Simplified assay for lysyl oxidase activity.

A simplified method for the assay of lysyl oxidase activity was developed. The method is based on the measurement of tritiated water released by enzyme action from labeled protein-bound lysine and hydroxylysine. Trichloroacetic acid (TCA) supernates of the incubation mixtures are passed through small Dowex 50 (H⁺) columns and the effluents are counted. For rapid screening purposes an indication of the presence of enzyme activity in enzyme preparations can be obtained by measuring the radioactivity present in aliquots of the TCA supernates as such and by measuring the radioactivity after drying at 60°C, taking the difference between the two as a measurement of enzyme activity.     

The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.

Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.     

Regeneration of enzymatic activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and zinc acetate staining: the example of inositol 1,4,5-trisphosphate 5-phosphatase.

Regeneration of enzyme activity after sodium dodecyl sulfate-gel electrophoresis was investigated with a purified inositol 1,4,5-trisphosphate 5-phosphatase. In order to avoid silver or Coomassie blue staining, we have used zinc acetate. This staining procedure was sensitive, rapid, and reversible provided that zinc cations are chelated and activity is extracted after diffusion out of the gel. The method allows some gel lane staining and identification of the enzyme based on catalytic activity.     

用固定化方法研究超氧化物歧化酶的活性与结构的关系

 采用吸附、包埋、共价交联等方法固定化超氧化物歧化酶(SOD)所得固定化酶活力回收都不高,表明酶的催化反应速率受超氧阴离子自由基(O_2~-)扩散速率的控制。用海藻酸钠包埋SOD,固定化酶不表现活力,破固定化后所得的糊状物却有很高的活力。用戊二醛交联所得的固定化酶活力回收率也很低,表明ε-NH_3~+的正电荷是酶活力所必需。     

The effect of high pressure homogenization on the activity of a commercial β-galactosidase

High pressure homogenization (HPH) has been proposed as a promising method for changing the activity and stability of enzymes. Therefore, this research studied the activity of β-galactosidase before and after HPH. The enzyme solution at pH values of 6.4, 7.0, and 8.0 was processed at pressures of up to 150?MPa, and the effects of HPH were determined from the residual enzyme activity measured at 5, 30, and 45?°C immediately after homogenization and after 1?day of refrigerated storage. The results indicated that at neutral pH the enzyme remained active at 30?°C (optimum temperature) even after homogenization at pressures of up to 150?MPa. On the contrary, when the β-galactosidase was homogenized at pH 6.4 and 8.0, a gradual loss of activity was observed, reaching a minimum activity (around 30?%) after HPH at 150?MPa and pH 8.0. After storage, only β-galactosidase that underwent HPH at pH 7.0 retained similar activity to the native sample. Thus, HPH did not affect the activity and stability of β-galactosidase only when the process was carried out at neutral pH; for the other conditions, HPH resulted in partial inactivation of the enzyme. Considering the use of β-galactosidase to produce low lactose milk, it was concluded that HPH can be applied with no deleterious effects on enzyme activity.     

Simple purification and some properties of beef spleen exonuclease

A method for purification of beef spleen exonuclease is described, leading to electrophoretically homogeneous enzyme preparation. The method consists of three step fractionation of crude enzyme (after ammonium sulfate precipitation) as follows - ion exchange chromatography on ECTEOLA-cellulose, affinity chromatography on Concanavalin A-Sepharose and molecular sieving. The enzyme thus obtained is practically free of any contaminating activities - endonuclease or phosphomonoesterase. The molecular weight of the exonuclease was determined (98 000 +/- 3 000 daltons) and some other parameters of the enzyme were calculated. The investigation of the pH and thermo-stabilities showed significantly narrow limits of the exonuclease activity. The effect of the urea on the enzyme activity has also been evaluated.     

Reduction of 5'-nucleotidase activity in rat thyroid and adenohypophysis following methylthiouracil treatment.

5' -Nucleotidase activity was determined in rat thyroid and some other organs employing a specific assay method. During the course of methylthiouracil (MTU) treatment, thyroid 5'-nucleotidase activity decreased significantly. This decrease was specific for this enzyme since the activity of neutral phosphatase did not change and the activity of alkaline phosphatase and Mg2+-activated adenosine triphosphatase increased markedly. The 5'-nucleotidase activity of the adenohypophysis also decreased following MTU treatment. This enzyme activity of the liver, heart and whole brain remained unchanged after the treatment. The role of this enzyme was discussed in relation to tissue growth and increased contents of RNA and DNA in the thyroid and adenohypophysis.     

The analysis of low levels of γ-glutamyltransferase activity by high-performance liquid chromatography with electrochemical detection

A method for the analysis of low levels of the enzyme γ-glutamyltransferase in biological samples by high-performance liquid chromatography with electrochemical detection is described. A γ-glutamyl moiety from glutathione is transferred by the enzyme to glycylglycine to produce a tripeptide which is assayed directly after a purification step using octadecylsilica. Confirmation of the method is by use of the inhibitor AT-125. The method is used to measure the level of enzyme activity in rodent tissues and in cultured cells.     

Enzyme crystal embedded in latex film as immobilized enzyme

Summary A simple and effective method for enzyme crystals immobilization is developed. The water- based acrylic latex mixed with enzyme crystals is coated on a porous membrane. When dried, the latex produces a continuous and strong film in which enzyme crystals are embedded. Latex of three different compositions are synthesized to immobilize urease. The urease crystals embedded in latex film shows a good thermal stability that the activity remains at 60% of its initial activity after 5 days' incubation at 50°C. The film containing amorphous urease powder, on the other hand, has a very poor thermal stability that urease activity decreases to 50% and 3% of its initial activity after 8 hrs' and 3 days' incubation, respectively. The diffusion limitation in the lattices of urease crystal is the main reason for the low activity retention of urease crystals embedded in the latex film.     

Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones

The potential of using immobilized Heat Shock Protein 70 (HSP 70) in combination with other molecular chaperones to ameliorate problems of enzyme denaturation was investigated. Firefly luciferase was used as a model enzyme due to its sensitivity to thermal denaturation, and the availability of a sensitive chemiluminescent assay method for determination of relative activity of this enzyme. Control experiments and development of effective combinations of HSP with other chaperones involved re-activation of enzyme in bulk solution. A combination of HSP 70, alpha-crystallin and reticulocyte lysate (RL) in bulk solution were found to re-activate soluble firefly luciferase to about 60% of the initial activity after the enzyme activity had been reduced to less than 2% by thermal denaturation. HSP 70 that was covalently immobilized onto glass surfaces was also able to re-activate denatured enzyme that was in bulk solution. Over 30% of the initial activity could be regained from heat denatured enzyme when using immobilized HSP in the presence of other chaperones. The activity of soluble enzyme decayed to negligible values in a period of days when stored at room temperature. In the presence of immobilized HSP and chaperones, activity stabilized at about 10% of the initial activity even after many weeks. The results suggest that immobilized molecular chaperones such as HSP 70 may provide some potential for stabilization and re-activation of enzymes that are trapped in thin aqueous films for applications in biosensors and reactors.     

An automated continuous assay of membrane-bound and solube ATPases and related enzymes.

A simple, rapid, and precise method is described for the continuous automated determination of the activity of membrane-bound enzymes which deliberate inorganic phosphate, e.g., ATPases and phosphatases. The characteristics of this method, which is based on the determination of liberated phosphate in the presence of nucleotides, are: (A) the enzyme reaction can be followed continuously during a certain period, thus providing a higher precision, as compared to other methods in which the enzyme reaction is measured by few distinct determinations; (B) the enzyme protein and other (membrane) proteins of the enzyme preparation have not to be removed during the continuous determination of enzyme activity because they remain solubilized after denaturation; and (C) low or moderate concentration of nonionic detergents do not disturb the reading of the absorbancy.     

Stabilisation and immobilisation of penicillin amidase

Penicillin amidase was coupled to a periodate-oxidised dextran by reductive alkylation in the presence of sodium cyanoborohydride. A loss of activity (25%) was observed but the conjugate enzyme dextran was more thermostable than the native enzyme. Native and dextran-conjugated penicillin amidase were immobilised on amino activated silica (Promaxon, Spherosil, Aerosil) by a classical method using glutaraldehyde for the native enzyme and reductive alkylation for the modified enzyme. Good relative activity of the enzymes was obtained after insolubilisation. Immobilisation of both native and modified enzymes resulted in the thermostabilisation of the penicillin amidase.     

Differences in thermal sensitivities of the regulatory-catalytic and catalytic forms of cyclic AMP-dependent protein kinases from various tissues.

The cAMP-dependent protein kinase from various tissues was more thermally sensitive when activated by cAMP than the non-activated enzyme. For example, when the activity ratio (the activity of protein kinase assayed -cAMP/+cAMP) was 0.40, 80% and 76% of total hepatic cAMP dependent protein kinase activity was recoverable after incubations at 45 degrees C for 15 and 30 minutes, respectively. However, when the activity ratio was elevated to about 0.80 - 0.90 by increasing cAMP levels in vivo or adding exogenous cAMP to soluble liver extracts, the total protein kinase activity recoverable after incubations at 45 degrees C for 15 minutes was 34-44% and 19-22%, respectively. This observation was used to estimate the degree of activation of the enzyme in vivo and in vitro, since the loss of enzyme activity at 45 degrees C was directly related to the degree of activation of the enzyme in tissue extracts. The regulatory-catalytic form of cAMP-dependent protein kinase was thermally resistant at 45 degrees C unless activated by incubation with exogenous cAMP, histones or NaCl, while the catalytic form of the enzyme was highly thermally sensitive at this same temperature. These data describe a new property of the cAMP-dependent protein kinase and suggest an alternative method which measure the degree of activation of the enzyme.     

Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes.

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.     

