Coupled reaction of immobilized aspartate aminotransferase and malate dehydrogenase. A plausible model for the cellular behaviour of these enzymes

To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.     

Enzymatic synthesis of polyaniline film using a copper-containing oxidoreductase: bilirubin oxidase.

Electroactive polyaniline films have been synthesized by using a copper-containing oxidoreductase, bilirubin oxidase (BOD). Enzymatic polymerization took place on the surface of BOD-adsorbed solid matrix which was in contact with a buffer solution containing aniline. Optimum conditions for enzymatic polymerization of aniline were investigated. Elemental analysis and IR spectroscopy indicated that the enzymatically synthesized film was polyaniline. The cyclic voltammetric studies demonstrated that the polyaniline film was electrochemically reversible in the redox properties in acidic aqueous solutions. Since the film retained enzymatic activity of BOD which was employed as a catalyst for polymerization, enzymatic polymerization seems promising in preparation of immobilized enzyme membranes.     

A mild methods of general use for covalent coupling of enzymes to chemically activated collagen films

Films of highly polymerized collagen, prepared in industrial conditions, were chosen for surface covalent binding of enzymes because of their insolubility, mechanical resistance, proteic nature, hydrophilic properties and for their abundance in chemically activable ? COOH. Untanned films, previously acid- methylated, were activated by acyl azide formation. After removal of reagents by repeated washing, the coupling of enzyme was performed by immersion of the activated film in the enzyme solutions (2 to 3 hr, 0°C). The procedure is particularly mild since the enzymes never come into contact with chemical reagents, and thus avoid all denaturing processes. All the enzymes tested were successfully bound: glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, glutamate pyruvate transaminase, creatine kinase, hexokinase, trypsin, and urease. As tested with aspartate amino transferase, enzymatic activity remained constant for months (100% after 5 months) in spite of repeated use of the film at 30°C, washing and storage in buffer at 4°C between assays.     

Chemically activated collagen for amyloglucosidase attachment. Use in a helicoidal reactor.

Amyloglucosidase was covalently bound to collagen sheets by a previously described method. The time of acidic methylation (first step of the collagen activation process) was important to obtain a good enzymatic surfacic activity. Homogeneity of the coupling procedure on the surface of collagen films was shown. Some properties of free enzyme were not affected after grafting; optimum pH and temperature, activation energy, and Km for maltose. Heat stability of the bound enzyme was slightly better; Km for soluble starch increased fivefold. In contrast, the maximal velocity in the presence of soluble starch remained four times that of maltose hydrolysis. Amyloglucosidase collagen membranes were used in a helicoidal reactor to produce glucose from maltose or soluble starch solutions. Tracer studies have shown that the helicoidal reactor behaved as a CSTR. The influence of maltose concentration and flow rate on conversion was studied and confirmed the absence of diffusional limitations for maltose. Recycling of concentrated solutions of maltose and soluble starch indicated strong diffusional restrictions for soluble starch. The catalytic support kept all its activity for 18 days continuous operation at 40 degrees C and 80% after 17 months storage at 4 degrees C.     

Aspartate aminotransferase immobilized on collagen films. Activity of dissociated subunits.

Aspartate aminotransferase has been covalently bound to insoluble films of collagen. The immobilized subunits of holoenzyme obtained after alkaline dissociation were inactive but their activity was recovered by incubation with either active enzyme, reduced holoenzyme, or apoenzyme. It was also possible to reduce the bound subunits by sodium borohydride and to reassociate them with native subunits of holoenzyme.     

Immobilization of glycoenzymes through carbohydrate side chains.

Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N-hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.     

Immobilization of RNase Rs via its carbohydrate moiety to aminoethyl-Bio-Gel P-2 and its application for the hydrolysis of RNA to 2′,3′ cyclic nucleotides

Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35–40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 μM periodate oxidized enzyme are allowed to react with 1 ml (packed volume) of AE-Bio-Gel P-2 at 6±1°C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K_m but resulted in a 2·5-fold decrease in the V_max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2′,3′ cyclic nucleotides in 14 h, at pH 5·5 and 37°C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity.     

