Calcium is required for the increase of dark respiration during diurnal nitrogen fixation by Synechococcus RF-1

Under 12/12 h light/dark cycles, 1 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, pH 8.0) added at the start of the dark period, inhibited the increase of dark respiration which was associated with nitrogen fixation in Synechococcus RF-1. Twenty-five millimolar NaNO₃ added 30 min before the start of dark period suppressed this respiratory increase. If 1.25 mM CaCl₂ was added to the EGTA-treated sample from 3 to at least 10 h later in the dark period, a quick rise in respiratory rate was observed. This rise was also reduced by 25 mM NaNO₃. Extracellular Ca²⁺ appears to be required for the increase in dark respiration associated with the rhythmic appearance of nitrogenase activity in the dark cycle.     

外源褪黑素与钙离子互作对高温胁迫下黄瓜幼苗过氧化伤害的缓解效应

为了探明褪黑素(MT)和钙离子(Ca²⁺)在调控植物耐热性中是否存在互作关系,以黄瓜幼苗为试材,分析了内源MT和Ca²⁺对高温胁迫的响应;并通过叶面喷施100 μmol·L^-1 MT、10 mmol·L^-1 CaCl₂、3 mmol·L^-1乙二醇二乙醚二胺四乙酸(EGTA,Ca²⁺螯合剂)+100 μmol·L^-1 MT、0.05 mmol·L^-1氯丙嗪(钙调素拮抗剂,CPZ)+100 μmol·L^-1 MT、100 μmol·L^-1氯苯丙氨酸(p-CPA,MT合成抑制剂)+10 mmol·L^-1 CaCl₂和去离子水(H₂O),研究高温下(42/32 ℃)外源MT和Ca²⁺对黄瓜幼苗活性氧积累、抗氧化系统及热激转录因子(HSF)和热激蛋白(HSPs)等的影响。结果表明: 黄瓜幼苗内源MT和Ca²⁺均受高温胁迫诱导;外源MT可上调常温下钙调素蛋白(CaM)、钙依赖蛋白激酶(CDPK5)、钙调磷酸酶B类蛋白(CBL3)、CBL结合蛋白激酶(CIPK2)mRNA表达;CaCl₂处理的MT合成关键基因色氨酸脱羧酶(TDC)、5-羟色胺-N-乙酰转移酶(SNAT)和N-乙酰-5-羟色胺甲基转移酶(ASMT)水平也显著升高,MT含量快速增加。MT和CaCl₂可显著增强高温下黄瓜的抗氧化能力,减少活性氧(ROS)积累,同时上调HSF7、HSP70.1和HSP70.11 mRNA表达,从而减轻高温胁迫引起的过氧化伤害,植株热害症状明显减轻,热害指数和电解质渗漏率显著降低。加入EGTA和CPZ后,MT对黄瓜幼苗抗氧化能力和热激蛋白表达的促进效应明显减弱,Ca²⁺对高温下黄瓜幼苗过氧化伤害的缓解效应也被p-CPA逆转。可见,MT和Ca²⁺均可诱导黄瓜幼苗的耐热性,二者在热胁迫信号转导过程中存在互作关系。     

Protease inhibitors do not prevent the killing of cultured hepatocytes by cystamine

A study was made of the conditions of the killing of cultured hepatocytes by the reactive disulfide cystamine. Six to 12 mM cystamine killed up to 60% of the hepatocytes within 3 hours. The cytosolic calcium ion concentration rose prior to the loss of viability. Treatment with EGTA in a Ca2+-free medium lowered the initial Ca2+ concentration and prevented the rise in response to cystamine. However, there was no change in the number of dead cells. Furthermore, the sensitivity of cultured hepatocytes to cystamine was unaffected by the concentration of calcium in the culture medium. Addition to the culture medium of 3 protease inhibitors, leupeptin, antipain, or chymostatin, did not reduce the extent of cell killing by cystamine despite an inhibition of protein degradation. These data do not support the hypothesis that the toxicity of cystamine is necessarily mediated by proteases activated by a rise in the cytosolic calcium ion concentration.     

