Crotonobetaine reductase fromEscherichia coli — a new inducible enzyme of anaerobic metabolization of L(-)-carnitine

Crotonobetaine reductase fromEscherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, -butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, -butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40–45 °C). The K _m value for crotonobetaine was determined to be 1.1×10^–2M. -Butyrobetaine,^D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For -butyrobetaine (K _i =3×10^–5M) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Production,purification, and properties of β-glucosidase from Candida wickerhamii

Summary Candida wickerhamii growing on cellobiose produced -glucosidase with high activity against -nitrophenyl glucoside (PNPG) but low activity against cellobiose. -glucosidase production was constitutive, and was repressed by -glucosides and glucose. -glucosides containing an aromatic moiety in the aglycon were the best substrates for -glucosidase indicating that the enzyme is an aryl--glucosidase. A -glucosidase from C. wickerhamii cells was purified by (NH₄)₂SO₄ precipitation, dialysis, ion-exchange chromatography and gel filtration. The purified enzyme was homogeneous as shown by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme hydrolysed PNPG but not cellobiose. The K_m of the enzyme was 0.185 mM. Glucose inhibited the enzyme competitively and the K_i was 7.5 mM. The apparent molecular mass was 97,000. The optimum pH and temperature for enzyme activity were between pH 7 and 7.4 and 40°C respectively. At temperatures of 45°C and greater the enzyme was inactivated. The activation energy of the enzyme was 29.4 kJ · mol^-1.     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Hydrodynamic characteristics of two-phase inverse fluidized bed

Hydrodynamic characteristics of a new mode of liquid-solid fluidization, termed as inverse fluidization in which low density floating particles are fluidized with downward flow of liquid, are experimentally investigated. The experiments are carried out with low density particles (<534 kg/m³) which allow high liquid throughputs in the system. During the operation, three regimes, namely, packed, semi-fluidization and fully fluidization are encountered. Empirical correlations are proposed to predict the pressure drop in each regime. A computational procedure is developed to simulate the variation of pressure drop with liquid velocity.List of Symbols Ar modified Archimedes number, d _p ³ (– _s)g/² - d _p particle diameter, mm - f friction factor (eq. 2) - g acceleration due to gravity, m/s² - H total bed height, m - H _c height of the column, m - H_f height of fluidized bed, m - H₀ height of initial bed, m - H_p height of the packed bed, m - (p) pressure drop across the bed, N/m² - (p) _f pressure drop across fluidized bed section, N/m² - (p) _p pressure drop across the packed bed section, N/m² - (p) _sf total pressure drop in semifluidization regime, N/m² - Re Reynolds number, d _pU ₁/ - Re_m modified Reynolds number, d _pU ₁/(1– _p) - U ₁ superficial liquid velocity, m/s - U_mf minimum fluidization velocity, m/s - U_osf onset fluidization velocity, m/s Greek Letters _f voidage of fluidized bed - _p voidage of packed bed - liquid viscosity, kg/ms - liquid density, kg/m³ - _s particle density, kg/m³     

Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles

Summary Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H⁺ uptake as visualized by the pH indicator, acridine organge. The reoriented H⁺-pump is electrogenic because permeant extravesicular anions or intravesicular K⁺ plus valinomycin enhance H⁺ transport. ATP stimulates H⁺ uptake with an apparentK _m of 93 m. Support of H⁺ uptake andP _i liberation by ATP>GTPITP> UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H⁺ uptake,(K _i , 24 m). Mg²⁺ and Mn^2– support ATP-driven H⁺ uptake, but Ca²⁺, Ba²⁺ and Zn²⁺ do not. Imm Zn²⁺ inhibits MgATP-driven H⁺ transport completely. NEM-sensitiveP _i liberation is stimulated by Mg²⁺ and Mg^2– and, unlike H⁺ uptake, also by Ca²⁺ suggesting Ca²⁺-dependent ATP hydrolysis unrelated to H⁺ transport. The inside-out oriented H⁺-pump is relatively insensitive toward oligomycin, azide, N,N-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparentK _i , 0.77 m), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparentK _i , 0.39 m). Taken together, the H⁺-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of vacuolar type (V-type) H⁺-ATPases. Hence,V-type H⁺-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.     