反义trxs基因导入对小麦籽粒脱支酶活性的影响

以转反义硫氧还蛋白基因株系01TY34-73-9及其对照品种‘豫麦34’为材料,运用PCR检测和酶活性测定的方法,对转基因株系遗传稳定性以及转基因与对照种子中脱支酶活性进行测定。结果显示:(1)外源基因已经稳定遗传至后代;(2)转基因种子在不同成熟时期和不同萌发过程中的脱支酶活性与对照相比均有不同程度的降低平均降低10.3%,但仅花后25 d到收获后5 d脱支酶活性显著低于对照,其中最低值出现在花后30 d,平均比对照下降了12.0%;(3)在花后30 d和后熟5 d萌发过程中,转基因种子脱支酶活性始终低于对照,平均下降6.2%和22.2%。表明反义trxs基因的导入干扰了小麦trxh基因的表达,使trxh转录量减少,小麦籽粒中脱支酶的活性受抑。     

A new mechanochecmical method of enzyme immobilization

A new mechanochemical method for enzyme immobilization has been elaborated. The principle of this method consists of the following precepts. Partially hydrolyzed nylon fiber, the surface of which is known to be strewn with micro-cracks, is reversibly stretched (～25%) and placed into an enzyme solution. Then, in the same solution, the fiber is made to relax and is taken out. The fiber retains considerable enzymatic activity even after numerous thorough washings (in a similar procedure without fiber stretching, equivalent washing removed all the enzymatic activity from the fiber). Immobilization on the fiber proceeds due to trapping of enzyme molecules by the microcavities on the surface of the support. The catalytic activity of mechanochemically immobilized chymotrypsin and trypsin is commensurable with their activity on covalent immobilization on nylon (calculated per unit of the macrosurface). A wide range of commercial polymers may be made of use as supports in the mechanochemical method of immobilization.     

大鼠脊髓不完全性损伤后前角运动神经元的酶细胞化学改变

以改良Ａｌｅｎ氏法造成Ｗｉｓｔａｒ大鼠不完全性脊髓损伤,采用神经学功能评分法评定大鼠运动功能,应用定量酶细胞化学方法观察脊髓前角运动神经元内乙酰胆碱酯酶（ＡＣｈＥ）和酸性磷酸酶（ＡｃＰ）活性变化。结果显示：１．脊髓损伤后大鼠运动功能障碍,随后逐渐恢复。２．前角运动神经元内ＡＣｈＥ活性减弱、ＡｃＰ活性增强;随后酶活性呈逐渐恢复,四周时ＡＣｈＥ活性基本恢复正常。结果说明：大鼠脊髓不完全性损伤后运动功能变化与前角运动神经元的功能状态具有较强的相关性;前角运动神经元在不完全性脊髓损伤运动功能恢复中起重要作用。     

Immunoenzyme Analysis of Cytokinins as an Assay for Cytokinin Oxidase Activity

A new highly sensitive method of measuring cytokinin oxidase activity was worked out on the basis of an immunoenzyme test-system for isopentenyladenosine (IPA) assay. The enzyme activity is determined by immunoenzyme assay as the difference between the IPA quantities in the reaction mixture before and after incubation. The specificity of the assay was verified by using the well-known enzyme activators and inhibitors and by monitoring the formation of other cytokinins.     

Characterization of molecular-sieve-bound inulinase

The enzyme inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7), prepared from Kluyveromyces marxianus has been immobilized using an inorganic solid support, molecular sieve 4A via the metal link method. The immobilized enzyme had around 22 units of inulinase activity per g of the support with retention of 72% of the original activity. The optimum protein to molecular sieve ratio for the maximum retention of inulinase activity was 9 mg/g molecular sieve. The properties of soluble and immobilized enzyme differed in many respects. The optimum pH of the enzyme shifted from 6 to 5 and the optimum temperature of enzyme activity changed from 50 to 55°C. K_m values were 6.7 mM for soluble enzyme and 10 mM for immobilized enzyme. The heat stability of the enzyme was improved by immobilization. Immobilized enzyme retained about 76% of the original activity after 40 days of storage at room temperature (30±2°C).     

A rapid and simple spectrophotometric assay of angiotensin-converting enzyme.

A rapid, simple, and accurate method for the chemical assay of anglotensin-converting enzyme has been developed. The method relies on previously published method for spectrophotometric assay of angiotensin-converting enzyme activity and on the use of 2,4,6-trichloro-s-triazine (TT) as a colorimetric reagent of hippuric acid (N-benzoylglycine). When 3% TT in dioxane was added to the incubation medium of the angiotensin-converting enzyme after stopping the incubation by the immersion of the test tubes in a boiling-water bath, the absorbance at 382 nm increased linearly as a function of both enzyme concentration and incubation time. Neither hippuryl-l-histidyl-l-leucine (HHL, substrate for this assay system) nor histidyl-leucine was positive in color reaction with TT. Accordingly, this method does not require any procedures for separation of hippuric acid from HHL. The enzyme activity was found to be highest at pH 8.3, at chloride ion concentration of 600 mm, and at HHL concentration of 3 mm, when the 5000g supernatant fluid of the rat lung was used.