Twitches in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid

Single muscle fibers continue to twitch for up to 20 min when immersed in ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) solutions containing less than 10⁻⁸ M free calcium. Failure of the twitch results from reversible depolarization, which occurs after 15–20 min in EGTA. The results make it clear that external calcium or calcium in the transverse tubules play no essential part in action potential propagation or excitation-contraction coupling.     

Utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora for risk assessment of environmental chemicals.

The utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora crassa is compared with that of other eukaryotic assay systems for the evaluation of the mutagenic effects of environmental chemicals. In contrast to other in vitro specific-locus assays, the Neurospora assay can detect mutations not only at the ad-3A and ad-3B loci but also recessive lethal mutations elsewhere in the genome. Mutational damage in this system can be characterized readily by means of classical genetic techniques involving heterokaryon tests to determine genotype, and allelic complementation among ad-3B^R mutations. The percentages of ad-3B^R mutations showing allelic complementation with polarized or nonpolirized complementation patterns provide a presumptive identification of the genetic alterations at the molecular level in individual mutants. Dikaryon and trikaryon tests (using 3 strains carrying multilocus deletion mutations as tester strains) distinguish ad-3 mutations resulting from gene/point mutation, multilocus deletion mutation, and various types of multiple-locus mutation.

The array of ad-3 mutations recovered from forward-mutation experiments can be expressed in terms of Mutational Spectra, which make it possible to make comparisons of mutational types between different doses of the same mutagen, different mutagens, or the effects of the same mutagen on different strains.

Another important feature of this specific-locus assay system is that the effects of mutagens can be studied in both DNA excision repair-proficient (H-12) and -deficient (H-59) two-component heterokaryons to evaluate both quantitative and qualitative differences between the spectra of induced d-3     

Effect of the uvrD3 mutation on ultraviolet radiation-induced DNA-repair replication in Escherichia coli K12

Ultraviolet-radiation-induced DNA-repair replication was measured in wild-type, polA1, uvrD3, and uvrD3 strains of Escherichia coli K12. A large stimulation of repair replication was observed in the uvrD3 strain, compaired to the wild-type and polA1 strains. This enhanced repair replication was reduced in the polA1 uvrD3 strain. Therefore, a uvrD3 mutation appears to affect the amount of repair replication performed by DNA polymerase I. In the polA1 strain, there also appears to be an effect of the uvrD3 mutation on the amount of repair replication performed by DNA polymerase III (and/or II). The enhanced repair replication observed for the uvrD3 strains appears to be in response to the enhanced DNA degradation observed for these strains.     

Calcium-activated thiol-proteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol.

1. Rat erythrocytes were fused by incubation with benzyl alcohol and Ca2+. 2. Cell fusion was inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, Tos-Lys-CH2Cl, and to a lesser extent by Tos-Phe-CH2Cl. Phenylmethanesulphonyl fluoride, Tos-Arg-OMe and histamine did not inhibit cell fusion. 3. Gel electrophoresis of membrane proteins from "ghosts" of the erythrocytes treated with benzyl alcohol showed that a high-molecular-weight polymer was present: this was consistent with the entry into the cells of Ca2+ and the activation of a transglutaminase enzyme. 4. In the treated cells the proteins corresponding to bands 2 and 3 in human erythrocytes were decreased, and a polypeptide with a slightly greater mobility than band 3 was produced. 5. These changes were inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, and Tos-Lys-CH2Cl, but not by phenylmethanesulphonyl fluoride, Tos-Arg-OMe, or histamine. 6. The intramembraneous particles of the P-fracture face of cells treated with benzyl alcohol to induce fusion were decreased in number and were susceptible to cold-induced aggregation; both of these phenomena were markedly inhibited to EGTA, and partially inhibited by Tos-Lys-CH2Cl and N-ethylmaleimide. 7. These several observations indicate that a Ca2+-activated thiol-proteinase, which acts to degrade membrane proteins and to give freedom of lateral movement to intramembranous particles, may be essential feature of membrane fusion in this system. 8. It is suggested that this proteinase may act to degrade spectrin-binding proteins that attach band-3 protein to the erythrocyte cytoskeleton.     

