Hydroxyl-radical-induced iron-catalysed degradation of 2-deoxyribose. Quantitative determination of malondialdehyde.

The degradation of 2-deoxyribose to thiobarbituric acid-reactive material was investigated with two hydroxyl-radical-generating systems: (i) a defined gamma-radiolysis method and (ii) incubation with FeSO4 in phosphate buffer. In each case the thiobarbituric acid-reactive material can be accounted for by malondialdehyde, as measured by an h.p.l.c. method for free malondialdehyde. In the radiolysis system there is a large post-irradiation increase in free malondialdehyde if iron ions are added to the samples. It is proposed that this is due to iron ions catalysing the formation of hydroxyl radicals from radiolytically generated H2O2 as well as stimulating the breakdown of an intermediate deoxyribose degradation product. A mechanism for the formation of malondialdehyde during deoxyribose degradation is proposed.     

人己糖胺酶D的原核表达、纯化及酶学特性研究

糖基化修饰是一种重要的蛋白质翻译后修饰,参与生物体中的信号传导、细胞识别等多种细胞活动,糖基缀合物的正常水解是生物体代谢的必需途径.人己糖胺酶D( Hexosaminidase D)是新发现的一种存在于人细胞质中的切除GalNAc糖基化修饰的外切酶,但该酶的酶学特性尚不清楚.利用PCR的方法,将Hex D的cDNA序列构建到质粒pET3C中,重组质粒转化大肠杆菌BL21( DE3) plysS后,通过优化异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度(0.1mmol/L)和诱导时间(10 h)获得了高可溶性表达的重组蛋白酶.采用Ni-NTA亲和层析对重组蛋白进行了纯化,SDS-PAGE检测分子量的大小(58 kDa)和纯度(95％以上).以4-甲基伞形酮-2-乙酰氨基-2-脱氧半乳糖(4-MU-O-GalNAc)为荧光底物,测定该酶的最适反应pH值为5.5,最适反应温度为37℃,且该酶的热稳定性较好,在50℃下放置半小时仍有较高活性,1mmol/L的金属离子(CuSO4、FeSO4·7H2O、MgCl2· 6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)及EDTA对该酶活性影响不大,10mmol/L AlCl3、CuSO、FeSO4·7H2O对该酶有不同程度的抑制.在最适条件下(pH 5.5,37℃)下,该酶的Km为0.16mmoL/L,最大反应速率为3.06 μmol/( min·mg).     

Age-dependent effect of oxidative stress on cardiac sarcoplasmic reticulum vesicles

The oxidative stress hypothesis of aging suggests that accumulation of oxidative damage is a key factor of the alterations in physiological function during aging. We studied age-related sensitivity to oxidative modifications of proteins and lipids of cardiac sarcoplasmic reticulum (SR) isolated from 6-, 15- and 26-month-old rats. Oxidative stress was generated in vitro by exposing SR vesicles to 0.1 mmol/l FeSO4/EDTA + 1 mmol/l H2O2 at 37 degrees C for 60 min. In all groups, oxidative stress was associated with decreased membrane surface hydrophobicity, as detected by 1-anilino-8-naphthalenesulfonate as a probe. Structural changes in SR membranes were accompanied by degradation of tryptophan and significant accumulation of protein dityrosines, protein conjugates with lipid peroxidation products, conjugated dienes and thiobarbituric acid reactive substances. The sensitivity to oxidative damage was most pronounced in SR of 26-month-old rat. Our results indicate that aging and oxidative stress are associated with accumulation of oxidatively damaged proteins and lipids and these changes could contribute to cardiovascular injury.     

