the first intron. Analysis with the EMBOS-Align program showed that the nucleotide sequence of the first intron of the mammalian rpS13 pre-mRNA is conserved to a greater extent as compared with the other introns. It was assumed that the first intron plays an important role in regulating the expression of the rpS13 gene at the splicing level in all mammals.     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

Interaction of human S26 ribosomal protein with fragments of mRNA and pre-mRNA for this protein in Hela nuclear cell extracts

As shown by nitrocellulose filtration assays with RNA fragments transcribed from various regions of the human ribosomal protein (rp) S26 gene, recombinant rpS26 binds to the first intron of the rpS26 pre-mRNA (apparent association constant (Ka) approximately 5.0 x 10(7) M-1) and, to a lesser extent, to the rpS26 mRNA (Ka approximately 2.0 x 10(7) M-1). The binding was specific, since human rpS19 had an order of magnitude lower Ka with the first intron and did not bind with the rpS26 mRNA. Immunoassays with specific antibodies showed that rpS26 contained in the nuclear extract of HeLa cells binds to the first intron of its pre-mRNA and, less efficiently, to its mRNA. In either case, RNA binding substantially increased in the presence of recombinant rpS26. Along with other (48 K, 59 K) nuclear proteins, rpS26 was assumed to form complexes, the functional role of which is storage of pre-mRNAs inactive in splicing.     

Changes in carp myosin ATPase induced by temperature acclimation

Summary Myosins were isolated from dorsal ordinary muscles of carp acclimated to 10°C and 30°C for a minimum of 5 weeks and examined for their ATPase activities. Ca²⁺-ATPase activity was different between myosins from cold-and warm-acclimated carp, especially at KCl concentrations ranging from 0.1 to 0.2 M, when measured at pH 7.0. The highest activity was 0.32 mol P_i·min^-1·mg^-1 at 0.2 M KCl for cold-acclimated carp and 0.47 mol P_i·min^-1·mg^-1 at 0.1 M KCl for warm-acclimated fish. The pH-dependency of Ca²⁺-ATPase activity at 0.5 M KCl for both carp was, however, similar exhibiting two maxima around 0.3 mol P_i·min^-1·mg^-1 at pH 6 and 0.4 mol P_i·min^-1·mg^-1 at pH 9. K⁺(EDTA)-ATPase activity at pH 7.0 neither exhibited differences between both myosins. It increased with increasing KCl concentration showing the highest value of about 0.4 mol P_i·min^-1·mg^-1 at 0.6–0.7 M KCl. Actin-activated myosin Mg²⁺-ATPase activity was markedly different between cold-and warm-acclimated carp. The maximum initial velocity was 0.53 mol P_i·min^-1·mg^-1 myosin at pH 7.0 and 0.05 M KCl for cold-acclimated carp, which was 1.6 times as high as that for warm-acclimated carp. These differences were in good agreement with those obtained with myofibrillar Mg²⁺-ATPase activity between both carp. No differences were, however, observed in myosin affinity to actin. Differences in myosin properties between cold- and warm-acclimated carp were further evidenced by its thermal stability. The inactivation rate constant of myosin Ca²⁺-ATPase was 25·10^-4·s^-1 at 30°C and pH 7.0 for cold-acclimated carp, which was about 4 times as high as that for warm-acclimated carp. Light chain composition did not differ between both carp myosins. The differences in a primary structure of the heavy chain subunit was, however, clearly demonstrated between both myosins by peptide mapping.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5 dithio-bis-2-nitrobenzoic acid - DTT dithiothreitol - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Some regulatory aspects of [14C]methylamine influx intoPisum sativum L. cv. Feltham First seedlings

