马鹿茸血免疫活性肽的制备及其活性研究

本文以免疫活性和DPPH·的清除能力为指标,研究了用木瓜蛋白酶与中性蛋白酶水解马鹿茸血制备活性肽的条件,并初步探讨了此活性肽的免疫活性与对DPPH·清除能力之间的关系.实验结果表明:当[E/S]为9000 U/g时,中性蛋白酶和木瓜蛋白酶的水解温度各为50和45 ℃,pH各为7.1和6.8,分别水解2和1.5 h可以获得最佳的酶解效果.在相同蛋白含量的情况下,两酶复合水解物的淋巴细胞增殖率比单酶水解增加了20.8%.免疫活性与DPPH·清除能力之间存在一定的相关性(r=0.957,P<0.01).     

微生物酶不对称水解合成S(+)-布洛芬的研究 Ⅱ.反应条件和产物提取

皮状丝孢酵母具有较强不对称水解底物专一性.在试验的五种布洛芬消旋酯中,水解甲酯和异丙酯生成s(+)-布洛芬ee可达97%,乙酯为93%以上;而水解活性以乙酯最强,转化率高于30%.不对称水解最适pH6.5—7.0;温度在28—37℃范围内拆分能力无明显差别.该酵母的水解酶为胞内酶,将酵母细胞制成两酮干粉进行水解可提高立体专一性.产物S(+)-布洛芬可借助于酸碱反应和有机溶剂提取得到,同时回收未水解的酯.     

大鲵（Andrias davidianus）4种生殖器官的蛋白水解酶种类和活性分析

用蛋白水解酶活性电泳方法（G-PAGE）分析了大鲵卵巢,输卵管,精巢,输精管的蛋白水解酶种类和活性,结果表明：1）精巢和输精管的蛋白水解酶种类（分子量）相似,活性有差异。主要的蛋白水解酶分子量为240,85,73,61,51,42,37和23kD;2）输精管的蛋白水解酶在碱性条件下活性最强,在中性条件下活性次之,在酸性条件下活性最弱。精巢的蛋白水解酶在中性和碱性条件下活性相似,在酸性条件下活性很弱,推测精巢蛋白水解酶活性的最适pH为中性,输精管蛋白水解酶活性的最适pH为碱性;3）卵巢和输卵管的蛋白水解酶种类（分子量）相似,主要的蛋白水解酶分子量为73、61、51和37kD,它们在酸性条件下活性最强,在中性条件下几乎无活性,推测它们活性的最适pH为酸性;4）与卵巢和输卵管相比,精巢和输精管的蛋白水解酶种类多,活性强,活性的最适pH高,推测这种差别可能有利于受精和发育中所需的蛋白水解酶快速灭活或活化。     

水稻氨基酰化酶的分离纯化与性质分析

氨基酰化酶（N－acylamino－acidamidohydrolase或acylaseⅠ,EC3．5．1．14）是专一水解N-酰基化L-氨基酸的蛋白酶．从水稻黄化苗得到的抽提液,经过硫酸铵分级沉淀、丙酮分级沉淀和阴离子交换层析三个步骤,纯化得到了该酶,比活达到100U／mg蛋白,在无还原剂存在的SDS-聚丙烯酰胺凝胶电泳上显单一条带,分子量为40kD．而凝胶层析分析表明活性分子的分子量约90kD,因此可推测它的活性分子由两个亚基通过非共价键作用组合而成．进一步研究此酶的性质,在所测的五种乙酰化氨基酸中,最适底物为N-乙酰-L-甲硫氨酸．该酶的最适温度为50℃,最适pH为7．0～8．0．Co2＋和Zn2＋能增强酶活性,但烷基化试剂对酶活性没有影响,表明酶活性中心不含活化的巯基或羟基基因     

米糠蛋白抗氧化活性肽的制备

以水解度(DH％)和对DPPH自由基清除率为指标,筛选出制备米糠蛋白抗氧化活性肽的最适蛋白酶.研究最适蛋白酶的酶解条件,探讨底物浓度、蛋白酶的加入量、pH值、酶解时间等因素对水解度(DH％)和DPPH自由基清除率的影响;在单因素基础上采用Box-Behnken响应曲面中心组合设计法,对酶解米糠蛋白的工艺进行优化.试验结果表明,在加酶量13970.82 U/g,时间3.05h,底物浓度4.97％的水解条件下,米糠蛋白的水解度能够达到23.67％,活性肽对DPPH自由基清除率达到64.26％.     

眼镜蛇毒中一个弱纤维蛋白原水解活性金属蛋白酶的纯化和性质研究

通过蛋白层析从中华眼镜蛇毒中分离纯化出一个新的纤维蛋白原水解酶atrase A. Atrase A是一个分子量为64.6 kD,等电点为pH 9.6和中性糖含量为4.16%的碱性单链糖蛋白.它具有弱的纤维蛋白原α链水解活性.该活性能被金属螯合剂EDTA, EGTA,1,10 phenanthroline和还原剂DTT完全抑制,而PMSF只能部分抑制该活性,大豆胰蛋白酶抑制剂对其没有影响, 表明atrase A属于金属蛋白酶. Atrase A具有水肿活性和金黄色葡萄球菌抑制活性.它对A549 和K562 细胞没有细胞毒性,但能使贴壁生长的A549细胞解离悬浮. Atrase A没有纤维蛋白、azocasein 、BAEE水解活性,对ADP、胶原诱导的血小板聚集没有明确的抑制作用. 经小鼠皮下注射后没有发现其有出血毒活性.     

东北羊草草原羊草种群生长与环境关系的研究

本文研究了东北单草草原单草种群的生长, 并利用灰色系统理论考查了环境因子对单草生长的作用规律. 结果表明:羊草种群的生长具有明显的季节进程, 7-8月份生长最快, 生长速率为0.20-0.403g/m²·d, 相对单草的生长. 土壤水解氮、速效钾、活性有机质、温度、pH值和有效磷具有较大的灰色关联度, 其中水解氮、速效钾和活性有机质是羊草生长的优势因子, pH值是限制因子. 羊草生长的最优模型为y=4.74x₁+6.15x₂+2.48x₃-73.64.     

胰蛋白酶活性的定量测定方法

对甲苯磺酰基精氨酸甲酯(TAME)是胰蛋白酶的专一性底物. TAME经胰蛋白酶水解释放出的对甲苯磺酰基精氨酸与活性测定混合物中的NaOH反应, 导致溶液pH值的下降. 以酚红为指示剂, 通过测定555nm处光吸收值的降低可以监测pH的变化. 在0.001～0.3μg的范围内, 胰蛋白酶含量与555nm处光吸收值的降低呈线性关系.     

Alcalase碱性蛋白酶对中华稻蝗蛋白水解条件的研究

本试验采用Alcalase碱性蛋白酶对中华稻蝗蛋白进行水解,研究其蛋白酶解条件和酶解物的抗氧化性(用抑制邻苯三酚自氧化率来表示).结果表明,实验室最佳酶解条件为:底物浓度1%,pH值8.0,温度55℃,水解时间4 h,加酶量(V/V,%)为10%.在此条件下其酶解物具有明显的抗氧化活性,对邻苯三酚自氧化的抑制率可达40%,水解度为51%.     