羟自由基诱导的烟草原生质体的凋亡:流式细胞法的新证据

用1.0 mmol/L FeSO4/0.5 mmol/L H202处理烟草(Nicotiana tabacum L.cultivar BY 2)原生质体,发现羟自由基能够诱导烟草原生质体的凋亡.具体表现为细胞核皱缩、DNA Ladder、TUNEL阳性反应等典型的凋亡特征.在动物细胞凋亡过程中,线粒体起着非常重要的作用,其中膜电位(△ψm)的变化以及由其引起的位于线粒体膜上的通透性孔(PTP)的开放与Cyt c的释放有关.另外,在动物凋亡细胞中,磷脂酰丝氨酸(phosphatidyl serine,PS)会从细胞膜内侧向外翻转.为了判断植物细胞凋亡过程中膜电位的变化情况以及PS的外翻程度,我们采用了流式细胞法.结果表明,随着处理时间的延长,烟草原生质体线粒体的膜电位逐渐降低;膜内PS大量外翻.说明由羟自由基和烟草原生质体组成的凋亡体系是一种可靠的凋亡组合,可以用来对植物细胞凋亡机理做进一步研究.     

Species and Organ Diversity in the Effects of Hydrogen Peroxide on Superoxide Dismutase Activity In Vitro

Superoxlde dlsmutase （SOD） is ubiquitous in aerobic organisms and constitutes the first link In the enzyme scavenging system of reactive oxygen species. In the present study, species and organ diversity of SOD activity In a solution and In an in-gel assay system, as well as the effects of hydrogen peroxide （H202） on SOD activity, were Investigated. In a solution assay system, SOD activity of jackfruIt root, shoot, leaves, axes, and cotyledons, of maize embryos and endosperms, of mung bean leaves and seeds, of sacred lotus axes and cotyledons, and of rice and wheat leaves was Increased by 1-15 mmol/L H2O2. However, SOD activity In rice root and seeds, maize roots and leaves, mung bean roots and shoots, and wheat seeds was decreased by 1-15 mmol/L H2O2. The SOD activity of wheat root and soybean roots, leaves, axes, and cotyledons was Increased by 1-4 mmol/L H2O2, but was decreased by concentrations of H2O2 〉4 mmol/L. The SOD activity of soybean shoots was not affected by 1-15 mmol/L H2O2. The SOD activity In crude mltochondrla of jackfruIt, maize, and upas seeds, as well as In purified mitochondria of jackfruIt, was also Increased by 1-15 mmol/L H2O2. In the In-gel assay system, the SOD In jackfruIt cotyledons was comprised of Mn-SOD, Cu/Zn-SOD, and Fe-SOD, the crude mltochondria of jackfruit seeds and maizes embryo was comprised of Mn-SOD and Cu/ Zn-SOD, and the crude mltochondria of maize seeds was comprised of Mn-SOD only. In the present study, H2O2 markedly Inhibited Cu/Zn-SOD and Fe-SOD activity.     

Gossypol-induced free radical toxicity to isolated islet cells

1. The cytotoxicity of the polyphenolic potential male antifertility agent gossypol was investigated on isolated mouse islets cells. 2. Gossypol shared many properties with the diabetogenic agent alloxan. 3. Gossypol (0.1-1.0 mmol/l) induced a concentration-dependent increase of Trypan Blue uptake by the cells, indicating an increase of membrane permeability to the dye. 4. Trypan Blue uptake induced by 0.5 mmol/l gossypol was inhibited by concomitant incubation of the cells with enzymatic (200 mg/l superoxide dismutase, 200 mg/l catalase, 3 mmol/l cytochrome-c), or low-molecular weight (50 mmol/l D-mannitol) scavengers of oxygen radicals, and the metal chelator diethylenetriaminepentacetic acid (DTPA) (50 mumol/l). 5. The results support the hypothesis that gossypol is B-cytotoxic by generation of noxious free radicals and that when proposing gossypol as a male antifertility agent, studies to exclude gossypol as a diabetogenic agent should first be performed in vivo.     

