Inactivation and proteolysis of heat-sensitive adenylate kinase of Escherichia coli CR341 T28

Adenylate kinase from Escherichia coli K12 (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. Denaturation by heat above 60 degrees C of pure or crude preparations of adenylate kinase from both strains of bacteria was shown to be "reversible" if the enzyme was converted to the random coiled state by guanidinium chloride after heat treatment. Like other small monomeric proteins, adenylate kinase refolded rapidly to the native active state by dilution of guanidinium chloride. Adenylate kinase from the mutant strain was irreversibly inactivated by exposure of crude extracts at 40 degrees C. This inactivation is due to proteolysis which follows thermal denaturation (or transconformation) of mutant adenylate kinase at 40 degrees C. ATP, P1, P5-di(adenosine 5')-pentaphosphate, and anti-adenylate kinase antibodies protected the thermosensitive adenylate kinase in crude extracts against denaturation and proteolysis at 40 degrees C.     

BspLS2I--a new site-specific endonuclease from the thermophilic bacteria Bacillus species LS2]

A new restriction endonuclease BspLS2I was isolated from the thermophilic bacterium Bacillus species LS2 and purified by blue sepharose and hydroxyapatite chromatographies. The enzyme is an isoschizomer of SduI from Streptococcus durans. BspLS2I recognizes the sequence 5' G(G/A/T)GC(C/T/A) decreases C 3' on double-stranded DNA and cleaves it is indicated by the arrow to yield sticky-ended DNA fragments. Maximum catalytic activity of endonuclease was found in 10 mM tris-HCl (pH 7.9) in the presence of 15-30 mM MgCl2 at 50 degrees C. The phage T4 glucosylated DNA is not cleaved by the enzyme.     

Tsp49I (ACGT/), a thermostable neoschizomer of the Type II restriction endonuclease MaeII (A/CGT), discovered in isolates of the genus Thermus from the Azores, Iceland and New Zealand.

One hundred and forty eight isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (SM49) from the island of Sao Miguel, in the Azores, showed a high level of restriction endonuclease activity when a cell-free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Tsp49I) has been partially purified from this isolate and the recognition and cleavage site determined. Tsp49I recognizes the four base sequence ACGT, which is the same as the recognition sequence of the mesophilic Type II restriction endonuclease MaeII. However, unlike MaeII, which cleaves DNA between the first and second bass of the recognition sequence (A/CGT), Tsp49I hydrolyses the phosphodiester bond in both strands of the substrate after the last base of the recognition sequence 5'-ACGT/-3', producing four base 3'-OH overhangs (sticky ends). The enzyme has a pH optimum of 9.0, requires 2 mM MgCl2 for maximum activity and retains full enzyme activity following incubation for 10 min at temperatures up to 8O degrees C. Two further examples of the same restriction endonuclease specificity as Tsp491 were detected in Thermus isolates from Iceland (TspIDSI) and New Zealand (TspWAM8AI). The three MaeII neoschizomers, Tsp49I, TspIDSI and TspWAM8AI, exhibit similar pH optima, heat stabilities and MgCl2 requirements, but differ in their requirements for NaCl and KCl.     

Taq52 I, a novel and thermostable type II restriction endonuclease from the genus Thermus, recognising the pentanucleotide sequence GC(A or T)GC and cleaving DNA between the first and second bases of the recognition sequence: G decreased or reduced C(A or T)GC.

127 isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (YS52) from Yellowstone National Park, USA, showed a high level of restriction endonuclease activity when a cell free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Taq52 I) has been partially purified from this isolate and the recognition and cleavage site determined. Taq52 I has a novel interrupted palindromic tetranucleotide recognition sequence GCWGC, where W can be either adenine (A) or thymine (T). It hydrolyses the phosphodiester bond in both strands of the substrate between the first and second bases of the recognition sequence: 5'G decreased or reduced CWGC3', producing three-base 5'-OH overhangs (sticky ends). The enzyme has a pH optimum of 7.0, requires 8 mM MgCl2 for maximum activity and is thermally stable, retaining full enzyme activity following incubation at 79 degrees C for 10 min. Taq52 I not only represents a new addition to the Type II restriction endonucleases, but also increases the small list of thermostable restriction endonucleases.     

