Chemical modification of ribonuclease T1 with ozone.

The single tryptophan residue in ribonuclease T1 [EC 3.1.4.8] was selectively oxidized by ozone to N'-formylkynurenine, which was then converted to kynurenine by acid-catalyzed deformylation in the frozen state. The two enzyme derivatives thus formed, NFK- and Kyn-RNase T1, lost enzymatic activity at pH 7.5, at which native RNase T1 most efficiently catalyzes the hydrolysis of RNA. At pH 4.75, the modified enzymes retained a decreased but distinct enzymatic activity toward RNA without alteration of substrate specificity, and Kyn-RNase T1 was four times more active than NFK-RNase T1. The binding of 3'-GMP to these modified enzymes decreased remarkably at pH 5.5, the optimum pH for binding to the intact enzyme. The gamma-carboxyl group of glutamic acid 58 was still reactive to iodoacetic acid after modification of tryptophan 59. The amounts of the carboxymethyl group introduced into NFK- and Kyn-RNase T1 were 0.36 and 0.59 mol, respectively, under conditions such that quantitative esterification of native RNase T1 takes place. CD spectroscopy indicated that the tertiary structure of the molecule was disordered in NFK-RNase T1, but not significantly in Kyn-RNase T1. It is concluded that tryptophan 59 functions in maintaining the active conformation of the protein structure, particularly in constructing the active environment for a functionally important set of groups involved in the binding of the substrate at the active site, although direct participation of in tryptophan the catalytic function of ribonuclease T1 is unlikely.     

Spermine stabilization of folded ribonuclease T1

The interaction of ribonuclease T1 with tetraprotonated spermine (SPM4+), Mg2+, phosphate and other ionic ligands at pH 6.0 was investigated in binding experiments at 25 degrees C and/or by their effects on the midpoint temperature for thermal unfolding of the enzyme. SPM4+ binding with the native protein at 25 degrees C was characterized by an association constant of approximately 2 x 10(4) M-1. This ligand also binds to the unfolded protein but with a approximately 35-fold lower affinity. Phosphate binds at the active site whereas Mg2+ and SPM4+ cations compete for binding at a polyanionic locus that probably involves residues Glu-28, Asp-29, and Glu-31 at the C-terminal end of the alpha-helix. Steady-state kinetic studies using minimal RNA substrates demonstrated that SPM4+ binding with the enzyme does not affect its catalytic activity. SPM4+ also preferentially binds with the folded form of the disulfide-reduced enzyme which has the same or slightly enhanced catalytic properties compared with native ribonuclease T1. The unfolding rate for the native protein in 8 M urea was approximately 8-fold lower in the presence of 0.05 M SPM4+. SPM4+ appears to increase the amplitude of an unobserved fast phase(s) for refolding of the native enzyme. A single kinetic phase characterized refolding of the reduced enzyme which was slightly faster than the slowest refolding phase for the native form.     

Conformational stability of ribonuclease T1. II. Salt-induced renaturation.

In the presence of high concentrations of the monovalent salts, sodium chloride and potassium fluoride, disulfide-reduced RNase T1 having four cysteinyl residues intact regenerates the spectral properties characteristic of native RNase T1, e.e., the fluorescence spectrum of the aromatic side chains and the ultraviolet circular dichroism spectrum. The folding of the polypeptide chain proceeded without formation of disulfide bonds to yield an enzymatically active species having an activity toward RNA equivalent to 25% of that of the native enzyme at the same salt concentration of 2 m. Unfolding of RNase T1 by a denaturant, urea, was suppressed in the presence of salts, and the salt-induced chain folding was observed spectroscopically even in 6.9 m urea solution. The salts also induced the chain folding of disulfide reduced and modified (carboxymethylated or carboxamidomethylated) RNase T1 into the native conformation, as indicated by its spectroscopic properties, but did not restore the enzymatic activity.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.     

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.     

The structure and function of ribonuclease T1. XXIII. Inactivation of ribonuclease T1 by reversible blocking of amino groups with cis-aconitic anhydride and related dicarboxylic acid anhydrides.