Age and Growth of the Whiskery Shark, Furgaleus macki, from Southwestern Australia

Age and growth of the whiskery shark, Furgaleus macki, from southwestern Australia were examined using vertebral ageing and tag-recapture data. The readability of bands on the vertebral centra varied markedly between individuals. Four readers were used to make band counts, with the most experienced reader having the lowest index of average percent error and the highest level of agreement with final counts. Marginal increment analysis indicated that opaque bands form in January. With parturition occurring from August to October, size data suggests that the first band is probably formed 15–17 months after birth. The age at maturity was estimated to be 4.5 years for males, and 6.5 years for females. The oldest male was 10.5 years, and oldest female was 11.5 years. Von Bertalanffy growth parameters for males were L =121.5cm fork length, K=0.423 year^–1, t ₀=–0.472 years, were L =120.7cm fork length, K=0.369 year^–1, t ₀=–0.544 years for females, and were L =118.1cm fork length, K=0.420 year^–1, t ₀=–0.491 years for combined sexes. Data from a tag recapture study were analysed using a maximum likelihood method to verify the estimates of growth parameters from vertebral ageing. Von Bertalanffy growth parameters from the tag recapture study were L =128.2cm fork length, K=0.288 year^–1, t ₀=–0.654 years. The two methods of estimating growth parameters produced similar results, with rapid growth until approximately 5 years of age, after which there was little increase in length.     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

The quantitative analysis of chromosome pairing and chiasma formation based on the relative frequencies of M I configurations

Whilst reliable estimates of chiasma frequencies can usually not be obtained, the probability (b) of a chromosome arm to be bound by at least one chiasma can often be determined. In the absence of interference this probability equals (1–e ^–2), where 2 is the average chiasma frequency of the chromosome arm and the average crossover frequency or map length. In the presence of interference is shown to retain its genetic meaning as an additive metric that may describe the chromosome arm or other distinctive chromosome segment in terms of genetic recombination. It is a form of potential map length, comparable to, but numerically different from the regular map length. It is termed provisionally crossing-over potential.A chromosome with armsm andn with crossing-over potentials and will form ring bivalents with a frequency (1–e ^–2).(1–e ^–2); open bivalents with a frequency (1–e ^–2).e ^–2+(1–e ^–2).e ^–2; univalent pairs with a frequencye ^–2.e ^–2. Estimates of these frequencies yield equations from which and may be solved. In rye (Secale cereale) their ratio (q) is approximately two and differs from the mitotic arm length ratio of 1.4, indicating localization of chiasmata in the long arms.Graphs are given to show how, with constantq, the relation between the probabilitiesb _m andb _n of the two arms being bound changes with changing averageb.Data are presented on chiasma frequencies in M I, and compared with the frequencies expected in the absence of interference to give an impression of the degree of interference. Apparent fusion of chiasmata simulates interference.     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

Purification and properties of gammagamma-enolase from pig brain

Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as - (pI = 6.5), - (pI = 5.6), and -enolase (pI = 5.2). The pI of purified -enolase was also 5.2. The -enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55°C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%–80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, -enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The K _m values for 2-PGA, PEP, and Mg²⁺ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to K _m values of other vertebrate enolases. The amino acid composition of pig -enolase, as determined by amino acid analysis, shows strong similarity to the compositions of -enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig -enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig -enolase and the other -enolases are almost identical. Li⁺ proved to be a noncompetitive inhibitor with either 2-PGA or Mg²⁺ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P2₁. An R _symm <5% was obtained for data between 50 and 3.65 Å, but was a disappointing 30% for data between 3.65 and 3.10 Å, indicating crystal disorder.     

Action potentials inAcetabularia: Measurement and simulation of voltage-gated fluxes

Summary Amounts and temporal changes of the release of the tracer ions K⁺ (⁸⁶Rb⁺),²²Na⁺, and³⁶Cl^– as well as of H⁺ in the course of action potentials inAcetabularia have been recorded. New results and model calculations confirm in quantitative terms the involvement of three major ion transport systemsX in the plasmalemma: Cl^– pumps, K⁺ channels, and Cl^– channels (which are marked in the following by the prefixes,P, K andC) with their equilibrium voltages _X V _e and voltage/time-dependent conductances, which can be described by the following, first approximation. Let the maximum (ohmic) conductance of each of the three populations of transporter species be about the same (_P L, _KL,_C L=1) but voltage gating be different: the pump ( _{p V e} about –200 mV) being inactivated (open,oclosed,c) at positive going transmembrane voltages,V _m; the K⁺ channels (_K V _e about –100 mV) are inactivated at negative goingV _m; and the Cl^– channels (_C V _e: around 0 mV), which are normally closed (c) at a restingV _m (near_PV_e) go through an intermediate open (o) state at more positiveV _m before they enter a third shut state (s) in series. Model calculations, in which voltage sensitivities are expressed by the factorf=exp(V _mF/(2RT)), simulate, the action potential fairly well with the following parameters (_PK_co10/f ks^–1,_PK_oc1000·f ks^–1,_KK_co200·f ks^–1,_Kk_oc2/f ks^–1,_cK_co500·f ks^–1,_CK_oc5/f ks^–1,_CK_so0.1/f ks^–1,_Ck_os20·f ks^–1). It is also shown that the charge balance for the huge transient Cl^– efflux, which frequently occurs during an action potential, can be accounted for by the observation of a corresponding release of Na⁺.     

