青年男性高原移居者最大摄氧量计算公式的推导

最大摄氧量（Ｖｏ２ｍａｘ）是评价人体体力的重要指标,其测定方法分直接法和间接法两种。目前所推导的间接计算公式都是在平原、或是在进入高原初期推导的,不适用于高原习服人群。本研究采用逐步回归的方法,推导出移居高原７－２７个月、不同高度的青年男性Ｖｏ２ｍａｘ间接计算公式。在海拔３６８０ｍ地区,Ｖｏ２ｍａｘ（Ｌ／ｍｉｎ）＝１．１５３１＋０．００７３２７身高（ｃｍ）＋０．０１６１３体重（ｋｇ）－０．００５８８３晨脉（ｂ／ｍｉｎ）－０．００４５３４运动心率（６０Ｗ,６／ｍｉｎ）,Ｒ＝０．７４５,Ｐ＜０．０１,ＳＳ＝３．７７９９;或Ｖｏ２ｍａｘ（Ｌ／ｍｉｎ）＝１．２１８６＋０．０１９８４体重（ｋｇ）＋０．０７２５９肺活量（Ｌ）－０．００６６５９晨脉（ｂ／ｍｉｎ）,Ｒ＝０．７１３,ｐ＜０．０１,ｓｓ＝３．９６３６。在４３５０ｍ地区,Ｖｏ２．ｍａｘ（Ｌ／ｍｉｎ）＝０．４９１７＋０．０１６８７体重（ｋｇ）＋０．１１０９肺活量（Ｌ）＋０．００１９８３屏气时间（Ｓ）,Ｒ＝０．７８１,Ｐ＜０．０１,ＳＳ＝２．１３５６。计算值与实测值比较,变异系数在１３％以内,结果准确可靠,适用于青年男性高原习服移居者。     

大肠杆菌精氨酰－tRNA合成酶的变种ArgRS306KR的纯化和基本性质

本文从含ＡｒｇＲＳ３０６ＫＲ基因ａｒｇｓ３０６ＫＲ的ｐＵＣ１８重组质粒的大肠杆菌ＴＧ１转化子中经ＤＥＡＥ－Ｓｅｐｈａｃｅｌ和Ｂｌｕｅ－Ｓｅｐｈａｒｏｓｅ两步柱层析,得到电泳一条带的ＡｒｇＲＳ３０６ＫＲ。纯酶的比活为２７９０单位／毫克。该酶氨酰化和ＡＴＰ～ＰＰｉ交换活力的最适ｐＨ分别为ｐＨ８．３和ｐＨ７．５。氨酰化活力对ＡＴＰ、Ａｒｇ和ｔＲＮＡ的Ｋｍ分别为２．６ｍｍｏｌ／Ｌ、１４．０μｍｏｌ／Ｌ和５．０μｍｏｌ／Ｌ：Ｖｍａｘ为７６３０单位／毫克;ｋｏａｔ为９Ｓ－１。ＡＴＰ～ＰＰｉ交换活力对ＡＴＰ和Ａｒｇ的Ｋｍ分别为８．３ｍｍｏｌ／Ｌ和９９μｍｏｌ／Ｌ;Ｖｍａｘ为１６３２０单位／毫克;ｋｃａｔ为１８Ｓ－１。     

粘质赛氏菌胞外蛋白酶的氨基酸组成、N端序列及其部分性质

终浓度为５ｍｍｏｌ／Ｌ的ＥＤＴＡ可彻底抑制粘质赛氏菌４１００３（２）胞外蛋白酶活力,而Ｃａ２＋可以回复部分酶活力,表明Ｃａ２＋为酶活力所必需;Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋等金属离子对酶具有不同程度的抑制作用;５－磷酸吡哆醛及对甲氧基苯甲醛对酶具有明显的激活作用;以酪蛋白为底物,采用双倒数法求得酶的Ｋｍ值为６．６７ｍｇｍｌ－１;Ｎ,Ｎ－二甲基酪蛋白也是酶的理想底物;经酸水解,测定出酶的氨基酸组成;利用蛋白质自动分析仪测定了酶的Ｎ端９个氨基酸序列．比较来源不同的粘质赛氏菌胞外蛋白酶的Ｎ端序列,发现它们之间存在一定程度的同源性,并且存在特定的通读结构Ａｌａ…Ａｓｐ…Ｇｌｙ．     