Ribonuclease T1 [EC 3.1.4.8] was inactivated rapidly by treatment at pH 8.0 and 0 degrees C with cis-aconitic anhydride and related dicabroxylic acid anhydrides, including citraconic, maleic, and succinic anhydrides. Under reaction conditions used, roughly 90% inactivation occurred within 30 min. Analyses of the inactivated enzymes indicated that the reaction took place fairly specifically at the alpha-amino group of the N-terminal alanine and the epsilon-amino group of lysine-41. Upon incubation of these inactivated enzymes at pH 3.6 and 37 degreeC, the activity was regenerated to various extents, depending on the nature of the introduced acyl groups. Under these conditions, the enzyme modified with cis-aconitc anhydride or citraconic anhydride recovered much of the origninal activity after 48 h whereas the enzyme modified with maleic anhydride recovered its activity only partially. Practically no activity was regenerated in the case of the enzyme modified with succinic anhydride under these conditions. The inactivation appears to be due mainly to the effect of the carboxyl group introduced at the epsilon-amino group of lysine-41. The results suggest the usefulness of cis-aconitic anhydride as a reversible blocking reagent for amino groups in proteins.     

Degradation of ribonucleic acid by immobilized ribonuclease.

An immobilized enzyme (pancreatic ribonuclease bound to porous titania) was investigated for the degradation of purified yeast ribonucleic acid as a substrate. The immobilized enzyme is active and stable in the pH range 4--8. Dependence of enzymatic activity on ionic strength, pH, temperature, fluid flow rate, and substrate concentration were investigated. A cumulative fluid residence time of 6 sec is sufficient for 50% substrate conversion at 25 degrees C and pH 7.0. The critical flow rate (i.e., the fluid flow rate necessary to remove film diffusion resistance) approximately doubles with each 10 degree C rise in reaction temperature. The critical flow rates obtained in this study are about 40 times greater than those obtained for a similar study on immobilized glucose oxidase. Arrhenius plots gave activation energies of -9.6 and -7.1 kcal/g mol at pH 4.6 and 7.0, respectively. The work reported herein is a bench-scale investigation of an immobilized enzyme with primary emphasis on the mass transfer and kinetic characteristics of the system. The rapid reaction rates obtainable at relatively low temperatures offer a potential alternative method of purifying yeast single cell protein (SCP) with miminum loss of desired protein. The key questions are how such a system would react in a yeast homogenate, what conditions in such a system must be controlled, and what type of immobilized reactor should be utilized, if such further work continued to show promise.     

The structure and function of ribonuclease T1. XXII. Tryptic cleavages of the single lysyl and arginyl bonds in ribonuclease T1.

1. When ribonuclease T1 [EC 3.1.4.8] was treated with trypsin [EC 3.4.21.4] at pH 7.5 and 37 degrees, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50 degrees and 1 h at 60 degrees. 3'-GMP and guanosine protected the enzyme significantly from tryptic inactivation. 2. Upon tryptic digestion at 50 degrees, the Lys-Tyr (41-42) and Arg-Val (77-78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains cross-linked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104. 3. When the trinitrophenylated enzyme, in which the alpha-amino group of alanine-1 and the episolone-amino group of lysine 41 were selectively modified, was treated with trypsin at 37 degrees, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.     

The interaction of ribonuclease T 1 with DNA.

The interaction of RNase T1 with calf thymus DNA was studied using uv difference spectroscopy and the effect of the enzyme on DNA melting. There was no indication of RNase T1 binding with native DNA. A prominent difference spectrum for RNase T1 binding with denatured DNA (d-DNA) was observed at pH 5, 25 degrees and low ionic strength (mu = .01 M) which was depressed at higher ionic strength and pH. The normalized difference spectrum at mu = .01 M, pH 5 and 25 degrees can be interpreted as indicating an interaction of an exposed guanine residue directly with the enzyme and a coupling of this process with the "melting" of short folded segments of d-DNA. The apparent association constant calculated per M guanine residues was 2.4 X 10-4 M-1 under these conditions. The results are discussed in reference to comparable studies on the interaction of RNase T1 with RNA and small guanine ligands.     

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.     

Some characteristics of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli: comparison with the native enzyme and its processing in E. coli and in vitro.