Competition between β-ketothiolase and citrate synthase during poly(β-hydroxybutyrate) synthesis inMethylobacterium rhodesianum

The enzymes -ketothiolase and citrate synthase from the facultatively methylotrophicMethylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The -ketothiolase had a relatively highK _m for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (K _i 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (K _m 0.07 mM) and was significantly inhibited by NADH (K _i 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined inM. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)^-1 during growth to the detection limit when poly(-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6–0.7, and 1.0 nmol (mg dry mass)^-1, respectively, were determined during growth and PHB synthesis. During growth, the -ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the -ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (K _mNADPH 18 M). PHB synthesis is probably mainly controlled by the CoA concentration inM. rhodesianum MB 126.Abbreviation PHB Poly(-hydroxybutyrate)     

Current chemical concepts of acids and bases and their application to anionic (“acid”) and cationic (“basic”) dyes

Summary In biomedical studies, dyes are divided into acid and basic dyes. This classification cannot be reconciled with current chemical definitions of acids and bases. Brönsted-Lowry acids are compounds that can donate protons; bases are proton acceptors. The definition of acids and bases is independent of the electric charge, i.e. acids and bases can be neutral, anionic or cationic. Reactions between acids and bases result in formation of new acid-base pairs. Lewis acids and bases do not depend on a particular element, but are characterized by their electronic configurations. Lewis bases are electron donors; Lewis acids are electron acceptors. This classification is also unrelated to the electric charge. Lewis acids and bases interact by formation of coordinate covalent bonds.In histochemistry and histology, dyes containing SO ₃ ^– , –COO^– and/or –O^– groups are classified as acid dyes. However, such compounds are electron pair donors and hence Brönsted-Lowry and Lewis anionic bases. Dyes carrying a positive charge are termed basic dyes. Chemically, many cationic dyes are Lewis acids because they can add a base, e.g. OH^–, acetate, halides. The hypothesis that transformation of –NH₂ into ammonium groups imparts basic properties to dyes is untenable; ammonium groups are proton donors and hence acids. Furthermore, conversion of an amino into an ammonium group blocks a lone electron pair and the color of the dye changes drastically, e.g. from violet to green and yellow. It appears therefore highly unlikely that ammonium groups are responsible for binding of cationic (basic) dyes. In histochemistry, it is usually not of critical importance whether anionic or cationic dyes are chemically acids or bases. It is therefore suggested to substitute the terms anionic for acid and cationic for basic dyes; this nomenclature will always be chemically correct.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

Beta-Adrenergic modulation of K+ current in human T lymphocytes

Summary The whole-cell voltage-clamp technique was employed to study the -adrenergic modulation of voltage-gated K⁺ currents in CD8⁺ human peripheral blood lymphocytes. The -receptor agonist, isoproterenol, decreased the peak current amplitude and increased the rate of inactivation of the delayed rectifier K⁺ current. In addition, isoproterenol decreased the voltage dependence of steady-state inactivation and shifted the steady-state inactivation curve to the left. Isoproterenol, on the other hand, had no significant effect on the steady-state parameters of current activation. The isoproterenol-induced decrease in peak current amplitude was inhibited by the -blocker propranolol. Bath application of dibutyryl cAMP (1mm) mimicked the effects of isoproterenol on both K⁺ current amplitude and time course of inactivation. Furthermore, the reduction in the peak current amplitude in response to isoproterenol was attenuated when PKI_5–24 (2–5 m), a synthetic peptide inhibitor of cAMP-dependent protein kinase, was present in the pipette solution. The increase in the rate of inactivation of the K⁺ currents in response to isoproterenol was mimicked by the internal application of GTP--S (300 m) and by exposure of the cell to cholera toxin (1 g/ml), suggesting the involvement of a G protein. These results demonstrate that the voltage-dependent K⁺ conductance in T lymphocytes can be modulated by -adrenergic stimulation. The effects of -agonists, i.e., isoproterenol, appear to be receptor mediated and could involve cAMP-dependent protein kinase as well as G proteins. Since inhibition of the delayed rectifier K⁺ current has been found to decrease the proliferative response in T lymphocytes, the -adrenergic modulation of K⁺ current may well serve as a feedback control mechanism limiting the extent of cellular proliferation.     

Effect of culture medium ions on chromate reduction by resting cells of Agrobacterium radiobacter

The influence of some ions in pre-growth culture medium on chromate reduction by resting cells of Agrobacterium radiobacter strain EPS-916 was investigated. The reduction was dependent on the Fe²⁺ content of the culture medium: the higher the iron content, the lower the reduction rate. The cells showed maximum chromate reduction when pre-grown in the presence of 0.243 m Mg²⁺, 20 m Ca²⁺ and 3.6 m Mn²⁺. Chromate reduction was not affected by the addition of MgCl₂, CdCl₂, ZnCl₂, MnCl₂, Na₂SO₄ (1000 m), and Na₂MoO₄ (100 m) to the activity assays. However, activity was inhibited by the presence of Na₂SO₄ (10 mm), Na₂MoO₄ (200 m) and ferric citrate.