Pyruvic Transaminase Research Articles

Aspartate aminotransferase from a thermophilic formate-utilizing methanogen, Methanobacterium thermoformicicum strain SF-4: relation to serine and phosphoserine aminotransferases, but not to the aspartate aminotransferase family.

The primary structure of the aspartate aminotransferase (AspAT) of an archaebacterium, Methanobacterium thermoformicicum strain SF-4, has been determined by cloning and sequencing of the gene for the enzyme. The gene had a consensus promoter and a ribosome binding sequence of methanogens in the 5' untranslated region, followed by an open reading frame starting with ATG and terminating with TGA. The deduced amino acid sequence was identical with the partial amino acid sequences of the enzyme including the N-terminal sequence, and the deduced molecular weight of 41,684 was virtually identical to that reported earlier for this enzyme [Tanaka, T., Yamamoto, S., Taniguchi, M., Hayashi, H., Kuramitsu, S., Kagamiyama, H., & Oi, S. (1992) J. Biochem. 112, 811-815]. The gene was expressed in Escherichia coli by inserting it into an expression vector just downstream of the lacZ promoter, and this verified that the cloned gene really encodes the Methanobacterium AspAT. The primary structure of the Methanobacterium AspAT showed extremely low homology, 5%, with AspATs of eubacteria, eukaryotes, and a thermoacidophilic arachaebacterium, Sulfolobus solfataricus. On the other hand, the Methanobacterium AspAT showed remarkable amino acid sequence homology, 31.5%, with rat serine:pyruvate aminotransferase and, 13.5%, with E. coli phosphoserine aminotransferase. Thus, the Methanobacterium AspAT apparently belongs to subgroup IV of the aminotransferases [Mehta, P.K., Hale, T.I., & Christen, P. (1993) Eur. J. Biochem. 214, 549-561], but not to subgroup I, in which all the AspATs known so far are included.

THE UNRELIABILITY OF THE LIDOCAINE/MONOETHYLGLYCINEXYLIDIDE TEST FOR ASSESSMENT OF LIVER DONORS

The serum monoethylglycinexylidide (MEGX) level 15 min (t15) after i.v. administration of lidocaine (1 mg/kg) in liver donors was retrospectively correlated with graft outcome and early hepatic function. Among the 35 orthotopic liver transplants studied, 4 recipients had to be retransplanted within 10 days post-OLT because of early graft nonfunction or dysfunction, and 3 recipients died, with a median (range) donor MEGX t15 (ng/ml) of 100 (86-119) and 169 (146-182), respectively. The remaining 28 OLT patients living with functioning grafts had a donor MEGX of 87 (18-245). No significant correlations could be found between donor MEGX t15 and recipient mean and peak glutamic-oxaloacetic and -pyruvic transaminases, total serum bilirubin, or mean and minimum prothrombin time values studied from day 1 to day 5 post-OLT. Moreover, categorization of donors using the MEGX t15 cut-off point of 80 ng/ml could not predict liver graft quality, as previously suggested. In summary, MEGX t15 in liver donors correlated neither with graft outcome nor with early functional parameters. Accordingly, the MEGX test should not be used as an isolated discriminatory evaluation for organ utilization.

Effect of feeding her (Zizyphus mauritianaLam.) juice on growth, blood composition and histology of albino rats

Ber juice was fed to groups of growing albino rats at the level of 10 ml/day in a 15% protein diet for four weeks. Diets containing ber juice showed significantly higher feed efficiency ratios when compared with control rats. No marked differences were observed in blood composition, serum glucose, proteins, cholesterol, serum glutamate oxaloacetate and pyruvate trans-aminases, alkaline phosphatase and organ weights in the different groups. The organs of animals fed ber juice with or without seeds or the casein control diet showed a normal histopathological picture. The results indicate that consumption of ber juice does not result in any adverse effects in rats.

Caffeine reduces the hepatotoxicity of paracetamol in mice.

Paracetamol causes extensive liver damage when taken in overdose quantities; however, it is less hepatotoxic when administered in combination with caffeine. The present work in mice was undertaken to study the effect of caffeine on mortality rates and biochemical and histological parameters of liver damage after administration of toxic doses of paracetamol. It was found that caffeine markedly increased the survival rate after administration of a dose of paracetamol that was lethal to 50% and 100% of mice, reduced liver damage as assessed by serum glutamic pyruvic and glutamic oxaloacetic transaminase activities, partially prevented the depletion of reduced glutathione and reduced histological changes to the liver accompanying paracetamol intoxication. The results support the possibility that caffeine might be useful for the treatment of paracetamol intoxication in humans.

A single serine:pyruvate aminotransferase gene on rat chromosome 9q34-q36

It was found in our previous study (Oda et al., 1990. J. Biol. Chem. 265: 7513–7519) that in the rat two mRNAs encoding mitochondrial and peroxisomal serine:pyruvate aminotransferase (SPT/AGT) are formed from a single SPT/AGT gene through alternative transcription initiation in exon 1. In an attempt to analyze the mechanisms underlying this unique phenomenon, we have isolated genomic clones harboring the entire rat SPT/AGT gene. In the present study, the location of the rat SPT/AGT gene was determined to be in the q34–q36 region of chromosome 9 by fluorescence in situ hybridization. Southern blot analysis of rat genomic DNA revealed an allelic BamHI restriction fragment length polymorphism among three different inbred rat strains. These results indicated that a single copy SPT/AGT gene is located on chromosome 9q34-q36 in the rat genome. This locus has been assigned the gene symbol Spat.

The primary structure of omega-amino acid:pyruvate aminotransferase.

The complete amino acid sequence of bacterial omega-amino acid:pyruvate aminotransferase (omega-APT) was determined from its primary structure. The enzyme protein was fragmented by CNBr cleavage, trypsin, and Staphylococcus aureus V8 digestions. The peptides were purified and sequenced by Edman degradation. omega-ATP is composed of four identical subunits of 449 amino acids each. The calculated molecular weight of the enzyme subunit is 48,738 and that of the enzyme tetramer is 194,952. No disulfide bonds or bound sugar molecules were found in the enzyme structure, although 6 cysteine residues were determined per enzyme subunit. Sequence homologies were found between an omega-aminotransferase, i.e. mammalian and yeast ornithine delta-aminotransferases, fungal gamma-aminobutyrate aminotransferase and 7,8-diaminoperalgonate aminotransferase, and 2,2-dialkylglycine decarboxylase. The enzyme structure is not homologous to those of aspartate aminotransferases (AspATs) including the enzymes of Escherichia coli and Sufolobus salfactaricus, though significant homology in the three-dimensional structures around the cofactor binding site has been found between omega-APT and AspATs (Watanabe, N., