各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans.

Methylamine dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methylamine-grown cells. The enzyme exhibited a pI value of 4.3 and was composed of two 46,700-dalton subunits and two 15,500-dalton subunits. Each small subunit possessed a covalently bound pyrrolo-quinoline quinone prosthetic group. The amino acid compositions of the large and small subunits are very similar to those of other methylamine dehydrogenases which have been isolated from taxonomically different sources. The enzyme was able to catalyze the oxidation of a wide variety of primary aliphatic amines and diamines, but it did not react with secondary, tertiary, or aromatic amines. The enzyme exhibited optimal activity at pH 7.5, with Km values of 12.5 microM for methylamine and 156 microM for phenazine ethosulfate and a Vmax of 16.9 mumol/min per mg of protein. No loss of enzyme activity was observed after incubation for 48 h at pH values ranging from 3.0 to 10.5, and the enzyme was very stable to thermal denaturation. Enzyme activity and immunological detection of each subunit were only observed with cells which had been grown on methylamine as a carbon source.     

Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase

The small subunits of spinach ribulosebisphosphate carboxylase-oxygenase were isolated by mild acid precipitation of the hexadecameric holoenzyme. About one-third of the small subunits remained in the supernatant while the remainder, and all of the large subunits, were precipitated and irreversibly denatured. The spinach small subunits were able to reassemble with the large subunit octamer of ribulosebisphosphate carboxylase-oxygenase from the cyanobacterium, Synechococcus ACMM 323, prepared as described previously (Andrews, T. J., and Ballment, B. (1983) J. Biol. Chem. 258, 7514-7518) to produce a catalytically active, hybrid enzyme. The heterologous small subunits bound an order of magnitude less tightly than homologous small subunits and the specific activity of the hybrid, when fully saturated with foreign small subunits, was about half that of the homologously reassembled or native Synechococcus enzyme. In addition, the Km(CO2) of the hybrid was about twice as high. However, the degree of partitioning between carboxylation and oxygenation was identical for the hybrid, the homologously reassembled, and the native Synechococcus enzymes and clearly less in favor of carboxylation than partitioning by the spinach enzyme. Therefore, this important facet of catalysis by ribulosebisphosphate carboxylase-oxygenase appears to be specified exclusively by the large subunit.     

Subunit dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase from Chromatium vinosum

The large and small subunits of ribulose bisphosphate carboxylase from Chromatium vinosum were dissociated and separated at pH 9.6 by sucrose density gradient centrifugation. After further purification by gel filtration, the small subunit fraction contained no carboxylase activity. The large subunit fraction was highly depleted of small subunit based on analysis by denaturing polyacrylamide gel electrophoresis. Carboxylase activity of the large subunit fraction was approximately 1% of the untreated native enzyme. Addition of purified small subunit to the large subunit fraction yielded increases of up to 67-fold in carboxylase activity, further indicating that both subunit types are required for catalysis by this enzyme. The isolated large subunit was fully capable of high-affinity activator 14CO2 binding in the presence of Mg2+ and 2-carboxyarabinitol bisphosphate, indicating that the activator and catalytic sites were not grossly denatured by the depletion of small subunit. Kinetic constants of the native C. vinosum enzyme defined a new class of ribulose bisphosphate carboxylase, which permits the detection of possible kinetic differences if the large and small subunits can be favorably reassembled with those of another kinetic class. From experiments with the enzymes from tobacco and spinach leaves it is concluded that the enzyme from higher plant sources is not suitable for such dissociation/reconstitution-type experiments.     

Extracytoplasmic phosphatases of selected species of Saccharomyces, Kluyveromyces and Rhodotorula

Phosphatase activities of yeasts belonging to the genera Saccharomyces, Kluyveromyces and Rhodotorula were studied. Rhodotorula rubra exhibited activities at acid, neutral and alkaline pH; the other yeasts only had activity at acid pH. Growing yeasts in a constant pH (4.5) medium decreased phosphatase activities in Saccharomyces and Kluyveromyces, while neutral activity was enhanced in Rhodotorula rubra which excreted more enzyme under these conditions. Washing cells with sucrose solutions lowered phosphatase activities in all yeasts, due to enzyme liberation. Acid phosphatase activities in isolated and purified cell walls were very small. Phosphatases thus appear not to be strongly bound to yeast cell walls.     