Properties of the main endonuclease specific for apurinic sites of Escherichia coli (endonuclease VI). Mechanism of apurinic site excision from DNA.

The main endonuclease for apurinic sites of Escherichia coli (endonuclease VI) has no action on normal strands, either in double-stranded or single-stranded DNA, or on alkylated sites. The enzyme has an optimum pH at 8.5, is inhibited by EDTA and needs Mg2+ for its activity; it has a half-life of 7 min at 40 degrees C. A purified preparation of endonuclease VI, free of endonuclease II activity, contained exonuclease III; the two activities (endonuclease VI and exonuclease III) copurified and were inactivated with the same half-lives at 40 degrees C. Endonuclease VI cuts the DNA strands on the 5' side of the apurinic sites giving a 3'-OH and a 5'-phosphate, and exonuclease III, working afterwards, leaves the apurinic site in the DNA molecule; this apurinic site can subsequently be removed by DNA polymerase I. The details of the excision of apurinic sites in vitro from DNA by endonuclease VI/exonuclease III, DNA polymerase I and ligase, are described; it is suggested that exonuclease III works as an antiligase to facilitate the DNA repair.     

The effectiveness of hand cooling at reducing exercise-induced hyperthermia and improving distance-race performance in wheelchair and able-bodied athletes.

The purpose of this study was to examine the effectiveness of reducing core temperature in postexercise hyperthermic subjects and to assess if hand cooling (HC) improves subsequent timed distance performance. Following a detailed measurement check on the use of insulated auditory canal temperature (T(ac)), eight wheelchair (WA) athletes and seven male able-bodied (AB) athletes performed two testing sessions, comprising a 60-min exercise protocol and 10-min recovery period, followed by a performance trial (1 km and 3 km for WA and AB, respectively) at 30.8 degrees C (SD 0.2) and 60.6% (SD 0.2) relative humidity. In a counterbalanced order, HC and a no-cooling condition was administered during the 10-min recovery period before the performance trial. Nonsignificant condition x time interactions for both WA (F(15,75) = 1.5, P = 0.14) and AB (F(15,90) = 1.2, P = 0.32) confirmed that the exercise-induced changes (Delta) in T(ac) were similar before each intervention. However, the exercise-induced increase was evidently greater in AB compared with WA (2.0 vs. 1.3 degrees C change, respectively). HC produced DeltaT(ac) of -0.4 degrees C (SD 0.4) and -1.2 degrees C (SD 0.2) in comparison (WA and AB, respectively), and simple-effects analyses suggested that the reductions in T(ac) were noteworthy after 4 min of HC. HC had an impact on improving AB performances by -4.0 s (SD 11.5) (P < 0.05) and WA by -20.5 s (SD 24.2) (P > 0.05). In conclusion, extraction of heat through the hands was effective in lowering T(ac) in both groups and improving 3-km performance in the AB athletes and trends toward positive gains for the 1-km performance times of the WA group.     

The inactivation of foot and mouth disease, Aujeszky's disease and classical swine fever viruses in pig slurry

The aim of the study was to investigate the decontamination of pig slurry containing exotic viruses of pigs, foot AND mouth disease virus (FMDV), Aujeszky's disease virus (ADV) AND classical swine fever virus (CSFV). Laboratory-scale decontamination experiments showed that FMDV, ADV and CSFV were heat inactivated in slurry within 3 min at 67 degrees C, 3 min at 62 degrees C and 3 min at 60 degrees C and in Glasgow Eagles medium within 5 min at 67 degrees C, 4 min at 65 degrees C and 2 min at 65 degrees C, respectively. At pilot scale, FMDV was heat inactivated at 66 degrees C in water and 61 degrees C in slurry, ADV at 61 degrees C in water or slurry and CSFV at 62 degrees C in water and 50 degrees C in slurry. Treatment of pig slurry for the inactivation of exotic viruses may be achieved through the use of a thermal pilot plant operating in continuous mode. The work demonstrates the suitability of thermal treatment in ensuring the safety of pig slurry following a disease outbreak.     