地衣芽孢杆菌β－甘露聚糖酶的纯化及酶学性质

地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＮＫ－２７菌株发酵产生的β－甘露聚糖酶（β－ｍａｎｎａｎａｓｅ）经硫酸铵盐析沉淀,两次ＤＥＡＥ纤维素和ＳｅｐｈａｄｅｘＧ－１００离子交换柱层析以及制备ＰＡＧＥ筹步骤,获得了凝胶电泳均一的样品。用ＳＤＳ－凝胶电泳测得纯化后的β－甘露聚糖酶分子量为２６ｋＤ,用凝胶聚焦电泳测得等电点ＰＩ为５．０。酶反应的最适ｐＨ为９．０,最后温度为６０℃,稳定ｐＨ为６．０—９．０,稳定温度为４０℃。金属离子中Ｍｇ￣（２＋）、Ｃａ￣（２＋）、Ｆｅ￣（２＋）、Ｎｉ￣（２＋）对该酶有一定的激活作用;而Ｓｎ￣（２＋）、Ｚｎ￣（２＋）、Ａｌ￣（３＋）、Ａｇ￣＋和Ｈｇ￣（２＋）对该酶有强烈的抑制作用。ＮＫ－２７菌株的β－甘露聚糖酶对魔芋葡萄甘露聚糖和角豆胶半乳甘露聚糖的Ｋｍ值分别为７．１４和５．５６ｍｇ·ｍｌ￣（－１）;Ｖ＿（ｍａｘ）分别为２００．５３和１５７．４５μｍｏｌ·ｍｇ￣（－１）·ｍｉｎ￣（－１）。     

小鼠腹腔巨噬细胞不完全失活的外向K~+电流的基本特性

采用膜片钳技术以全细胞方式在小鼠腹腔渗出巨噬细胞（ＰＥＭ）中记录到一种不完全失活的外向Ｋ＋电流（Ｉｏ）,该电流在膜电位正于－１０ｍＶ时激活,对Ｋ＋具有高度特异性,其半值电导电位Ｖ１／２为７９．５ｍＶ,在膜电位正于３０ｍＶ时,该电流失活,在６０－１２０ｍＶ的膜电位范围内,其失活时间常数τｉ与膜电位无关．随着胞外Ｋ＋离子浓度（［Ｋ＋］ｏ）升高,该电流的失活过程减慢。在生理电压范围内（－８０－０ｍＶ）,该电流缺乏稳态失活,且其失活不具有频率依赖性。胞外４－ＡＰ（３ｍｍｏｌ／Ｌ）、Ｂａ２＋（３ｍｍｏｌ／Ｌ）及ＴＥＡ（５ｍｍｏｌ／Ｌ）可抑制该电流,抑制率分别为８５％,６６％及３１％。胞外Ｚｎ２＋（１ｍｍｏｌ／Ｌ）可影响该电流活性,对该电流的抑制具有电压依赖性     

转BmK IT4基因烟草的抗虫性

用酶切方法从「ｐＢＳ－ＢｍＫ ＩＴ４质粒中获得ＢｍＫ ＩＴ４基因片段,并构建ＣａＭＶ ３５Ｓ启动子下的ＢｍＫ ＩＴ４基因表达质粒ｐＥ３－ＢｍＫ ＩＴ４,以根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｒｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导的叶盘法转化云烟（Ｎｉｃｏ－ｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）Ｋ３２６叶片,获得４５株抗卡那霉素的再生植株。用这些再生植株进行抗虫     

重组家蚕病毒表达传染性法氏囊病病毒VP2蛋白

将传染性囊病病毒ＨＺ９６株主要宿主保护性抗原ＶＰ２的ｃＤＮＡ基因克隆到杆状病毒转移载体ｐＢａｃ－ＰＡＫ８中,获得重组转移载体ｐＢａｃＰＡＫ－ＶＰ２,载体ｐＢａｃＰＡＫ－ＶＰ２与修饰病毒Ｂｍ－ＢａｃＰＡＫ６线性化基因组ＤＮＡ共转染单层家蚕Ｂｏｍｂｙｘ ｍｏｒｉ（Ｂｍ）Ｎ细胞,经细胞内同源重组,筛选到重组病毒。ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ免疫鲩迹结果表明,ＶＰ２在家蚕培养细胞和家蚕幼虫中均得到了表达。     