Limited autolysis of calcium-activated neutral protease (CANP): reduction of the Ca2+-requirement is due to the NH2-terminal processing of the large subunit

Calcium-activated neutral protease (rabbit mCANP), composed of large and small subunits, was converted to a lower-Ca2+-requiring form (derived microCANP) by limited autolysis in the presence of Ca2+. The NH2-terminal regions of the two subunits of mCANP were cleaved by autolysis, but the COOH-termini remained intact after autolysis. When native mCANP or derived microCANP was dissociated into subunits, the proteolytic activity of the large subunit was reduced to 2-5% of that of the native dimeric enzyme. The Ca2+-sensitivity of one hybrid CANP reconstituted from the large subunit of derived microCANP and the small subunit of native mCANP was similar to that of derived microCANP. However, the other hybrid molecule composed of the large subunit of native mCANP and the small subunit of derived microCANP required a high concentration of Ca2+ for activity, like native mCANP. These results indicate that the Ca2+-sensitivity of derived microCANP is determined by the structural change of the large subunit resulting from loss of its NH2-terminal region. The autolysis of the small subunit apparently has no effect on the reduction of the Ca2+-requirement.     

The two-step reversible denaturation of lactate dehydrogenase at low pH.

Upon exposure to conditions of low pH, beef B4 lactate dehydrogenase rapidly loses enzymatic activity, but this process can be completely reversed yielding 100% of the original activity if the enzyme is immediately returned to neutral conditions. As the time of exposure to low pH is increased, the fraction of activity recovered declines to a values of 50--60% and remains nearly constant over a period of many hours. Correlated with this behavior is a change in the kinetics of the recovery of activity. Recovery of activity has been shown to be a second-order process for enzyme exposed to low pH for brief periods of time (Anderson, S., and Weber, G. (1966), Arch. Biochem. Biophys. 116, 207). After several minutes at low pH recovery of activity is found to become first order and to occur at a considerably slower rate. Gel filtration chromatography at low pH separates the protein into two fractions. The lower molecular weight fraction is found to be primarily monomeric, as indicated by equilibrium ultracentrifugation, and is capable of recovering enzymatic activity. The higher molecular weight fraction is generated from the lower molecular weight fraction, and is incapable of recovering activity. These results are interpreted to indicate that the enzyme exists sequentially in three denatured forms at low pH, the first two capable of being restored to the native state, and the third irreversibly denatured.     

Rat small-intestinal β-galactosidases. Studies on the fractionation of `acid'' β-galactosidase with isoelectric focusing, gel filtration and ion-exchange chromatography

1. Different forms of the rat small-intestinal ;acid' beta-galactosidase were separated by using the isoelectric-focusing technique. The isoelectric points of the different forms were at pH4.2, 4.6, 5.4, 6.1 and approx. 8. 2. The two forms of ;acid' beta-galactosidase isoelectric at pH4.2 and 4.6 were completely excluded from the Sephadex G-200 gel, whereas the form isoelectric at pH8 had K(av.) 0.4. The concentration and pH of the elution buffer influenced the distribution of enzyme activity between different forms. Thus, under certain conditions of ionic strength and pH, the enzyme seems to form high-molecular-weight aggregates with low isoelectric points. These may be homopolymeric aggregates or the result of binding of enzyme to, for example, membrane fragments. The forms isoelectric at pH5.4 and 6.1 are probably aggregates of intermediate size. 3. During ion-exchange chromatography at pH6.0 one fraction of ;acid' beta-galactosidase was not retained on the column and was isoelectric at pH8 and another fraction was eluted when the buffer concentration in the eluate had increased to about 50mm. The main part of enzyme eluted in this second fraction was also isoelectric at pH8, indicating that the elution of this fraction is not a simple ion-exchange procedure but probably also involves a splitting of high-molecular-weight aggregates, originally retained because of their low isoelectric points. The enzyme subunits have a higher isoelectric point, and are therefore no longer bound to the ion-exchange resin.     

Lactate dehydrogenase associated with the mitochondrial fraction and with a mitochondrial inhibitor--II. Enzyme interaction with a mitochondrial inhibitor

A LDH inhibitor has been isolated from the LDH-free crude mitochondrial fraction of rabbit skeletal muscle. The inhibitor is only released after solubilization of the particle bound enzyme. It only interacts with soluble LDH, since the enzyme bound to the mitochondrial fraction was not inhibited. The inhibitor molecular weight is above 10,000 dalton, it is precipitated by 7.5% trichloroacetic acid or 80% (NH4)2SO4 saturation. It is highly stable to heat and pH variations. The inhibitor only interacts with the enzyme at ionic strengths below 20 mM and at pH 6.0 or less. The kinetic behavior of the inhibited enzyme is non-hyperbolic and is similar to the mitochondrial fraction bound enzyme.     