Optimization of medium compositions favoring butanol and 1,3-propanediol production from glycerol by Clostridium pasteurianum

Medium compositions favoring butanol and 1,3-propanediol (1,3-PDO) production from glycerol by Clostridium pasteurianum DSM525 were investigated using statistical experimental designs. Medium components affecting butanol and 1,3-PDO production were screened using a fractional factorial experimental design. Among the six tested variables (phosphate buffer, MnSO4·H2O, MgSO4·7H2O, FeSO4·7H2O, (NH4)2SO4, and yeast extract), FeSO4·7H2O, (NH4)2SO4, and yeast extract were found to be significant variables for further optimization of medium using a Box-Behnken design. Optimal butanol (0.98 g/L/h) and 1,3-PDO (1.19 g/L/h) productivities were predicted by the corresponding quadratic model for each product and the models were validated experimentally under optimized conditions. The optimal medium composition for butanol production was significantly different from that for 1,3-PDO production (0.06 vs. 0 g/L for FeSO4·7H2O, 7.35 vs. 0 g/L for (NH4)2SO4, and 5.08 vs. 8.0 g/L for yeast extract), suggesting that the product formation from glycerol by C. pasteurianum DSM525 can be controlled by changing medium compositions.     

氧化亚铁硫杆菌中磁小体形成相关基因在不同亚铁浓度刺激下的差异表达

氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)的生物控制矿化作用可以使其在胞内形成黑色电子致密颗粒—磁小体。本研究利用生物信息学方法对氧化亚铁硫杆菌标准菌株ATCC 23270的全基因组进行分析, 并通过Real-time PCR技术研究氧化亚铁硫杆菌中与磁小体形成相关的mpsA、magA、thy和mamB四个基因在不同亚铁浓度刺激下的差异表达, 结果发现它们在转录层面的表达量受亚铁浓度的影响, 当亚铁浓度达到150~200 mmol/L范围内达到最高表达,这对进一步深入研究氧化亚铁硫杆菌中磁小体的形成机理有积极的意义。     

High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring

Dark fermentative hydrogen production from glucose by a thermophilic culture (33HL), enriched from an Icelandic hot spring sediment sample, was studied in two continuous-flow, completely stirred tank reactors (CSTR1, CSTR2) and in one semi-continuous, anaerobic sequencing batch reactor (ASBR) at 58 degrees C. The 33HL produced H2 yield (HY) of up to 3.2 mol-H2/mol-glucose along with acetate in batch assay. In the CSTR1 with 33HL inoculum, H2 production was unstable. In the ASBR, maintained with 33HL, the H2 production enhanced after the addition of 6 mg/L of FeSO4 x H2O resulting in HY up to 2.51 mol-H2/mol-glucose (H2 production rate (HPR) of 7.85 mmol/h/L). The H2 production increase was associated with an increase in butyrate production. In the CSTR2, with ASBR inoculum and FeSO4 supplementation, stable, high-rate H2 production was obtained with HPR up to 45.8 mmol/h/L (1.1 L/h/L) and HY of 1.54 mol-H2/mol-glucose. The 33HL batch enrichment was dominated by bacterial strains closely affiliated with Thermobrachium celere (99.8-100%). T. celere affiliated strains, however, did not thrive in the three open system bioreactors. Instead, Thermoanaerobacterium aotearoense (98.5-99.6%) affiliated strains, producing H2 along with butyrate and acetate, dominated the reactor cultures. This culture had higher H2 production efficiency (HY and specific HPR) than reported for mesophilic mixed cultures. Further, the thermophilic culture readily formed granules in CSTR and ASBR systems. In summary, the thermophilic culture as characterized by high H2 production efficiency and ready granulation is considered very promising for H2 fermentation from carbohydrates.     

阳离子及ATP对绿豆线粒体膨胀与收缩的影响

本文报道了一价阳离子 K~+、Na~+及两价阳离子 Mg~(++)、Ca~(++)以及 ATP 对绿豆线粒体膨胀和收缩的影响。K~+、Na~+在低渗条件下引起线粒体瞬时的迅速膨胀。在同样离子强度下K~+引起的膨胀大于 Na~+。ATP 和 Mg~(++)能诱发低渗条件下膨胀线粒体的收缩,但对等渗和高渗 KCl 或 Nacl 溶液中膨胀的线粒体无明显作用。生理浓度的 Mg~(++)、Ca~(++)在低渗条件下引起线粒体缓慢的但幅度较大的膨胀,5mmol/l ATP 引起这种膨胀线粒体的部分收缩。1mmol/lca~(++)在含0.125mmol/l KCl 或在含0.25mol/l甘露醇的等渗介质中几乎不引起膨胀,而ATP 促进大幅度膨胀,10mmol/l MgCl_2引起这种膨胀线粒体的部分收缩。2mmol/l MgCl_2在含有0.25mol/l 甘露醇的等渗介质中引起明显膨胀,ATP 促进这种膨胀。0.125mol/lKCl+2mmol/l MgCl_2为肌动蛋白从单体聚合成多聚体所必须的条件。在此条件下,线粒体几乎不膨胀,而加入 ATP 后则促进大幅度膨胀。在电子显微镜下观察了等渗及低渗条件下线粒体形态变化。     