Cytochemical model system for microsomal rat liver glucose-6-phosphate.

A method is described for the incorporation of a microsomal rat liver fraction into polyacrylamide films without significant loss of its glucose-6-phosphatase activity. The enzymatic activity was completely lost when the films were prepared with ammonium persulfate as initiator of the polymerization as previously described for alkaline phosphatase, but modification of this method showed that about 90% of the glucose-6-phosphatase activity could be retained. The enzyme in the films prepared with the new method was completely inhibited by alloxan, HgCl2, and preincubation in 0.05 M acetate buffer (pH 5.0) at 37 degrees C, as determined biochemically. Similar results were obtained for the enzyme in films determined histochemically according to the lead method of Wachstein and Meisel. In this respect the behavior of the incorporated enzyme is similar to that in suspension. Films fixed with 1.5% glutaraldehyde showed rapid inactivation of glucose-6-phosphatase. There was good correlation between the biochemical and histochemical activity determined after fixation. A method to embed polyacrylamide films in Epon for electron-microscopical investigation is also described. Dimethyl sulfoxide was used as the dehydrating agent instead of ethanol/acetone.     

Partial purification and immobilization of ribonuclease T2.

A simple procedure, consisting of water extraction, heat treatment at pH 2.0, negative adsorption on DEAE-cellulose at pH 4.9, and concanavalin A-Sepharose chromatography, was developed for the partial purification of ribonuclease (RNase) T2 from taka-diastase powder with an overall yield of 5.5%. The partially purified enzyme when coupled to aminoethyl Bio-Gel P-60, retained 12-16% of the activity of the soluble enzyme. Temperature stability studies on RNase T2 bound to matrices, activated with increasing concentrations of glutaraldehyde, and the influence of lysine modification on the activity of the soluble enzyme revealed that the low activity observed for the gel-bound enzyme is probably due to the masking of the active site of the enzyme as a result of the involvement of lysine residues, situated near the active site, during coupling. Immobilization did not affect the pH and temperature optima of RNase T2. On repeated use, the bound enzyme retained approximately 55% of its initial activity after six cycles. These results are discussed, taking into consideration the factors affecting immobilized enzymes.     

Siloxane-based biocatalytic films and paints for use as reactive coatings

We have developed a new methodology for preparing films and paints suitable for use as biocatalytic coatings. The hydrolytic enzymes pronase and alpha-chymotrypsin were immobilized by either sol-gel entrapment or by covalent attachment into a polydimethylsiloxane (PDMS) matrix and cast into thin films or incorporated into an oil-based paint formulation. All of the coatings retained enzymatic activity and adhered to several different materials. The enzymatic films and paints also exhibited higher thermostability than enzyme free in solution or covalently attached to the outer surface of PDMS. A porous membrane based on a PDMS-immobilized enzyme was also prepared by an immersion precipitation process. Protein adsorption measurements showed that the enzyme-containing films and paints adsorbed less protein than enzyme-free controls, and that protein adsorption decreased with increasing proteolytic activity of the coating. These coatings thus provide the means to apply a stable enzymatic surface to a wide range of materials, and may be generally useful as biocatalytic paints and films.     

Studies on Horseradish Peroxidase Immobilized onto Arylamine and Alkylamine Glass

Peroxidase from horseradish has been immobilized onto zirconia coated arylamine and alkylamine glass through the process of diazotization and glutaraldehyde coupling, respectively. Arylamine glass bound enzyme retained 77% of the initial activity with a conjugation yield of 18 mg g^-1 support, while alkylamine glass bound enzyme retained 38% of the initial activity with a conjugation yield of 16 mg g^-1 support. The immobilized enzyme showed an increase in optimum pH, temperature for maximum activity, energy of activation (Ea), and thermal stability but decrease in time for linearity and Km for H₂O₂. Vmax value of arylamlne conjugated enzyme decreased but Vmax of alkylamine conjugated enzyme was unaltered compared to free enzyme. Both arylamine and alkylamine bound enzyme showed higher stability in cold compared to that of free enzyme. The application of glass bound peroxidase in discrete analysis of serum urate is demonstrated.     