一种来源于Leifsonia shinshuensis DICP 16菌体的β-D-木糖苷酶的分离纯化及其性质

采用盐析、DE 52、Q-Sepharose Fast Flow阴离子交换层析、Toyopearl Butyl 650C疏水层析以及Sephacryl S-300 HR凝胶过滤层析联用的方法, 从Leifsonia shinshuensis DICP 16菌体中纯化出一种β-木糖苷酶.分离后该酶在SDS-PAGE 上呈单一蛋白质条带, 通过SDS-PAGE和凝胶过滤层析法, 测得该酶是一个由两个分子量约为91 kD的相同亚基组成的同源二聚体.其水解对硝基苯酚木糖苷(pNPX)的最适反应温度为55°C, pH值为7.0.该木糖苷酶在45°C以下, pH 6.0～11.0之间具有很好的稳定性.在45°C, pH值为7.0的条件下, 水解pNPX的Km, Vmax分别为1.04 mmol/L, 0.095 mmol/(min·mg).研究不同的金属离子对该酶的活性影响, 发现Fe2+和Cu2+是很强的抑制剂.通过对天然木糖苷化合物的水解测试, 发现该酶可以水解人参皂苷Rb3的木糖基, 产生人参皂苷Rd, 却不能水解紫杉烷木糖苷的木糖基.     

Studies on the Mechanism of Activation of Pepsinogen

The mechanism of activation of pepsinogen was studied. It was found that no peptide bond cleavage occurred in the molecule of denatured pepsinogen at pH 2. It was inferred from this that a specific secondary and tertiary structure is formed in the molecule of pepsinogen in acid and that it might be necessary for the hydrolysis of the peptide bond. From the circular dichroism studies on pepsinogen and pepsin, it was found that there is a conformational change in the molecule of pepsinogen at pH 4.3~4.5 and that this change is followed by a gradual formation of pepsin.     

Mechanism of intramolecular activation of pepsinogen. Evidence for an intermediate delta and the involvement of the active site of pepsin in the intramolecular activation of pepsinogen.

Intramolecular pepsinogen activation is inhibited either by pepstatin, a potent pepsin inhibitor, or by purified globin from hemoglobin, a good pepsin substrate. Also, pepsinogen at pH 2 can be bound to a pepstatin-Sepharose column and recovered as native zymogen upon elution in pH 8 buffer. Kinetic studies of the globin inhibition of pepsinogen activation show that globin binds to a pepsinogen intermediate. This interaction gives rise to competitive inhibition of intramolecular pepsinogen activation. The evidence presented in this paper suggests that pepsinogen is converted rapidly upon acidification to the pepsinogen intermediate delta. In the absence of an inhibitor, the intermediate undergoes conformational change to bind the activation peptide portion of this same pepsinogen molecule in the active center to form an intramolecular enzyme-substrate complex (intermediate theta). This is followed by the intramolecular hydrolysis of the peptide bond between residues 44 and 45 of the pepsinogen molecule and the dissociation of the activation peptide from the pepsin. Intermediate delta apparently does not activate another pepsinogen molecule via an intermolecular process. Neither does intermediate delta hydrolyze globin substrate.     

Monkey pepsinogens and pepsins. VI. One-step activation of Japanese monkey pepsinogen to pepsin

When Japanese monkey pepsinogen was activated at pH 2.0 in the absence of pepstatin, the activation segment of the amino(N)-terminal 47 residues was released as a single intact polypeptide. This clearly shows that the pepsinogen was activated to pepsin directly. This direct activation was called a 'one-step' process. On the other hand, when pepsinogen was activated at pH 2.0 in the presence of pepstatin, an appreciable amount of pepsinogen was converted to an intermediate form between pepsinogen and pepsin, although a part of pepsinogen was activated directly to pepsin. The intermediate form was generated by releasing the N-terminal 25 residues of pepsinogen. This activation through the intermediate form is thought to be a 'two-step' or 'stepwise-activating' process involving a bimolecular reaction between pepstatin-bound pepsinogen and free pepsin.     

Activation of human pepsinogens

Human pepsinogen A3 and A5 have been purified to chromatographic and electrophoretic homogeneity. At pH 2 pepsinogen A3 activates at a much faster rate than pepsinogen A5. Leu-23-Lys-24 is the first bond cleaved during activation of pepsinogen A3. This bond is also cleaved in pepsinogen A5, but together with the cleavage of Asp-25-Phe-26. Amino acid sequencing shows that pepsinogen A3 has Glu at position 43, whereas pepsinogen A5 has Lys.     