Effects of butyrate precursors on electron relocation when methanogenesis is inhibited in ruminal mixed cultures

AIMS: Inhibition of ruminal methanogenesis often causes accumulation of H(2), formate and ethanol, which are not energy substrates for ruminants. It was hypothesized that the addition of butyrate precursors would avoid the formation of these products and relocate electrons into butyrate. METHODS AND RESULTS: In four ruminal 24-h incubations, two inhibitors of methanogenesis, each at three different initial concentrations (0, 2 or 4 mmol l(-1) for propynoic acid, and 0, 4 or 8 mmol l(-1) for ethyl 2-butynoate), were combined with two butyrate precursors at two different initial concentrations (0 or 4 mmol l(-1) for crotonic acid or 3-butenoic acid). Ground lucerne hay was the substrate. Propynoic acid at 4 mmol l(-1) decreased CH(4) formation by more than two-thirds. Ethyl 2-butynoate at 8 mmol l(-1) suppressed methanogenesis by more than 90%. Butyrate precursors generally did not decrease the accumulation of H(2) and formate or ethanol production. CONCLUSIONS: Butyrate precursors were ineffective as electron acceptors because they were not completely converted to butyrate and were also metabolized through other pathways. SIGNIFICANCE AND IMPACT OF THE STUDY: Effectiveness of butyrate precursors may be improved by adding them to the fermentation continuously or by enhancing the kinetics of their conversion into butyrate.     

Oxalate modulates thiobarbituric acid reactive species (TBARS) production in supernatants of homogenates from rat brain, liver and kidney: effect of diphenyl diselenide and diphenyl ditelluride

The aim of this paper was to investigate the mechanism(s) involved in the sodium oxalate pro-oxidative activity in vitro and the potential protection by diphenyl diselenide ((PhSe)(2)) and diphenyl ditelluride ((PhTe)(2)) using supernatants of homogenates from brain, liver and kidney. Oxalate causes a significant increase in the TBARS (thiobarbituric acid reactive species) production up to 4mmol/l and it had antioxidant activity from 8 to 16mmol/l in the brain and liver. Oxalate had no effect in kidney homogenates. The difference among tissues may be related to the formation of insoluble crystal of oxalate in kidney, but not in liver and brain homogenates. (PhSe)(2) and (PhTe)(2) reduced both basal and oxalate-induced TBARS in rat brain homogenates, whereas in liver homogenates they were antioxidant only on oxalate-induced TBARS production. (PhSe)(2) showed a modest effect on renal TBARS production, whereas (PhTe)(2) did not modulate TBARS in kidney preparations. Oxalate at 2mmol/l did not change deoxyribose degradation induced by Fe(2+) plus H(2)O(2), whereas at 20mmol/l it significantly prevents its degradation. Oxalate (up to 4mmol/l) did not alter iron (10micromol/l)-induced TBARS production in the brain preparations, whereas at 8mmol/l onwards it prevents iron effect. In liver preparations, oxalate amplifies iron pro-oxidant activity up to 4mmol/l, preventing iron-induced TBARS production at 16mmol/l onwards. These results support the antioxidant effect of organochalcogens against oxalate-induced TBARS production. In addition, our results suggest that oxalate pro- and antioxidant activity in vitro could be related to its interactions with iron ions.     