EGTA和La3+对几种逆境诱导辣椒样半萜环化酶基因表达的影响

用钙离子螯合剂EGTA及细胞膜钙离子通道拮抗剂La³⁺预处理辣椒叶片,以破坏辣椒叶片中的钙信使系统,再用紫外线、CuCl₂、HgCl₂处理辣椒叶片,研究表明EGTA和La³⁺预处理未能降低CuCl₂、HgCl₂、UV诱导辣椒倍半萜环化酶活化的作用,EGTA预处理反而对CuCl₂、HgCl₂、UV的诱导辣椒倍半萜环化酶活性作用有一定的促进效应.单独用EGTA处理也能诱导离体辣椒叶片表现出倍半萜环化酶活性.Northen Blot分析结果表明,EGTA能诱导辣椒倍半萜环化酶基因转录.研究表明,在辣椒倍半萜环化酶基因表达过程中,还存在钙信使系统以外的信号传递途径;非生物诱发因子对倍半萜环化酶基因表达诱导作用与生物Elicitor的诱导作用在信号传递上有差异.     

The Use of Alizarin Red S To Detect and Localize Calcium in Gametophyte Cells of Ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca²⁺ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KC1 to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca²⁺. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca²⁺ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

拟南芥IAA酰胺合成酶GH3-6负调控干旱和盐胁迫的反应

生长素是一种重要的植物激素, 几乎参与了植物所有的生命活动过程。GH3-6具有IAA酰胺合成酶活性, 催化氨基酸与IAA形成IAA的氨基轭合物, 发挥暂时或永久灭活IAA的作用。该文探讨了GH3-6基因在拟南芥(Arabidopsis thaliana)逆境适应过程中的功能。结果显示GH3-6基因受干旱、ABA和高盐的诱导表达。与野生型相比, GH3-6基因过表达突变体dfl1-D对干旱的抗性明显减弱, 叶片失水速率更快。在抗盐方面, dfl1-D也显著弱于野生型。在3种逆境(干旱、ABA和高盐)胁迫下, GH3-6基因的高表达抑制了逆境响应基因RD22、KIN1、RD29A和DREB1A的表达。而且在干旱胁迫下, dfl1-D中ABA的含量明显低于野生型。研究结果证明, 高表达GH3-6基因负调控拟南芥对逆境的抗性。     

The relationship of 2-acetamidofluorene mutagenicity in plate tests with its in vivo liver cell component distribution and its carcinogenic potential

1. Using a plating technique, the mutagenic potentials of 2-acetamidofluorence (AAF) and N-hydroxy-AAF were examined after metabolic activation by liver preparations from different animals. Animals used were: male and female rats; male rats treated with 3-methylcholanthrene (MC); male rats treated with AAF; hamsters; guinea pigs; cotton rats and baboons. Irrespective of the animal susceptibility to AAF carcinogenesis, mutation frequency was always increased in the Salmonella typhimurium TA 1538 tester strain. Indeed, the greater response was found in the presence of liver from cotton rats, a species which is resistant to AAF-induced carcinogenesis.

2. Carcinogen binding, with labelled molecules, was also studied in liver cell constituents of rats, guinea pigs and cotton rats. A much better correlation was found between carcinogenicity and carcinogen binding, at least in those species studied, than between carcogenicity and plate test mutagenicity. The difficulty which this new information poses for the interpretation of plate tests is discussed.

Affinities of ATP for the dinitrophenol-induced ATPase

1. 1. The effect of Mg²⁺ on ATP-dependent processes catalysed by intact rat-liver mitochondria can be explained quantitatively by the formation of Mg-ATP complexes that cannot act as a substrate for the adenine nucleotide translocator.

2. 2. The dinitrophenol-induced ATPase is characterized by two affinities of ATP: K_m(1) = 6.7 μM and K_m(2) = 63 μM, which contribute to the extent of 70% and 30%, respectively, to the total ATPase activity under the standard conditions employed.