Analysis of the activation of pepsinogen in the presence of protein substrates and estimation of the intrinsic proteolytic activity of pepsinogen

Monkey pepsinogen A, monkey progastricsin, and porcine pepsinogen A were activated in the presence of two different protein substrates, namely, reduced and carboxymethylated lysozyme and hemoglobin. In each case, an extensive delay in activation was observed. The intermolecular activation reaction required for the generation of pepsin or gastricsin was strongly inhibited and this inhibition was essentially responsible for the delay. However, the intramolecular reaction required for the generation of the intermediate forms of the proenzymes was scarcely affected. The delay was longer at pH 3.0 than at pH 2.0. Irrespective of the delay in activation of pepsinogen, the digestion of substrates proceeded rapidly, evidence of the significant proteolytic activity of pepsinogen itself. Kinetic experiments demonstrated that pepsinogen changed from an enzymatically inactive species to an active species before the release of the activation segment. The proteolytic activity of the active pepsinogen was highest at pH 2.0, at 37 degrees C and the activity under these conditions was comparable to that of pepsin.     

Occurrence of two different pathways in the activation of porcine pepsinogen to pepsin

Activation of porcine pepsinogen at pH 2.0 was found to proceed simultaneously by two different pathways. One pathway is the direct conversion process of pepsinogen to pepsin, releasing the intact activation segment. The isolation of the released 44-residue segment was direct evidence of this one-step process. At pH 5.5 the segment bound tightly to pepsin to form a 1:1 pepsin-activation segment complex, which was chromatographically indistinguishable from pepsinogen. The other is a stepwise-activating or sequential pathway, in which pepsinogen is activated to pepsin through intermediate forms, releasing activation peptides stepwisely. These intermediate forms were isolated and characterized. The major intermediate form was shown to be generated by removal of the amino-terminal 16 residues from pepsinogen. The released peptide mixture was composed of two major peptides comprising residues 1-16 and 17-44, and hence the stepwise-activating process was deduced to be mainly a two-step process.     

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.     

Evidence for the occurrence of one-step activation in porcine pepsinogen

Upon activation at pH 2.0 and 14°C, a significant portion of porcine pepsinogen was found to be converted directly to pepsin, releasing the 44-residue intact activation segment. The released segment was further cleaved to smaller peptides at pH 2.0, but at pH 5.5 it formed a tight complex with pepsin, and the complex was chromatographically indistinguishable from pepsinogen. This intact segment could be isolated for the first time. Thus one-step activation occurs in porcine pepsinogen along with the already known sequential activation.     

Molecular characteristics of pepsinogen and pepsin from duck glandular stomach

1. Two procedures were developed for the preparation of duck pepsinogen, an enzyme from the family of aspartic proteases (EC 3.4.23.1) and its zymogen. 2. The amino acid composition, sugar content and the partial N- and C-terminal sequences of both the enzyme and the zymogen were determined. These sequences are highly homologous with the terminal sequences of chicken pepsin(ogen). 3. Duck pepsinogen and pepsin are unlike other pepsin(ogen)s in being relatively stable in alkaline media: pepsinogen is inactivated at pH 12.1, pepsin at pH 9.6. 4. Duck pepsin is inhibited by diazoacetyl-D,L-norleucine methyl ester (DAN), 1,2-epoxy-3(p-nitrophe-noxy)propane (EPNP), pepstatin and a synthetic pepsin inhibitor Val-D-Leu-Pro-Phe-Phe-Val-D- Leu. The pH-optimum of duck pepsin determined in the presence of synthetic substrate is pH 4. 5. Duck pepsin has a marked milk-clotting activity whereas its proteolytic activity is lower than that of chicken pepsin. 6. The activation of duck pepsinogen is paralleled by two conformational changes. The activation half-life determined in the presence of a synthetic substrate at pH 2 and 14 degrees C is 20 sec.     

Denaturation studies on natural and recombinant bovine prochymosin (prorennin).

1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v.c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.