Activation of mitochondrial glycerol 3-phosphate dehydrogenase by cadmium ions

Mitochondrial glycerol 3-phosphate dehydrogenase (EC 1.1.2.1.) requires Ca2+ ions for its activity. Cadmium ions also have activatory effect on the enzyme. They activate the glycerol 3-phosphate dehydrogenase in a very narrow concentration range (1-2 mmol/l). As contrasted with calcium, strong inhibitory effect occurred at higher concentrations (3-4 mmol/l). The inhibition induced by cadmium ions was completely reversible by washing of the mitochondria.     

Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51

The nutritional medium requirement for biosurfactant production by Bacillus licheniformis K51 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were H3PO4, CaCl2, H3BO3, and Na-EDTA. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were (g/l): glucose, 1.1; NaNO3, 4.4; MgSO4 x 7H2O, 0.8; KCl, 0.4; CaCl2, 0.27; H3PO4, 1.0 ml/l; and trace elements (mg/l): H3BO3, 0.25; CuSO4, 0.6; MnSO4, 2.2; Na2MoO4, 0.5; ZnSO4, 6.0; FeSO4, 8.0; CoCl2, 1.0; and Na-EDTA, 30.0. Using this statistical optimization method, the relative biosurfactant yield as critical micelle dilution (CMD) was increased from 10x to 105x, which is ten times higher than the non-optimized rich medium.     

L-脯氨酸-4-羟化酶的异源表达和合成反式-4-羟基-L-脯氨酸的条件优化

L-脯氨酸-4-羟化酶(L-Proline-4-hydroxylase,P4H)是依赖α-酮戊二酸(α-KG)和Fe2+的双加氧酶成员之一,在反式-4-羟基-L-脯氨酸(trans-4-hydroxy-L-proline,t-4Hyp)等重要手性化合物的生物合成中发挥关键作用。本研究构建了来源于Bradyrhizobium japonicum USDA 6的P4H重组大肠杆菌Escherichia coli BL21(DE3)/p ET-28b-p4h BJ,SDS-PAGE和酶活检测结果表明,该菌株具有表达可溶性P4H和催化合成t-4Hyp的能力。通过优化,确定了该重组菌全细胞催化合成t-4Hyp较优的反应体系和条件:10 m L p H 6.5 80 mmol/LMES缓冲液、9 mmol/L L-Pro,6 mmol/L L-抗坏血酸,6 mmol/Lα-KG,0.8 mmol/L Fe SO4·7H2O,反应温度为35℃;在20 g/L湿细胞的催化反应中,t-4Hyp的合成量达到34.86 mg/L,比优化前(17.53 mg/L)提高了98.86%。该工作为进一步利用P4H生物催化法合成t-4Hyp奠定了一定的技术基础。     

Alterations in cardiac contractile proteins due to oxygen free radicals.

In view of the potential role of free radicals in the genesis of cardiac abnormalities under different pathophysiological conditions and the importance of contractile proteins in determining heart function, this study was undertaken to examine the effects of oxygen free radicals on the rat heart myofibrils. Xanthine plus xanthine oxidase (X + XO) which is known to generate superoxide anions (O2-) and hydrogen peroxide (H2O2), an activated species of oxygen, was found to decrease Ca(2+)-stimulated ATPase activity, increase Mg(2+)-ATPase activity and reduce sulfhydryl (SH) group contents in myofibrils; these effects were completely prevented by superoxide dismutase (SOD) plus catalase (CAT). Both H2O2 and hypochlorous acid (HOCl), an oxidant, produced actions on cardiac myofibrils similar to those observed by X + XO. The effects of H2O2 and HOCl were prevented by CAT and L-methionine, respectively. N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), inhibitors of SH groups, also produced effects similar to those seen with X + XO. Dithiothreitol (DTT), a well known sulfhydryl-reducing agent, prevented the actions of X + XO, H2O2, HOCl, NEM and DTNB. These results suggest that marked changes in myofibrillar ATPase activities by different species of oxygen free radicals may be mediated by the oxidation of SH groups.     