3. 3. K_m(1) of ATP is competitively increased by atractyloside, and is insensitive to changes in cation concentration or to oligomycin or aurovertin.

4. 4. K_m(2) is as sensitive to atractyloside as the K_m(1) and is also insensitive to oligomycin. However, it is increased by decreasing the cation concentration, and disappears in the presence of aurovertin.

5. 5. It is proposed that two conformations of the adenine nucleotide translocator exist, characterized by their different affinities for ATP. The distribution of the enzyme over these two conformations appears to be a function of the energy state of the mitochondria (coupled or uncoupled).

AUF1在胞质DNA抑制细胞葡萄糖代谢中的作用研究

目的 探讨AUF1在胞质DNA引起的细胞葡萄糖代谢应答中的作用及其机制。方法 (1)用核质分离技术分离细胞核与细胞质,并通过生物素-亲和素亲和层析技术分离细胞质中与胞质DNA(ISD)结合的蛋白质,然后通过“银染-质谱”和“复合物-质谱”技术鉴定出差异蛋白——AUF1。再利用体外结合实验验证AUF1与胞质DNA的相互作用。(2)在胞质DNA刺激后,通过ATP检测试剂盒和CCK8细胞氧还活力检测试剂,比较野生型细胞和基于CRISPR/Cas9技术的AUF1基因敲除细胞中葡萄糖代谢应答情况。(3)通过半定量PCR技术,在野生型、基因敲除AUF1、基因敲除后回补AUF1或空载体的四类细胞中检测葡萄糖转运蛋白GLUTs以及葡萄糖代谢相关酶的mRNA表达情况,筛选出与细胞糖代谢相关的AUF1下游效应分子——GLUT3。进而用实时荧光定量PCR进行验证。(4)通过半定量和荧光定量PCR分析胞质DNA刺激下GLUT3的mRNA变化情况,分析胞质DNA的刺激是否影响GLUT3的mRNA表达。结果 (1)两次质谱分析均发现AUF1能与ISD结合。体外结合实验也证实,不论是原核表达的GST-AUF1还是真核细胞表达的GFP-AUF1均能与单链和双链的ISD相结合。(2)基因敲除AUF1后的HEK293细胞在用胞质DNA刺激后,胞内的ATP水平和对CCK8的还原能力都明显高于野生型细胞。提示AUF1基因敲除细胞内的葡萄糖代谢不受胞质DNA刺激所抑制,说明AUF1很可能参与了胞质DNA对细胞糖代谢的调节。(3)半定量PCR技术检测发现在AUF1敲除的细胞中GLUT3的mRNA明显减少,而其他的GLUT家族成员和代谢酶则没有显著差异。实时荧光定量PCR证实上述现象,提示AUF1很可能通过稳定GLUT3的mRNA参与葡萄糖代谢的调节。(4)无论是单链还是双链ISD刺激后的细胞中,GLUT3的mRNA均减少,说明GLUT3可能是胞质DNA对糖代谢的调节过程中的一个下游效应分子。结论 AUF1能与胞质DNA结合,很可能通过调节下游GLUT3的mRNA稳定性参与胞质DNA引起的糖代谢应答反应。     