[¹⁴C]Methylamine influx intoPisum sativum L. cv. Feltham First seedlings showed Michaelis-Menten-type kinetics with apparentV _max=49.2 mol·g^-1 FW·h^-1 and apparentK _m=0.51 mM. The competitive interactions between ammonium and methylamine were most obvious when biphasic kinetics were assumed with saturation of the first phase at 0.05 mM. The inhibitor constant for ammonium (K _i)=0.027 mM. When [¹⁴C]methylamine was used in trace amounts with ammonium added as substrate, the influx of tracer showed Michaelis-Menten-type kinetics with apparentV _max=3.46 mol·g^-1 FW·h^-1 and apparentK _m=0.15 mM. The initial rate of net ammonium uptake corresponded with that found when [¹⁴C]methylamine was used to trace ammonium influx. The latter was also stimulated by high pH_o and inhibited by nitrate. Ammonium pretreatment±methionine sulphoximine or glutamine pretreatment of the seedlings inhibited subsequent [¹⁴C]methylamine influx, while methylamine or asparagine pretreatment stimulated [¹⁴C]methylamine influx. There was also a stimulatory effect of prior inoculation withRhizobium. The results are discussed in terms of current models for the regulation of ammonium uptake in plants.     

Ion-Exchange Properties of Cell Walls Isolated from Lupine Roots

Acid–base properties of cell walls isolated from various root tissues of 7-day-old lupine seedlings and 14-day-old lupine plants grown in various media were studied. The ion-exchange capacity of root cell walls was estimated at various pH values (from 2 to 12) and constant ionic strength (10 mM). The parameters determining the qualitative and quantitative composition of cell wall ionogenic groups along the root length and in its radial direction were estimated using Gregor's model. This model fits the experimental data reasonably well. Four types of ionogenic groups were found in the cell walls: an amino group (pK _a 3), two types of carboxylic groups (pK _a 5 and 7.3, the first being the carboxylic group of galacturonic acid), and a phenolic group (pK _a 10). The number of functional groups of each type was estimated, and the corresponding ionization constant values were calculated. It is shown that the chemical composition of the ionogenic groups was constant along the root length as well as in its radial direction and did not depend on either physiological state or root nutrition, while the number of different groups varied. The content of carboxylic groups of -D-polygalacturonic acid in the root cell walls of 14-day-old plants was shown to depend on the distance from the root tip, being maximal in the zone of lateral roots. The number of these groups was 10- and 2-fold less in the central cylinder compared to that of cortex for 14-day-old plants and 7-day-old seedlings, respectively.     

Passive ionic properties of frog retinal pigment epithelium

Summary The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was –88 mV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8–20mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductanceT _i of each membrane. For the apical membrane:T _K0.52,T _{HCO 3}=0.39 andT _Na=0.05, and its specific resistance was estimated to be 6000–7000 cm². From the basalT _K=0.90 and its specific resistance was estimated to be 400–1200 cm². From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110mm. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered tight. The shunt pathway did not distinguish between anions (HCO₃ ^–, Cl^–, methylsulfate, isethionate) but did distinguish between Na⁺ and K⁺.     

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

Endothelin-1 and insulin induce cellular inactivation of protein kinase FA/glycogen synthase kinase-3α in a common signaling pathway

In this study, we investigate the effects of endothelin-1 (ET-1) and insulin on the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) in rat adipocytes. The cellular activity of kinase F_A/GSK-3 is inhibited to 50% of control within 30 min when cells are treated with 1 nM ET-1 at 37°C; in addition, significant inhibition to 60% of control is observed at as low as 1 pM ET-1. Conversely, ET-1 at concentrations up to 1 nM has no direct effect on purified kinase F_A/GSK-3 in vitro. Immunoblotting analysis further reveals that the protein level of this kinase is not significantly changed when treated with 1 nM ET-1 for 30 min. Similar to ET-1, insulin as low as 10 nM can also induce inactivation of kinase F_A/GSK-3 to 50% of control in adipocytes when processed under identical conditions. Most importantly, when treated with both insulin and ET-1, the activity of kinase F_A/GSK-3 can be decreased only to 50% of control. Taken together, the results provide initial evidence that ET-1 and insulin may regulate this important multisubstrate/multifunctional protein kinase in a common signaling pathway in cells.     

Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump

A dual-wavelength fluorimeter was constructed, which used two light emitting diodes (LEDs) to excite the fluorescence dye RH 421 alternately with two different wavelengths. The ratio of the emissions at the two excitation wavelengths provided a drift-insensitive signal, which allowed detection of very small changes of the fluorescence intensity. Those small changes were induced by ion binding and release in conformation E₁ of the Na,K-ATPase. Titration experiments were performed to determine equilibrium dissociation constants (± standard deviation) for each step in the complete binding and release sequence: 0.12 ± 0.01 mM (E₂(K₂) KE₁), 0.08 ± 0.01 mM (KE₁ E₁), 3.0 ± 0.2 mM (NaE₁ E₁), 5.2 ± 0.4 mM (Na₂E₁ NaE₁) and 6.5 ± 0.4 m_M (Na₃E₁ Na₂E₁) at pH 7.2 and T=16°C. These numbers show that the affinities of the binding sites exposed to the cytoplasm, are higher for K⁺ than for Na⁺ ions, similar to what was found on the extracellular side. The physiological requirement for extrusion of Na⁺ from the cytoplasm, and for import of K⁺ from the extracellular medium seems to be facilitated not by favorable binding affinities in state E₁ but by the two ATP-driven reaction steps of the cycle, E₂(K₂) + ATP K₂E₁ · ATP and Na₃E₁ · ATP (Na₃) E_l-P, which border the ion exchange reactions at the binding sites in conformation E₁. Correspondence to: H.-J. Apell     

Intraparticle mass-transfer resistance and apparent time stability of immobilized yeast alcohol dehydrogenase

The stability and, consequently, the lifetime of immobilized enzymes (IME) are important factors in practical applications of IME, especially so far as design and operation of the enzyme reactors are concerned. In this paper a model is presented which describes the effect of intraparticle diffusion on time stability behaviour of IME, and which has been verified experimentally by the two-substrate enzymic reaction. As a model reaction the ethanol oxidation catalysed by immobilized yeast alcohol dehydrogenase was chosen. The reaction was performed in the batch-recycle reactor at 303 K and pH-value 8.9, under the conditions of high ethanol concentration and low coenzyme (NAD⁺) concentration, so that NAD⁺ was the limiting substrate. The values of the apparent and intrinsic deactivation constant as well as the apparent relative lifetime of the enzyme were calculated.The results show that the diffusional resistance influences the time stability of the IME catalyst and that IME appears to be more stabilized under the larger diffusion resistance.List of Symbols C _A, C_B, C_E mol · m^–3 concentration of coenzyme NAD⁺, ethanol and enzyme, respectively - C _p mol · m³ concentration of reaction product NADH - d _p mm particle diameter - D _eff m² · s^–1 effective volume diffusivity of NAD⁺ within porous matrix - k _d s^–1 intrinsic deactivation constant - K _A, K_A, K_B mol · m^–3 kinetic constant defined by Eq. (1) - K _A ^x mol · m^–3 kinetic constant defined by Eq. (5) - r _A mol · m^–3 · s^–1 intrinsic reaction rate - R m particle radius - R _v mol · m^–3 · s^–1 observed reaction rate per unit volume of immobilized enzyme - t _E s enzyme deactivation time - t _r s reaction time - V mol · m^–3 · s^–1 maximum reaction rate in Eq. (1) - V ^x mol · m^–3 · s^–1 parameter defined by Eq. (4) - V _f m³ total volume of fluid in reactor - w _s kg mass of immobilized enzyme bed - factor defined by Eqs. (19) and (20) - kg · m^–3 density of immobilized enzyme bed - unstableness factor - effectiveness factor - Thiele modulus - relative half-lifetime of immobilized enzyme Index o values obtained with fresh immobilized enzyme     

Über die Vitalfluorochromierung des Kernchromatins und der Chromosomen von HeLa-Zellen mit AMHA und die Bindung des Acridinfarbstoffs an DNA