Proteinase Ak.1 was produced during the stationary phase of Bacillus sp. Ak.1 cultures. It is a serine proteinase with a pI of 4.0, and the molecular mass was estimated to be 36.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 60 and 70 degrees C, with half-lives of 13 h and 19 min at 80 and 90 degrees C, respectively. Maximum proteolytic activity was observed at pH 7.5 with azocasein as a substrate, and the enzyme also cleaved the endoproteinase substrate Suc-Ala-Ala-Pro-Phe-NH-Np (succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanalide). Major cleavage sites of the insulin B chain were identified as Leu-15-Tyr-16, Gln-4-His-5, and Glu-13-Ala-14. The proteinase gene was cloned in Escherichia coli, and expression of the active enzyme was detected in the extracellular medium at 75 degrees C. The enzyme is expressed in E. coli as an inactive proproteinase at 37 degrees C and is converted to the mature enzyme by heating the cell-free media to 60 degrees C or above. The proproteinase was purified to homogeneity and had a pI of 4.3 and a molecular mass of 45 kDa. The NH2-terminal sequence was Ala-Ser-Asn-Asp-Gly-Val-Glu-, showing the exact signal peptide cleavage point. Heating the proenzyme resulted in the production of active proteinase with an NH2-terminal sequence identical to that of the native enzyme. The characteristics of the cloned proteinase were identical to those of the native enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

尼龙网固定化果胶酶的制备及其性质研究

用尼龙网作载体,经3-二甲氨基丙胺活化,用戊二醛将果胶酶固定化。所得固定化酶Km值与自然酶接近;对温度的稳定性有较大的提高,100℃保温30min才能使其失活。固定化酶在较宽的pH范围内能保持其正常活力,它对金属离子抑制剂的耐受性有较显著的提高,用0.5％果胶溶液作底物,重复使用10次后酶活力保留44％。固定化果胶酶与自然酶相比较,对不同果汁的澄清效果不同。固定化果胶酶在无保护剂存在的条件下,室温放置四个月活力不减少。     

Purification and physicochemical characterization of a human placental acid phosphatase possessing phosphotyrosyl protein phosphatase activity

A 17-kilodalton (kDa) human placental acid phosphatase was purified 21,400-fold to homogeneity. The enzyme has an isoelectric point of pH 7.2 and a specific activity of 106 mumol min-1 mg-1 using p-nitrophenyl phosphate as a substrate at pH 5 and 37 degrees C. This placental acid phosphatase showed activity toward phosphotyrosine and toward phosphotyrosyl proteins. The pH optima of the enzyme with phosphotyrosine and with phosphotyrosyl band 3 (from human red cells) were between pH 5 and 6 and pH 5 and 7, respectively. The Km for phosphotyrosine was 1.6 mM at pH 5 and 37 degrees C. Phosphotyrosine phosphatase activity was not inhibited by tartrate or fluoride, but vanadate, molybdate, and zinc ions acted as strong inhibitors. Enzyme activity was also inhibited by DNA, but RNA was not inhibitory. It is a hydrophobic nonglycoprotein containing approximately 20% hydrophobic amino acids. The average hydrophobicity was calculated to be 903 cal/mol. The absorption coefficient at 280 nm, E1% 1cm, was determined to be 5.7. The optical ellipticity of the enzyme at 222 nm was -5200 deg cm2 dmol-1, which would correspond to a low helical content. Free sulfhydryl and histidine residues were necessary for the enzyme activity. The enzyme contained four reactive sulfhydryl groups. Chemical modification of the sulfhydryls with iodoacetate resulted in unfolding of the protein molecule as detected by fluorescence emission spectroscopy. Antisera against both the native and the denatured protein were able to immunoprecipitate the native enzyme. However, upon denaturation, the acid phosphatase lost about 70% of the antigenic determinants. Both antisera cross-reacted with a single 17-kDa polypeptide on immunoblotting.     

Chemical modifications of ribonuclease U1.

In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.     

交联葡聚糖结合无花果蛋白酶的制备与性质

本文报道了将Sephadgx G-200与对β-硫酸酯乙砜基苯胺(SESA)首先醚化制备对氨基苯砜乙基交联葡聚糖(ABSE-Sephadex G-200),然后经重氮化固定无花果蛋白酶。固定化酶的活力回收达69％。BANA对该酶固定化过程中的活性变化有保护作用。天然酶与固定化酶都具有良好的耐热性,在69°～70℃,80min固定化酶较天然酶稳定。 用苯甲酰-DL-精氨酰-β-荼胺(BANA)为底物,在半胱氨酸存在下,测定了两种形式酶的动力学性质。在pH7.7的磷酸盐缓冲系统中,37℃,天然无花果蛋白酶的K_m=0.32mol/L;在间歇振摇下固定化酶的表观K′m=1.02mmol/L。最适pH无明显改变,均为pH7.7。     

The structure and function of ribonuclease T1. XX. Specific inactivation of ribonuclease T1 by reaction with tosylglycolate.