Proteomic analysis of MOLT-4 cells treated by valproic acid

The effect of valproic acid (VA) on protein expression in human T-lymphocytic leukemia cells MOLT-4 was studied. VA is an inhibitor of histonedeacetylases and has a potential use as antitumor agent in leukemia treatment. The authors in this work prove that 4 h long incubation with 2 mmol/l VA causes phosphorylation of histone H2A.X and its colocalization with 53BP1 in nuclear foci. Their co-localization is typical for DSB signaling machinery. These foci were detected in cells after 4 h exposure without increase of Annexin V positive apoptotic cells. Slight increase in apoptosis (Annexin V positivity) after 24 h is accompanied by more intensive increase in phosphorylation of H2A.X and also by formation of nuclear foci containing γH2A.X and 53BP1. Treatment of cells with 2 mmol/l VA resulted in induction of apoptosis affecting about 30% of cells after incubation for 72 h. The changes in protein expression were examined after cell incubation with 2 mmol/l VA for 4 h. Proteins were separated by two-dimensional electrophoresis and quantified using image evaluation system. Those exhibiting significant VA-induced abundance alterations were identified by mass spectrometry. Changes in expression of 22 proteins were detected, of which 15 proteins were down-regulated. Proteomic analysis resulted in successful identification of three proteins involving alfa-tubulin 3, tubulin-specific chaperone and heterogeneous nuclear ribonucloprotein F. Expression of seven proteins was up-regulated, including heterogeneous nuclear ribonucloprotein A/B. Identified proteins are related to microtubular system and hnRNP family. Suppression of microtubular proteins and changes of balance among hnRNPs can contribute to proliferation arrest and apoptosis induction.     

Aromatic hydroxylation in PBN spin trapping by hydroxyl radicals and cytochrome P-450

Phenyl N-tert-butylnitrone (PBN) is widely used as a spin trapping agent, but is not useful detecting hydroxyl radicals because the resulting spin adduct is unstable. However, hydroxyl radicals could attack the phenyl ring to form stable phenolic products with no electron paramagnetic resonance signal, and this possibility was investigated in the present studies. When PBN was added to a Fenton reaction system composed of 25 mM H(2)O(2) and 0.1 mM FeSO(4), 4-hydroxyPBN was the primary product detected, and benzoic acid was a minor product. When the Fe(2+) concentration was increased to 1.0 mM, 4-hydroxyPBN concentrations increased dramatically, and smaller amounts of benzoic acid and 2-hydroxyPBN were also formed. Although PBN is extensively metabolized after administration to animals, its metabolites have not been identified. When PBN was incubated with rat liver microsomes and a reduced nicotinamide adenine dinculeotide phosphate (NADPH)-generating system, 4-hydroxyPBN was the only metabolite detected. When PBN was given to rats, both free and conjugated 4-hydroxyPBN were readily detected in liver extracts, bile, urine, and plasma. Because 4-hydroxyPBN is the major metabolite of PBN and circulates in body fluids, it may contribute to the pharmacological properties of PBN. But 4-hydroxyPBN formation cannot be used to demonstrate hydroxyl radical formation in vivo because of its enzymatic formation.     

5′-磷酸腺苷对小鼠脾细胞氧化应激损伤的保护作用

目的研究5′-磷酸腺苷（5′-AMP）体外抗氧化和对体外氧化损伤脾细胞的损伤修复能力。方法用化学比色法测定5′-AMP体外清除二苯代苦味酰基自由基（DPPH自由基）的能力;建立过氧化氢（H2O2）氧化损伤体外培养小鼠脾细胞模型,用MTT法检测5′-AMP修复受损伤脾细胞的作用,并分析其对细胞抗氧化体系及抗氧化能力的影响。结果5′-AMP具有剂量依赖性的体外抗氧化和清除活性氧能力,添加0.5mmol/L、1mmol/L、5mmol/L和10mmol/L5′-AMP均能显著修复H2O2诱导的脾细胞氧化损伤（P〈0.05）,总抗氧化能力和抗氧化酶类活力（P〈0.01）,5′-AMP添加量大于1mmol/L时,可显著降低丙二醛（MDA）含量（P〈0.01）。其细胞培养液的氧自由基（ROS）水平逐渐降低,5′-AMP添加量为10mmol/L时,ROS水平接近对照组水平。结论5′-磷酸腺苷能显著修复氧化损伤,具有显著的抗氧化作用。     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.