Apurinic and apyrimidinic DNA endonuclease of extremely thermophilic Thermothrix thiopara.

An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara. The enzyme has a molecular weight of approximately 26,000. It cleaves neither native nor UV- or gamma-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities. The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide. It shows maximal activity at 70 degrees C and a pH between 7.5 and 9.0. The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM. The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 70 degrees C but about 150 min in the presence of 0.5 M ammonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature. One cell of T. thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10(6) phosphodiester bonds per h at 70 degrees C. An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T. thiopara.     

An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA.

A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.     

Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus.

Deoxyribonucleic acid polymerase I was purified from Bacillus stearothermophilus to 50 to 70% homogeneity. Its molecular weight was 76,000. The enzyme was insensitive to sulfhydryl blocking agents and showed maximal activity at 60 degrees C, pH 8 to 9, 0.25 M KCl, and 0.02 M MgSO4. The rate of heat inactivation of the deoxyribonucleic acid polymerase followed first-order kinetics with a half-life of 90 min at 60 degrees C; the addition of 0.05% bovine serum albumin protected the enzyme, which could be heated for 180 min without loss of activity. The ratios of polymerase to nuclease activities were about 20 for 5'-3' exonuclease and more than 500 for 3'-5' exonuclease. The Km for deoxyribonucleoside-5'-triphosphates was 7 microM.     

Sse8387I, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5''-CCTGCAGG-3''.

A type II restriction endonuclease designated Sse8387I was partially purified from Streptomyces sp. 8387. This enzyme cleaved adenovirus 2 DNA at three sites, lambda phage DNA at five sites, and pUC18 and M13mp18 RF DNA at one site each, but did not cleave the DNAs from pBR322, SV40, or phi X174. Sse8387I recognized the octanucleotide sequence 5'-CCTGCA decreases GG-3', cleaving where shown by the arrow. Sse8387I is the first restriction endonuclease to be reported that recognizes an octanucleotide sequence consisting of all four nucleotides, G, A, T, and C. The frequency of occurrence of Sse8387I sites within sequenced regions of primate genomes was 2.4 times that of NotI sites.     

Sequence of the radioactive tryptic peptide obtained after inactivating the F1-ATPase of the thermophilic bacterium PS3 with 5'-p-fluorosulfonylbenzoyl[3H]adenosine at 65 degrees C

Following a lag of about 30 min, the F1-ATPase from the thermophilic bacterium, PS3 (TF1), was inactivated slowly by 0.8 mM 5'-p-fluorosulfonylbenzoyladenosine (FSBA) at 23 degrees C and pH 7.0. When the enzyme was treated with 0.2 mM FSBA at pH 7.0 and 23 degrees C for 15 min and gel-filtered, no enzyme activity was lost. However, the lag in inactivation was abolished when the enzyme was subsequently incubated with 2.0 mM FSBA at 23 degrees C in the pH range from 6.8 to 10.0. The pH-inactivation profile obtained under these conditions revealed a pK alpha of about 9.3 which was associated with the inactivation. When pretreated TF1 was inactivated at 23 degrees C with [3H]FSBA by about 90%, greater than 20 mol of [3H]SBA was incorporated per mole of enzyme. TF1 was inactivated rapidly by 0.8 mM FSBA at pH 6.4 and 65 degrees C, and no lag was observed. Following inactivation of TF1 with 0.8 mM [3H]FSBA at 65 degrees C and pH 6.4, about 10 mol of [3H]SBA was incorporated per mole of enzyme. When a tryptic digest of the labeled enzyme was fractionated by reversed-phase high-performance liquid chromatography, a single major radioactive peptide was isolated. When subjected to automatic Edman degradation, this peptide was shown to have the amino acid sequence: A-L-A-P-E-I-V-G-E-E-H-X-Q-V-A-R, where X indicates that a phenylthiohydantoin derivative was not detected in cycle 12. However, from the DNA sequence of the gene encoding the subunit of TF1 (Y. Kagawa, M. Ishizuka, T. Saishu, and S. Nakao (1985) Abstracts International Symposium on Energy Transducing ATPases, Kobe, Japan, p. 84), this position has been shown to be occupied by tyrosine. This tyrosine is homologous with beta-Tyr-368 of the bovine mitochondrial F1-ATPase (MF1) the modification of which is responsible for the inactivation MF1 by FSBA.     