Purification and partial characterization of pyruvate decarboxylase from Oryza sativa L

Pyruvate decarboxylase(PyrDC) was purified from rice bran to a specific activity of 1 mu kat/mg and partially characterized. The holoenzyme is a tetramer of two types of subunits with molecular masses 64 kDa and 62 kDa. Purified rice PyrDC exhibits positive cooperative kinetics with respect to pyruvate and functions with a significant lag phase. When compared to other plant PyrDC, the lag phase was shorter at low pyruvate concentrations and the S0.5 was smaller. The optimum pH (6.25) was also less acidic and the enzyme retained 30% of its maximal activity at neutral pH. In contrast to other plant PyrDC, rice PyrDC could be active at the onset of anoxia and would be activated by small changes in pyruvate concentration.     

Neutral hydrolases of rat brain. Preliminary characterization and developmental changes of neutral beta-N-acetylhexosamindases

The bulk of rat brain neutral beta-N-acetylhexosaminidases (2-acetamido-2-deoxy-beta-D-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) were present in the cytosol fraction. They were not bound by concanavalin A-Sepharose while the acid beta-N-acetylhexosaminidases were all bound. The neutral beta-N-acetylgalactosaminidase had a pH optimum of 5.2 and Km of 0.57 mM, while the neutral beta-N-acetylgalactosaminidase had the highest reaction rate at lost more than 90% of the activity in 30 min at 50 degrees C. The galactosaminidase pH 6.0 with a Km of 0.12 mM. No divalent ions activated either of the enzymes. The galactosaminidase was heat-stable and lost only 10--20% of its activity after 3 h at 50 degrees C. The neutral glucosaminidase was inhibited by free N-acetylglucosamine but not by N-acetylgalactosamine. The reverse was found for the neutral beta-galactosaminidase. Two enzymes were separated almost completely by hydroxyapatite chromatography. Heat stability of the separated activity peaks suggested that the neutral beta-N-acetylgalactosaminidase, which was not bound to hydroxyapatite, may be specific to the galactosaminide substrate. The neutral beta-N-acetylglucosaminidase may, on the other hand, have some activity toward the galactosaminide substrate. Both of the neutral enzyme activities were highest during the first postnatal week in rat brain in contrast to the acidic enzyme which showed peak activities during the second and third weeks. These results confirmed and expanded earlier observations by Frohwein and Gatt in calf brain. The relationship of these enzymes to the hexosaminidase C in human tissues is less certain at the present time.     

Modes of DNA cleavage by the EcoRV restriction endonuclease

The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.     

Bovine brain cathepsin D: inhibition by pepstatin and binding to concanavalin A.

1. Cathepsin D from bovine brain has been purified 1100-fold in 46% recovery. Three isozymes are present with pI (+/- 0.05) = 6.10, 6.30 and 6.40. 2. The isozymes are single polypeptide chains with apparent Mr = 42,000 and are similar with respect to substrate binding and cleavage; the pH-optimum is 3.5 with virtually no activity at neutral pH. 3. Pepstatin inhibits the enzyme and kinetic data are consistent with a "tight binding" mechanism. 4. The dissociation constant for the concanavalin A-enzyme complex is Kd = 19 nM at pH 5.0. 5. Under conditions where 90% of the enzyme is bound to soluble concanavalin A, full enzymatic activity is observed.     

Immobilization–stabilization of a new recombinant glutamate dehydrogenase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.     

Isolation and characterization of a thermostable intracellular enzyme with peroxidase activity from Bacillus sphaericus

During a screening for bacteria producing enzymes with peroxidase activity, a Bacillus sphaericus strain was isolated. This strain was found to contain an intracellular enzyme with peroxidase activity. The native enzyme had a molecular mass of above 300 kDa and precipitated at a salt concentration higher than 0.1 M. Proteolytic digestion with trypsin reduced the molecular mass of the active enzyme to 13 kDa (dimer) or 26 kDa (tetramer) and increased its solubility, allowing purification to homogeneity. Spectroscopic investigations showed the enzyme to be a hemoenzyme containing heme c as the covalently bound prosthetic group. The enzyme was stable up to 90 degrees C and at alkaline conditions up to pH 11, with a pH optimum at pH 8.5. It could be visualized by activity staining after SDS-PAGE and showed activity with a number of typical substrates for peroxidases, e.g., 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) diammonium salt, guaiacol and 2,4-dichlorophenol; however the enzyme had no catalase and cytochrome c peroxidase activity.     