1. Ribonuclease T1 [EC 3.1.4.8] was inactivated by reaction with tosylglycolate (carboxymethyl rho-toluenesulfonate). At pH 5.5 and 8.0, alkylation of the gamma-carboxyl group of glutamic acid-58 appeared to be the predominant reaction and the major cause of inactivation by tosylglycolate, as in the case of the iodoacetate reaction, although the rate of inactivation was slower than that by iodoacetate. At pH 8.0, histidine residues were also alkylated to some extent. 2. The maximal rate of inactivation was observed at around pH 5.5 and the pH dependence of the rate of inactivation suggested the implication of two groups in the reaction, with apparent pKa values of about 3-4 (possibly histidine residue(s)). 3. In the presence of substrate analogs, ribonuclease T1 was markedly protected from inactivation by tosylglycolate at pH 5.5. The extent of protection corresponded to the binding strength of the substrate analog, except for guanosine. Ribonuclease T1 was much less protected from inactivation by guanosine than by 3'-AMP or 3'-CMP, which has a lower binding strength toward ribonuclease T1. This may indicate that glutamic acid-58 is situated in the catalytic site, at which the phosphate moiety of these nucleotides directly interacts. 4. Enzyme which had been extensively inactivated with tosylglycolate at pH 5.5 scarcely reacted with iodoacetate at pH 5.5, suggesting that these reagents react at the same site, i.e. glutamic acid-58. On the other hand, enzyme which had been inactivated almost completely with tosylglycolate at pH 8.0 still reacted with iodoacetate to some extent at pH 8.0, and the modes of reaction of tosylglycolate and iodoacetate toward ribonuclease T1 appeared to be somewhat different.     

Conformational stability of ribonuclease T1. I. Thermal denaturation and effects of salts.

The thermal transition of RNase T1 was studied by two different methods; tryptophan residue fluorescence and circular dichroism. The fluorescence measurements provide information about the environment of the indole group and CD measurements on the gross conformation of the polypeptide chain. Both measurements at pH 5 gave the same transition temperature of 56 degrees C and the same thermodynamic quantities, delta Htr (= 120 kcal/mol) and delta Str (= 360 eu/mol), for the transition from the native state to the thermally denatured state, indicating simultaneous melting of the whole molecule including the hydrophobic region where the tryptophan residue is buried. Stabilization by salts was observed in the pH range from 2 to 10, since the presence of 0.5 m NaCL caused an increase of about 5 degrees C to 10 degrees C in the transition temperature, depending on the pH. The fluorescence measurements on the RNase T1 complexed with 2'-GMP showed a transition with delta Htr =167 kcal/mol and delta Str =497 eu/mol at a transition temperature about 6 degrees C higher than that for the free enzyme. The large value of delta Htr for RNase T1 indicates the highly cooperative nature of the thermal transition; this value is much higher than those of other globular proteins. Analysis of the CD spectrum of thermally denatured RNase T1 suggests that the denatured state is not completely random but retains some ordered structures.     

Catalytic acitivity of Ntau-carboxymethylhistidine-12 ribonuclease.

Ntau-Carboxymethylhistidine-12 RNase is active with both RNA and uridine cyclic 2':3'-monophosphate as substrates. Experimental evidence is presented to show that the activity cannot be due to contaminating RNase A, or other RNase-type protein, to the presence of a mixed dimer between 12- and 119-substituted RNases, or to the presence of trace amounts of Ntau-carboxymethylhistidine-12 RNase. The carboxymethyl derivative has approximately 1 and 13 per cent the specific activity of native enzyme against cyclic 2',3'-UMP at pH 5.0 and 8.5, respectively.     

Studies on the properties of immobilized urokinase: effects of pH and temperature

Human urokinase was immobilized on an ethylene vinyl acetate copolymer surface. Soluble urokinase showed its maximum activity at pH 8.5, while the immobilized enzyme was most active at pH 9.0. Apparently, the shift in optimal pH was due to the polyanionic nature of the carrier surface on which the enzyme was immobilized. Optimal temperatures of soluble urokinase and immobilized enzyme were identical, i.e., 37 degrees C. The stability of immobilized enzyme against thermal degradation was several times higher than that of the soluble enzyme. Its stability at higher temperatures is one of the main reasons for the clinical use of immobilized urokinase as an antithrombotic material.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.