Some restriction endonucleases tolerate single mismatches of the pyrimidine.purine type.

DNAs from phage mutants M13mp18 and M13mp18/MP-1 were used to construct two closed circular heteroduplexes. One of them carried the sequence 5'-CCTGGG-3' 3'-GGGCCC-5' with a T.G mismatch at the position 6248. The other carried the sequence 5'-CCCGGG-3' 3'-GGACCC-5' with a C.A mismatch at the same position. Heteroduplexes were exposed to 7 restriction endonucleases having recognition sites within the sequence 5'-CCCGGG-3' 3'-GGGCCC-5' and to 1 restriction endonuclease having a recognition site within the sequence 5'-CCTGGG-3' 3'-GGACCC-5'. All tested enzymes cleaved at least one mismatch-containing sequence although with reduced efficiency. Smal and Xmal tolerated both mismatch-containing sequences. Aval, Hpall, Mspl, Ncil and Nsplll were able to tolerate only the T.G containing sequence, while BstNl was able to tolerate only the C.A containing sequence. It is inferred that the tolerance displayed by Smal and Xmal depends on the presence of either the original purines or the original pyrimidines in mismatches of both the T.G and C.A type and that all other tested enzymes require the presence of the original purines in mismaches of both types.     

Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase

The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.     

Bme585 I [5'-CCCGC(4/6)-3'], a new isoschizomer of restriction endonuclease Fau I, isolated from a strain of Bacillus mesentericus

Bme585 I is a new member of the restriction endonuclease type IIS family. It was partially purified from the heterothrophic, mesophilic bacterial strain Bacillus mesentericus 585 by ammonium sulphate precipitation and phosphocellulose column chromatography. Bme585 I is a monomeric protein with a molecular mass of 62 kD. The enzyme is active over a broad pH range from 7.0 to 8.8, has a temperature optimum of 37 degrees C and tolerance of NaCl in reaction buffer from 0 to 400 mM. Bme585 I recognizes the asymmetric sequence 5'-CCCGC(4/6)-3' and is therefore an isoschizomer of restriction endonuclease Fau I.     

Restriction endonuclease Sst 12I from a strain of Streptomyces sp. recognizing the nucleotide sequence 5'-CTGCAG-3'

A new restriction endonuclease Sst12I belonging to the II type and recognizing the sequence 5'-CTGCAG-3' was isolated from the bacterial strain Streptomyces sp. St-12. The enzyme hydrolyzes DNA between adenine and guanine residues; thus, it is a true isoschizomer of restrictase PstI. In contrast to PstI, the restriction endonuclease Sst12I hydrolyses DNA both at 37 degrees and 55 degrees C and remains active after long-term storage.     

Inactivation of Bacillus piliformis spores by heat and certain chemical disinfectants

The inactivation of Tyzzer's organism (Bacillus piliformis) spore isolated from rats by heat and various chemical disinfectants was studied. The spores were from B. piliformis-infected rat liver tissues. The spore suspension (10(4) 50% of rat liver lesion producing dose with prednisolone treatment/ml) was treated with heart or disinfectants. Inactivation of the spores was examined in experimentally infected rats. Rats were inoculated perorally with a treated spore suspension and injected subcutaneously with prednisolone. On the sixth day after inoculation, rats were examined grossly for liver lesions. Spores were inactivated at 80 degrees C for 15 min but not at 60 degrees C for 30 min. Spores were inactivated by 0.4% peracetic acid, 0.015% sodium hypochrolite, 1% iodophol, 5% phenol. Alcide and 0.37% formaldehyde solution, but not by 0.037% formaldehyde solution, 70% ethanol, 0.3% benzethonium chloride solution, 3% cresol and soap solution, or 4% chlorhexidine digluconate. These findings suggest that B. piliformis spores are relatively sensitive to heat and certain chemical disinfectants.     