Summary The fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations C _F10^–4 M and short incubation periods t _I2 h cell growth is not affected by the drug. But at higher C _F and longer t _I the population doubling time of the cell cultures rapidly increases, and finally the cells die.In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm^–1 between the 0-0-transitions of the long wave length ¹ L _a absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm^–1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission.Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nuceleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant K _F1=1,1·10⁵ M ^–1, binding parameter n ₁=0,15; the pre-intercalative or external binding 2, K _F2=6,9·10⁵ M ^–1, n ₂=0,21; the external binding 3, K _F3=2,8·10⁵ M ^–1, n ₃=0,55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant K _F1=1,1·10⁵ M ^–1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, K _F1=(1–4)·10⁴ M ^–1. Therefore, the amount of intercalated AMHA is also relatively high, and it is possible to visualize the DNA-bound fluorochrome in the nuclei and chromosomes of the living cells under the fluorescence microscope.     

Fluorescence and circular dichroism spectroscopic studies on bovine lactoperoxidase*

Intrinsic steady-state fluorescence of lactoperoxidase (LPO) and its ligand-bound complexes has been characterized as a structural probe of its structure in solution. On excitation at 295 nm, a broad emission maximum is observed around 338 nm for LPO and for its ligand-bound complexes. The quantum yield is 0.0185±0.0005 for LPO and indicates tryptophan heme energy transfer. Tryptophan residues are located away from heme and are approximately equally distributed among hydrophobic and hydrophilic environments. From Förster resonance energy transfer equations, the average distance between tryptophans and heme within the enzyme is computed to be 25.1±0.2 Å. These fluorescence properties are consistent with the recent theoretical three-dimensional model for LPO and reveal that Trp337 and Trp404 dominate the intrinsic fluorescence, and together contribute 64% of the observed intensity. The effects of the denaturing agents guanidine hydrochloride and urea on the intrinsic fluorescence of LPO and CD of the backbone amide chromophores have been examined. The considerably red shifted emission maximum at 356 nm indicates that tryptophans, buried in the hydrophobic environment, are exposed to the solvent on denaturation. A simple two-state transition between the native and denatured forms of the protein has been used to explain the results. [Denaturant]_1/2 5.5 M, determined from both these experiments, indicates that LPO is relatively stable toward the denaturing agents. Quenching studies using. I^–, Cs⁺ and polar neutral acrylamide are consistent with this picture. Acrylamide can penetrate the protein matrix. It is an efficient quencher and the quenching process is essentially homogeneous with all the tryptophans being accessible. Cs⁺ ion is a very inefficient quencher but the iodide ion shows the quenching process to be predominantly heterogeneous with widely differing tryptophan accessibility. The Stern–Volmer constants deduced are K ^sv =8.4±1.4 M^–1 and K ^sv =4.05±0.65 M_–1 for acrylamide and iodide quenching, respectively. The fractional accessibility, f _a , deduced is f _a =0.52±0.03 for iodide quenching.     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.     

Life history,population dynamics and production of Pontoporeia hoyi (Crustacea,Amphipoda) in relation to the trophic gradient of Lake George,New York

The life history characteristics, population dynamics and production of Pontoporeia hoyi in Lake George, New York, were studied from May 1981 through October 1982. P. hoyi, in terms of both density and standing crop, is the most prevalent member of the deep water macrobenthos of Lake George. It reproduces in the winter, with young being released in the late winter-early spring. At the southernmost study site, young released in the spring grew to 6–7 mm in length and bred during their first winter. At the remaining sites, P. hoyi required two years to complete its life cycle. This difference in life history characteristics can be related to food availability and temperature differences. The open waters of the south end of Lake George are not only more productive but are also more closely associated with the littoral zone, providing a wealth of bacteria-rich detritus for benthic deposit feeders. The greater food availability in the south basin of Lake George is reflected in significantly larger brood sizes and smaller size at maturity for P. hoyi populations from the south end of the lake.The southernmost study site has significantly greater P. hoyi density and standing crop than all other sites. The cohort of the year dominated density and standing crop at the southern site while the cohort of the previous year dominated standing crop at the other sites. Peak abundance ranged from 600 · m^–2 at the north site to 2 900 · m^–2 at the south site. Cohort production ranged from 2g · m^–2 at the north site to 15g · m^–2 at the south site.