离体大鼠不均匀牵张膈肌标本上的神经肌肉传递

在离体大鼠不均匀牵张膈肌标木（ＩＮＳＭＰ）上选择合适终板,可用同一微电极记录同一终板的小终板电住（ＭＥＰＰ）和终板电位（ＥＰＰ）。膜电位为－６２±２ｍＶ,ＭＥＰＰ频率和幅度在正常值范围,ＥＰＰ幅度高于２０ｍＶ,用直接法计算的量子含量对细胞外Ｃａ２＋浓度双对数作图呈直线,斜率为４．０６。２、５０和１００Ｈ２串刺激诱发量子释放的统计学性质为二项分布。表明ＩＮＳＭＰ上合适终板可作为电生理学上接近正常生理状态的神经肌接头模型,并提示生理状态下神经肌接头突触前诱发量子释放的机率较高（５０Ｈｚ时ｐ＝０．９）,遵循二项分布规律,递质动员较快（５０ＨＺ时为２０５０ｓ－１）,以保证正常的神经肌肉传递。     

UV—B辐射对小麦叶片H2O2代谢的影响

研究了温室种植的小麦在０（ＣＫ）、８．８２ｋＪ／ｍ＾２（Ｔ１）和１２．６ｋＪ／ｍ＾２（Ｔ２）三种剂量的紫外线Ｂ（ＵＶ－Ｂ）辐射下Ｈ２Ｏ２含量的变化及其机理。ＵＶ－Ｂ辐射下Ｈ２Ｏ２、还原型抗坏血酸（ＡｓＡ）和还原型谷胱甘肽（ＧＳＨ）含量增加,抗坏血酸过氧化物酶（ＡＰｘ）和谷胱甘肽不原酶（ＧＲ）活性升高,脂肪酸不饱和度指数（ＩＵＦＡ）降低。ＳＤＳ－ＰＡＧＥ谱图没有质上的差异,但凝胶着色深浅有变化。分析     

蚯蚓体内一种纤溶酶原激活剂(e-PA)对BAEE的降解

以苯甲酰－Ｌ－精氨酸乙酯（ｂｅｎｚｏｙｌ－Ｌ－ａｒｇｉｎｉｎｅｅｔｈｙｌｅｓｔｅｒ,ＢＡＥＥ）为底物,研究了蚯蚓体内纤溶酶原激活剂（ｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒｆｒｏｍＥｉｓｅｎｉａｆｅｔｉｄａ,ｅ－ＰＡ）的酶学性质．酶促反应的最适ｐＨ为８．４,ｅ－ＰＡ降解ＢＡＥＥ的Ｋｍ为１．２４±０．１６×１０－５ｍｏｌ／Ｌ,Ｋｃａｔ为１３．８０±４．０２ｓ－１．测定了构成ｅ－ＰＡ的大,小亚基分别降解ＢＡＥＥ的Ｋｍ和Ｋｃａｔ．结果表明,大亚基的Ｋｍ与全酶的Ｋｍ相差不多,但比小亚基小约１０倍,即对底物的亲和力比小亚基强约一个数量级．大小亚基的Ｋｃａｔ比较接近,分别是全酶的１／６和１／３．研究了８种抑制剂对ｅ－ＰＡ降解ＢＡＥＥ活性的影响,其中ｐｅｐｓｔａｔｉｎ和Ｅ－６４（一种巯基抑制剂）对酶促反应有激活作用,ＴＰＣＫ,ＴＬ－ＣＫ,ＰＭＳＦ,ｃｈｙｍｏｓｔａｔｉｎ和ｌｅｕｐｅｐｔｉｎ对其有不同程度的抑制作用,ＥＤＴＡ对ｅ－ＰＡ的活性没有影响．对ｅ－ＰＡ的ＢＡＥＥ活性和ｅ－ＰＡ的纤溶活性之间作了比较．     

Mechanistic insights and functional determinants of the transport cycle of the ascorbic acid transporter SVCT2. Activation by sodium and absolute dependence on bivalent cations

We characterized the human Na(+)-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na(+)-dependent transporter. The properties of SVCT2 are modulated by Ca(2+)/Mg(2+) and a reciprocal functional interaction between Na(+) and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na(+) increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K(m) without affecting the V(max), thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na(+) cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na(+):ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na(+):ascorbic acid:Na(+). However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca(2+)/Mg(2+) for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V(max) without affecting the transport K(m) or the Na(+) cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca(2+)/Mg(2+). At least three active states can be envisioned, including a low affinity conformation at Na(+) concentrations below 20 mM and two high affinity conformations at elevated Na(+) concentrations whose Na(+) cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca(2+)/Mg(2+)-dependent transporter.     