The influence of calcium and pH on growth in primary roots of Zea mays

Hasenstein, K. H. and Evans, M. L. 1988. The influence of calcium and pH on growth in primary roots of Zea mays. - Physiol. Plant. 72: 466–470.
We investigated the interaction of Ca²⁺ and pH on root elongation in Zea mays L. cv. B73 × Missouri 17 and cv. Merit. Seedlings were raised to contain high levels of Ca²⁺ (HC, imbibed and raised in 10 m M CaCl₂) or low levels of Ca²⁺ (LC, imbibed and raised in distilled water). In HC roots, lowering the pH (5 m M MES/Tris) from 6.5 to 4.5 resulted in strong, long-lasting growth promotion. Surprisingly, increasing the pH from 6.5 to 8.5 also resulted in strong growth promotion. In LC roots acidification of the medium (pH 6.5 to 4.5) resulted in transient growth stimulation followed by a gradual decline in the growth rate toward zero. Exposure of LC roots to high pH (pH shift from 6.5 to 8.5) also promoted growth. Addition of EGTA resulted in strong growth promotion in both LC and HC roots. The ability of EGTA to stimulate growth appeared not to be related to H⁺ release from EGTA upon Ca²⁺ chelation since, 1) LC roots showed a strong and prolonged response to EGTA, but only a transient response to acid pH, and 2) promotion of growth by EGTA was observed in strongly buffered solutions. We also examined the pH dependence of the release of ⁴⁵Ca²⁺ from roots of 3-day-old seedlings grown from grains imbibed in ⁴⁵Ca²⁺. Release of ⁴⁵Ca²⁺ from the root into agar blocks placed on the root surface was greater the more acidic the pH of the blocks. The results indicate that Ca²⁺ may be necessary for the acid growth response in roots.     

Characterization of phospholipase A2 and acyltransferase activities in squid (Loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions.

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.

The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.

Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

Role of calmodulin in the effect of guanyl nucleotides on rat hippocampal adenylate cyclase: involvement of adenosine and opiates

Abstract: The adenylate cyclase activity of rat hippocampal plasma membranes can be stimulated by vaso-active intestinal polypeptide (VIP). Low concentrations (10⁻⁹ to 10⁻⁷M) of 5'-guanylyl-imido diphosphate (GppNHp) evoke a transient inhibition of the enzyme, which is followed by stimulation with increasing GppNHp concentrations (10⁻⁶ to 10⁻⁴M). Inclusion of ethyleneglycol - bis - (β - aminoethylether) - N,N' - tetraacetic acid (EGTA) during incubation abolishes the GppNHp inhibition while preserving GppNHp activation. The stimulation induced by GppNHp is amplified by VIP, but the inhibition is unaffected. Adenosine analogs and opiates are inhibitory ligands in the presence of GTP, and their effects can be reversed by the appropiate receptor antagonists, 3-isobutyl-1-methylxanthine and naloxone. Treatment of membranes with trypsin abolishes the GppNHp-induced inhibition without affecting the GppNHp stimulation. The inhibition induced by GppNHp is also abolished by EGTA treatment followed by washing, which coincides wtih a reduction in the adenosine- and opiate-mediated, GTP-dependent inhibition. The GppNHp inhibition can be restored in EGTA-treated but not in trypsin-treated membranes by addition of calcium-calmodulin but not by Ca²⁺ or Mg²⁺. Calcium-calmodulindepleted membranes lack calcium stimulation as well as GppNHp-induced inhibition, whereas untreated membranes and calcium-calmodulin-depleted membranes plus exogenous calcium-calmodulin showed calcium stimulation and GppNHp inhibition. These results suggest that calmodulin is involved in both Ca²⁺ stimulation and guanine nucleotide-mediated inhibition of rat hippocampal adenylate cyclase.     

The use of the borocaptate anion as a ligand. Synthesis and X-ray crystal structure of pentaammine (1-thiolato-closo-undecahydrododecaborane)ruthenium(III) dihydrate

The complex [Ru(SB₁₂H₁₁)(NH₃)₅]·2H₂O has been prepared by the reaction of Cs₂B₁₂H₁₁SH with [RuCl(NH₃)₅]Cl₂ in aqueous solution. The complex represents the first reported example of the borocaptate anion acting as a ligand. The structure of the complex has been determined by single crystal X-ray diffraction analysis. The crystal parameters are monoclinic, space group P2₁/c, A = 8.056(1), B = 14.240(2), C = 15.172(2) Å, β=98.48° and Z = 4. The ruthenium atom has a distorted octahedral coordination. The distortion is probably due to the high (3⁻) charge and the large bulk of the borocaptate ligand. These features can also be observed in the spectroscopic properties of the complex.     