Porphyrin biosynthesis: immobilized enzymes and ligands. VI. Studies on succinyl CoA synthetase from cultured soya bean cells

Soybean callus succinyl CoA synthetase (succinate: CoA ligase, (ADP-forming), EC 6.2.1.5), has been chemically bound to Sepharose 4B and some of its properties have been studied. The optimal conditions for binding have been determined. The immobilized enzyme retained 48% of the activity of the soluble enzyme and the coupling yield amounted to 50%. Sepharose-succinyl CoA synthetase can be stored at 4 degrees C for periods up to 90 days with only 25% loss of activity; it can also be repeatedly used without alteration of its enzymic activity. The complex showed enhanced thermal stability; pH optimum was between 7.0 and 8.0 for the bound enzyme, and 8.0 for the free enzyme. A general decrease in the Michaelis-Menten constants for the different substrates of the insoluble enzyme, as compared with values obtained for the free enzyme, was found. Plots of the rate product formation against ATP concentration changed from sigmoideal for the soluble succinyl CoA synthetase to hyperbolic for the immobilized enzyme.     

A model system of coupled activity of co-immobilized creatine kinase and myosin.

Myosin and creatine kinase were co-immobilized onto Immunodyne films to mimic the behaviour of creatine kinase bound to the M-line of myofilaments. The Mg-ATPase activity of bound myosin was studied by a coupled enzymatic assay, which detects Mg-ADP in the bulk solution by means of pyruvate kinase and lactate dehydrogenase. The competition for Mg-ADP between pyruvate kinase and creatine kinase either free in solution or co-immobilized with myosin was studied at various creatine phosphate concentrations. Bound creatine kinase competed efficiently when present in very low amounts, corresponding to an activity ratio higher than 1:20,000 between creatine kinase and pyruvate kinase and a molar ratio higher than 1:1000 between creatine kinase and myosin. The Mg-ADP produced by myosin ATPase in the vicinity of the film did not diffuse into the bulk solution but, in the presence of creatine phosphate, was recycled into Mg-ATP by the neighbouring creatine kinase. The existence of an unstirred layer near the surface of the film is sufficient to explain the channeling of ADP (or ATP) between co-immobilized myosin and creatine kinase, without direct interaction or 'intimate coupling' between the enzymes. The problem now is to determine the importance of this kind of facilitated diffusion in the myofilaments in vivo.     

Diffusion-limited kinetics of immobilized myosin ATPase

To test the possibility that ATP diffusion limits the kinetics of myosin ATPase (EC. 3.6.1.3) in situ, myosin was covalently bound to the surface of 2 kinds of films: collagen and Immunodyne. On collagen films, it was bound either with 1-ethyl-3 (3 dimethyl-aminopropyl)carbodiimide (EDC) or with dimethyl-3,3'-dithiobis(propionimidate) (DTP). The apparent Km for K+-ATP rose from 0.26 mM for free myosin in solution to 2-5 mM for covalently bound myosin, and maximum K+-ATPase activity was very low. With the other film, Immunodyne from Pall, the maximum activity of bound myosin was 170 nmol per min per 1.5 cm2 film. The apparent Km for K+-ATP was 2.1 mM when the incubation mixture was vigorously stirred, and the effect of stirring indicated that the kinetics of K+-ATP hydrolysis are limited by external diffusion. The large amount of myosin bound per unit of Immunodyne film surface permitted the study of Mg2+-ATPase activity, although it was 400-500 times less than the K+-ATPase activity. The apparently non-Michaelian kinetics of Mg2+-ATP hydrolysis are attributable to the external diffusion. The apparent Michaelis constant observed at low Mg2+-ATP concentrations rose from 0.27 microM for myosin in solution to 5 microM for myosin bound to Immunodyne film.     