Kinetically controlled enzymatic synthesis of dipeptide precursor of L-alanyl-L-glutamine

An important nutritional dipeptide precursor, benzoyloxycarbonyl protected L-alanyl-L-glutamine (Z-Ala-Gln), was successfully prepared through a kinetically controlled enzymatic peptide synthesis method. A commercially available and low-cost protease (papain) was used as biocatalyst with Z-Ala-OMe and Gln as acyl donor and nucleophile, respectively. The dipeptide yield was 35.5% under the optimized reaction conditions: 35°C, pH 9.5, and the ratio of acyl donor/nucleophile is 1:10. Based on the reaction mechanism and experimental data, the kinetic model was established, which was in accordance with the Michaelis-Menten equation, and the apparent Michaelis constant K(m)(app) and the apparent maximum reaction rate r(max)(app) were calculated as 1.71 mol/L and 6.09 mmol/(L Min), respectively.     

6BA诱导的带正电荷的葡萄叶过氧化物酶

河岸葡萄叶的带正电荷过氧化物酶（ｃａｔｉｏｎｉｃ ｐｅｒｏｘｉｄａｓｅ, ＣＰＯＤ） 可受６ＢＡ 诱导,但盐、Ｈ２Ｏ２ 或Ｆｅ２＋＋ Ｈ２Ｏ２ 均使叶片ＣＰＯＤ 活性明显下降,而高浓度的无机盐明显刺激纯ＣＰＯＤ 活性增加。在以愈创木酚为底物时ＣＰＯＤ 最适ｐＨ 为４ ．６０ ～５ ．７５ ,对Ｈ２Ｏ２ 的表观Ｖｍａｘ 和Ｋｍ 值分别为１１０ Ｕ／ｍｇ 蛋白和１ ．１５ｍ ｍｏｌ／Ｌ。     

Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology

The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K(m),V(max), K(cat), activation energy (E(a)) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K(m)) for casein decreased with corresponding increase in V(max), as reaction temperature was raised from 45-60 degrees C. The protease exhibited K(m) of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60 degrees C, respectively, whereas V(max) values at these temperatures were 74.07, 99.01, 116.28, and 120.48 microg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45 degrees C until 60-65 degrees C followed by thermal inactivation. Likewise, the energy of activation (E(a)) was more between 45-55 degrees C (9747 cal/mol) compared to E(a) between 50-60 degrees C (4162 cal/mol).     

Environmental and Kinetic Parameters for Cr(VI) Bioreduction by a Bacterial Monoculture Purified from Cr(VI)-Resistant Consortium

Hexavalent chromium, Cr(VI), is toxic to living systems. Widespread contamination of water and soil by Cr(VI) present a serious public health problem. Chromium-resistant bacteria can reduce and detoxify Cr(VI). Twelve bacteria resistant to high concentrations of Cr(VI) were isolated from soil enrichment cultures. Environmental parameters and kinetic parameters of Cr(VI) bioreduction by one monoculture isolate, identified by 16S rRNA gene sequence as Bacillus sp. PB2, were studied. The optimal temperature for growth and Cr(VI) reduction was 35 degrees C. The isolate grew luxuriantly and substantially reduced Cr(VI) at initial pH 7.5 to 9. Maximal Cr(VI) bioreduction occurred at initial pH 8.0. Substantial Cr(VI) bioreduction was observed in salt media, but removal efficiency was inversely related to salt concentration (1-9%). Michaelis-Menten hyperbolic equation and the Lineweaver-Burk double reciprocal plot were comparatively employed to determine the k (m) and V (max) of Cr(VI) bioreduction. A k (m) of 82.5 microg mL(-1) and V (max) of 7.78 microg mL(-1) h(-1) were calculated by nonlinear regression analysis of the hyperbola curve. Linear regression analysis of the double reciprocal plot revealed k (m) and V (max) of 80.9 microg mL(-1) and 10.6 microg mL(-1) h(-1), respectively. Time course studies displayed about 90% reduction of Cr(VI) at an initial concentration of 8,000 microg L(-1) in 8 h, with an estimated t (1/2) of 4 h. Data from time course analysis of the rate of Cr(VI) bioreduction fitted zero-order model, and the kinetic constant k was calculated to be 840 microg L(-1) h(-1). The monoculture isolate, Bacillus sp. PB2, strongly reduces Cr(VI) and could be used for bioremediation of Cr(VI)-contaminated aquatic and terrestrial environments.     