Release of proteinase from mycelium of Mucor hiemalis

When Mucor hiemalis NRRL 3103 was grown in soybean medium, only a small fraction of the proteinase produced by the organism appeared in the culture filtrate, whereas the bulk of the enzyme was bound to the mycelial surface. Optimal pH of the proteinase ranged from 3.0 to 3.5. Inclusion of sodium chloride or other ionizable salts in the growth medium, however, resulted in the liberation from the mycelium of the loosely bound enzyme as it was formed. Maximal release of proteinase was achieved at a sodium chloride concentration of 0.5 m. The loosely bound proteinase was eluted also from intact resting mycelium by ionizable salts but not by water or by nonionizable substances. The amount of enzyme eluted from the mycelium depended upon the concentration of sodium chloride up to 0.3 m. Since liberation took place rapidly even at 0 C, a loose ionic linkage must exist rather than a biochemical binding of the enzyme to the mycelium. The recovery of proteolytic activity from repeated salt extractions was greater than that originally detected in the intact mycelium, possibly owing to unmasking of more active enzymes or functional groups. Further proteinase activity was released when salt-extracted mycelium was ruptured. Part of the proteinase thus observed was firmly attached to the cell fraction, and part of it appeared in the supernatant fluid. These conditions implied the presence of intracellular or firmly attached proteinase which could be partially released.     

Production of an extracellular ribonuclease by Pseudomonas maltophilia.

As part of a screening program for pseudomonad enzymes having an industrial interest, we selected ribonuclease (RNase) producing strains. Of the 150 pseudomonads screened, 6 were found to produce an extracellular RNase activity when grown on solid medium. In broth culture, the RNase activity from these six species remained bound to the cells unless gelatin was added to the medium. Gelatin was essential for the release of RNase in the broth culture, but the pH of the medium, addition of potential inducers such as nucleic acids, or addition of cations did not affect this release. However, gelatin did not appear to induce the synthesis of the enzyme. Strain B-88, identified as Pseudomonas maltophilia, was selected for further study of the enzyme. The extracellular RNase isolated from B-88 broth cultures could be separated in two fractions on the basis of the molecular weight by the ultrafiltration technique. The low molecular weight fraction reacts optimally at temperatures between 55 and 60 degrees C and optimal pH values varying from 7.4 to 9.5. At neutral or alkaline pH, the enzyme was stable at temperatures below 37 degrees C but was inactivated at 55 degrees C. The RNase was inhibited by mercury and cobalt and stimulated by magnesium.     

Degradation of peroxisomal catalase and urate oxidase of rat liver.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Factors affecting the stability of methanol dehydrogenase from Paracoccus denitrificans

Methanol dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methanol-grown cells. The enzyme was composed of subunits of M(r) 67,000 and 12,000, and non-covalently bound pyrroloquinoline quinone. It exhibited a pH optimum at pH values of 9.0 and above. It was not stable at pH greater than 9.0, but exhibited little loss of activity after prolonged incubation at pH values as low as 4.5. Methyl dehydrogenase was relatively stable to thermal denaturation. The thermal stability was enhanced by the presence of Ca2+ and diminished by the presence of EDTA. These data suggest a structural role for Ca2+ in this enzyme, similar to what has been observed with quinoprotein glucose and ethanol dehydrogenases.     

Mouse submaxillary renin has a protease activity and converts human plasma inactive prorenin to an active form

The activation of inactive prorenin by active renin was investigated. Inactive prorenin extensively purified from human plasma was activated by active renin which had been purified from mouse submaxillary glands by multiple chromatographic steps. The apparent lack of protease activity in renin was puzzling in view of the close similarity of its active site structure with that of acid proteases. After a series of affinity chromatographic steps designed to eliminate minute contaminants, renin was found to contain a very low but finite level of a neutral protease activity which was equivalent to 1/40,000 of that of cathepsin D tested by hemoglobinolytic activity. The protease activity was considered as intrinsic to renin since it co-purified with renin persistently at a constant ratio to the renin activity, was precipitated by a monoclonal antibody specific for renin, showed a neutral pH optimum of the enzyme activity in the same pH range as that of renin, and was inhibited by pepstatin. The neutral protease activity is likely to mediate the activation of inactive prorenin.