Porphyrin biosynthesis. Immobilized enzymes IV. Studies on aminolaevulate dehydratase attached to Sepharose

Summary Bovine liver aminolaevulate dehydratase (ALAD) has been chemically attached to Sepharose 4B and its properties have been studied. The optimal conditions for coupling have been determined. It was found that the immobilized enzyme retained a significant percentage of the activity of the free enzyme. The coupling yield was rather high. The insolubilized enzyme requires both anaerobiosis and a thiol activator for maximal activity. It can be stored at 4 °C for long periods with little loss of activity and it can be repeatedly used without alteration of its enzymic capacity. Attachment of ALA-D to the gel has led to an enhanced thermal stability. pH optima of free and bound enzyme was the same while a small decrease in the Km of the matrix bonded ALA-D as compared to that of the soluble enzyme was observed. The use of the fixed-ALA-D for the preparation of PBG is described.Dedicated to Professor Luis F. Leloir on occasion of his 70th Birthday.     

三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。     

Immobilization of aspartate aminotransferase on agarose

Various methods for immobilization of aspartate aminotransferase (AspAT; from cytosolic fraction of pig heart) on agarose were tested. Aldehyde-, thiol-, and CNBr-activated agaroses were studied in detail. The capacity of the aldehyde support to firmly bind protein was less than 0.2 mg/ml, whereas the apparent remaining specific activity of the bound AspAT was high (50-63% of soluble AspAT). The maximum capacity of SH-agarose to bind enzymatic protein was 3 mg/ml; the apparent remaining activity was 30-40%, and the specific activity determined by Vmax was 51%. Chemical coupling on to thiol-agarose did not denature the enzyme, as 93% of protein and 83% of the activity were recovered after release of the enzyme from the support. Enzyme protein was quantitatively bound to CNBr-activated agarose (up to 10 mg/ml of the gel). The apparent specific activities were 27-35%, while the value calculated from Vmax was 46%. Active site-protecting agents within the CNBr-coupling were tested. Bromphenol blue increased the apparent specific activity to 60% and Vmax to 80% at 3-fold molar concentration at the active sites. Kinetic constants for immobilized preparations were determined.     

Influence of the conditions of trypsin immobilization onto Spherosil on coupling efficiency

Summary The effect of several parameters (pH, time of reaction, temperature, enzyme concentration) on trypsin immobilization onto glutaraldehyde-activated amine-Spherosil was investigated. This activated support could be stored over long periods of time without any important loss of capacity for trypsin coupling. When increasing the amount of trypsin bound to the carrier, enzymatic activity shows an optimal value, beyond which an augmentation of Spherosil enzyme content results in a lowered activity. The influence of the number of available reactive aldehyde groups on silica was investigated by coupling L-lysine to activated support either prior to or simulataneously with trypsin immobilization. In both cases, the activity of trypsin derivatives is decreased when L-lysine concentration is increased, yet the activity of trypsin derivatives is never equal to zero, even in presence of a large excess of L-lysine. This suggests the presence of two types of reactive groups on the activated support.     

Oxidation of NADH in Glyoxysomes by a Malate-Aspartate Shuttle

Glyoxysomes isolated from germinating castor bean endosperm accumulate NADH by β-oxidation of fatty acids. By utilizing the glutamate: oxaloacetate aminotransferase and malate dehydrogenase present in glyoxysomes and mitochondria, reducing equivalents could be transferred between the organelles by a malate-aspartate shuttle. The addition of aspartate plus α-ketoglutarate to purified glyoxysomes brought about a rapid oxidation of accumulated NADH, and the oxidation was prevented by aminooxyacetate, an inhibitor of aminotransferase activity. Citrate synthetase activity in purified glyoxysomes could be coupled readily to glutamate: oxaloacetate aminotransferase activity as a source of oxaloacetate, but coupling to malate dehydrogenase and malate resulted in low rates of citrate formation. Glyoxysomes purified in sucrose or Percoll gradients were permeable to low molecular weight compounds. No evidence was obtained for specific transport mechanisms for the proposed shuttle intermediates. The results support a revised model of gluconeogenic metabolism incorporating a malate-aspartate shuttle in the glyoxysomal pathway.     

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.