Kinetic analysis of a unique direct prothrombinase, fgl2, and identification of a serine residue critical for the prothrombinase activity

fgl2 prothrombinase, by its ability to generate thrombin, has been shown to be pivotal to the pathogenesis of viral-induced hepatitis, cytokine-induced fetal loss syndrome, and xeno- and allograft rejection. In this study, the molecular basis of fgl2 prothrombinase activity was examined in detail. Purified fgl2 protein generated in a baculovirus expression system had no measurable prothrombinase activity, whereas the activity was restored when the purified protein was reconstituted into phosphatidyl-L-serine-containing vesicles. Reconstituted fgl2 catalyzed the cleavage of human prothrombin to thrombin with kinetics consistent with a first order reaction, with an apparent V(max) value of 6 mol/min/mol fgl2 and an apparent K(m) value for prothrombin of 8.3 microM. The catalytic activity was totally dependent on calcium, and factor Va (500 nM) enhanced the catalytic efficiency of fgl2 by increasing the apparent V(max) value to 3670 mol/min/mol fgl2 and decreasing the apparent K(m) value for prothrombin to 7.2 microM. By a combination of site-directed mutagenesis and production of truncated proteins, it was clearly shown that residue Ser(89) was critical for the prothrombinase activity of fgl2. Furthermore, fgl2 prothrombinase activity was not inhibited by antithrombin III, soybean trypsin inhibitor, 4-aminobenzamidine, aprotinin, or phenylmethylsulfonyl fluoride, whereas diisopropylfluorophosphate completely abrogated the activity. In this work we provide direct evidence that fgl2 cleaves prothrombin to thrombin consistent with serine protease activity and requires calcium, phospholipids, and factor Va for its full activity.     

The comparison of the estimation of enzyme kinetic parameters by fitting reaction curve to the integrated Michaelis-Menten rate equations of different predictor variables

The estimation of enzyme kinetic parameters by nonlinear fitting reaction curve to the integrated Michaelis-Menten rate equation ln(S(0)/S)+(S(0)-S)/K(m)=(V(m)/K(m))xt was investigated and compared to that by fitting to (S(0)-S)/t=V(m)-K(m)x[ln(S(0)/S)/t] (Atkins GL, Nimmo IA. The reliability of Michaelis-Menten constants and maximum velocities estimated by using the integrated Michaelis-Menten equation. Biochem J 1973;135:779-84) with uricase as the model. Uricase reaction curve was simulated with random absorbance error of 0.001 at 0.075 mmol/l uric acid. Experimental reaction curve was monitored by absorbance at 293 nm. For both CV and deviation <20% by simulation, K(m) from 5 to 100 micromol/l was estimated with Eq. (1) while K(m) from 5 to 50 micromol/l was estimated with Eq. (2). The background absorbance and the error in the lag time of steady-state reaction resulted in negative K(m) with Eq. (2), but did not affect K(m) estimated with Eq. (1). Both equations gave better estimation of V(m). The computation time and the goodness of fit with Eq. (1) were 40-fold greater than those with Eq. (2). By experimentation, Eq. (1) yielded K(m) consistent with the Lineweaver-Burk plot analysis, but Eq. (2) gave many negative parameters. Apparent K(m) by Eq. (1) linearly increased, while V(m) were constant, vs. xanthine concentrations, and the inhibition constant was consistent with the Lineweaver-Burk plot analysis. These results suggested that the integrated rate equation that uses the predictor variable of reaction time was reliable for the estimation of enzyme kinetic parameters and applicable for the characterization of enzyme inhibitors.     

小麦胆色素原脱氨酶的纯化及部分性质研究

生物中四吡咯化合物合成的共同途径是由δ－氨基酮戊酸（δ－ａｍｉｎｏｌｅｖｕｌｉｎｉｃａｃｉｄ,ＡＬＡ）在δ－氨基酮戊酸脱水酶（δ－ａｍｉｎｏｌｅｖｕｌｉｎａｔｅｄｅｈｙｄｒａｔａｓｅ,ＡＬＡＤ）作用下合成胆色素原（ｐｏｒｐｈｏ－ｂｉｌｉｎｏｇｅｎ,Ｐ...     

Characterization of ribonucleotide reductase activity from mouse L cells

We describe some fundamental properties of the cytidine 5'-diphosphate (CDP) and guanosine 5'-diphosphate (GDP) reductase activity from mouse L cells. Both activities increased in a nonlinear fashion at low protein concentrations; this may be due to dissociation of two protein subunits of the enzyme at very low concentrations. CDP reductase activity was greatly stimulated in the presence of ATP and required magnesium and iron for maximum activity. GDP reductase required 2'-deoxythymidine 5'-triphosphate for maximum activity. Also apparent Km values of 0.14 mmol/l for CDP and 0.05 mmol/l for GDP were determined from double reciprocal plots of velocity against substrate concentrations. Activity in extracts of logarithmically growing mouse L cells was very high indicating that attempts to purify the enzyme from this source should be rewarding.     

Kinetic behaviour of free lipase and mica-based immobilized lipase catalyzing the synthesis of sugar esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K(m) and V(max), were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K(m) values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V(max,app)>V(max)). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.