Amino Acid esters prevent thermal inactivation and aggregation of lysozyme

Small potent inhibitors of aggregation are eagerly demanded for preventing the inactivation of proteins. This paper shows that amino acid esters (AAEs) prevent heat-induced aggregation and inactivation of hen egg lysozyme. Lysozyme was completely inactivated (<1% original activity) during heat treatment at 98 degrees C for 30 min in a solution containing 0.2 mg/mL lysozyme in 50 mM Na-phosphate buffer (pH 6.5). The residual activities only slightly increased (<5%) in the presence of 100 mM commonly used additives such as arginine, guanidine, urea, and sugars. However, in the presence of 100 mM AAEs, the residual activities were >60% and no aggregates were observed during the heat treatment at 98 degrees C for 30 min. This fact provides new information on the scaffold for designing additives to prevent heat-induced aggregation.     

Heat- and light-induced reorganizations in the phycobilisome antenna of Synechocystis sp. PCC 6803. Thermo-optic effect

By using absorption and fluorescence spectroscopy, we compared the effects of heat and light treatments on the phycobilisome (PBS) antenna of Synechocystis sp. PCC 6803 cells. Fluorescence emission spectra obtained upon exciting predominantly PBS, recorded at 25 degrees C and 77 K, revealed characteristic changes upon heat treatment of the cells. A 5-min incubation at 50 degrees C, which completely inactivated the activity of photosystem II, led to a small but statistically significant decrease in the F(680)/F(655) fluorescence intensity ratio. In contrast, heat treatment at 60 degrees C resulted in a much larger decrease in the same ratio and was accompanied by a blue-shift of the main PBS emission band at around 655 nm (F(655)), indicating an energetic decoupling of PBS from chlorophylls and reorganizations in its internal structure. (Upon exciting PBS, F(680) originates from photosystem II and from the terminal emitter of PBS.). Very similar changes were obtained upon exposing the cells to high light (600-7500 micromol photons m(-2) s(-1)) for different time periods (10 min to 3 h). In cells with heat-inactivated photosystem II, the variations caused by light treatment could clearly be assigned to a similar energetic decoupling of the PBS from the membrane and internal reorganizations as induced at around 60 degrees C. These data can be explained within the frameworks of thermo-optic mechanism [Cseh et al. 2000, Biochemistry 39, 15250]: in high light the heat packages originating from dissipation might lead to elementary structural changes in the close vicinity of dissipation in heat-sensitive structural elements, e.g. around the site where PBS is anchored to the membrane. This, in turn, brings about a diminishment in the energy supply from PBS to the photosystems and reorganization in the molecular architecture of PBS.     

Distribution, purification, and characterization of thermostable phenylalanine dehydrogenase from thermophilic actinomycetes.

Phenylalanine dehydrogenase (L-phenylalanine:NAD oxidoreductase, deaminating; EC 1.4.1.-) was found in various thermophilic actinomycetes. We purified the enzyme to homogeneity from Thermoactinomyces intermedius IFO 14230 by heat treatment and by Red Sepharose 4B, DEAE-Toyopearl, Sepharose CL-4B, and Sephadex G-100 chromatographies with a 13% yield. The relative molecular weight of the native enzyme was estimated to be about 270,000 by gel filtration. The enzyme consists of six subunits identical in molecular weight (41,000) and is highly thermostable: it is not inactivated by incubation at pH 7.2 and 70 degrees C for at least 60 min or in the range of pH 5 to 10.8 at 50 degrees C for 10 min. The enzyme preferably acts on L-phenylalanine and its 2-oxo analog, phenylpyruvate, in the presence of NAD and NADH, respectively. Initial velocity and product inhibition studies showed that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism. The Km values for L-phenylalanine, NAD, phenylpyruvate, NADH, and ammonia were 0.22, 0.078, 0.045, 0.025, and 106 mM, respectively. The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH was exclusively transferred to the substrate.