Treatment of YAC128 mice and their wild-type littermates with cystamine does not lead to its accumulation in plasma or brain: implications for the treatment of Huntington disease

Cystamine is beneficial to Huntington disease (HD) transgenic mice. To elucidate the mechanism, cystamine metabolites were determined in brain and plasma of cystamine-treated mice. A major route for cystamine metabolism is thought to be: cystamine --> cysteamine --> hypotaurine --> taurine. Here we describe an HPLC system with coulometric detection that can rapidly measure underivatized cystamine, cysteamine and hypotaurine, as well as cysteine and glutathione in the same deproteinized tissue sample. A method is also described for the coulometric estimation of taurine as its isoindole-sulfonate derivative. Using this new methodology we showed that cystamine and cysteamine are undetectable (< or = 0.2 nmol/100 mg protein) in the brains of 3-month-old HD transgenic (YAC128) mice (or their wild-type littermates) treated daily for 2 weeks with cystamine (225 mg/kg) in their drinking water. No significant changes were observed in brain glutathione and taurine but significant increases were observed in brain cysteine. Cystamine and cysteamine were not detected in the plasma of YAC128 mice treated daily with cystamine between the ages of 4 and 12 or 7 and 12 months. These findings suggest that cystamine is not directly involved in mitigating HD but that increased brain cysteine or uncharacterized sulfur metabolites may be responsible.     

Redox state of the partially reduced cytochrome aa3-cyanide complex

The low-spin ferric cyanide complex of beef heart cytochrome aa₃ can be partially reduced by stoichiometric additions of ferrous cytochrome c or by similar additions of N,N,N′,N′-tetramethyl-p-phenylene diamine. In both cases the initial ratio of cytochrome c oxidized: cytochrome a reduced or Wurster's Blue: cytochrome a reduced approximates the value 2. It is concluded that the binding of a single HCN prevents the reduction of both cytochrome a₃ and its associated EPR-invisible Cu atom.     

The activation by Ca of the ATPase of extracted muscle fibrils with variation of ionic strength, pH and concentration of MgATP

1. The alteration of the Ca²⁺ requirements of the ATPase activity of fibrils from rabbits and crabs at varying ionic strength, pH and concentration of MgATP (i.e. MgATP²⁻ + MgHATP⁻) was investigated.

2. Under physiological conditions, it was found that the ATPase activity of rabbit and crab fibrils after an initial increase decreased steeply when the Ca²⁺ concentration is raised above 1×10⁻⁴ M. This is a primary effect of the over-optimal Ca²⁺ concentration and not a secondary one caused by the influence of accompanying ions.

3. The Ca²⁺ requirements for ATP splitting by rabbit fibrils remain constant at an ionic strength from 0.1 to 0.2 and for a MgATP concentration in the range from 0.5 to 10 mM. At I = 0.05 it is about 5 times smaller than at 0.1. When the pH is decreased from 8 to 7, the Ca²⁺ requirements are increased some 10 times but only 3 times when the pH is varied between 7 and 6.

4. In crab fibrils, there is no alteration of the Ca²⁺ requirements when the ionic strength is varied between 0.05 and 0.2, but a reduction of the pH from 8.0 to 6.0 raises the Ca²⁺ requirements for half activation and for threshold by a factor of 10. Changing the MgATP concentration increases the Ca²⁺ requirements only in the range from 1 to 5 mM, while the concentration required in 0.5 mM is identical with that at 1 mM, and 10 mM corresponds to 5 mM.

5. It can be deduced from the experimental results that at a pH above 6.0 maximal activation is always obtained if the Ca²⁺ concentration is 5×10⁻⁵ M. By contrast, relaxation is only achieved when the Ca²⁺ concentration is below 1×10⁻⁷ M for pH 7.0 and I > 0.1 or below 1×10⁻⁸ for pH > 7.0 or I < 0.1.

6. To achieve complete relaxation, an ethyleneglycoldiaminotetraacetate (EGTA) concentration of 1 mM is sufficient, even when there is a large degree of contamination by Ca²⁺ as long as the pH stays above 6.5.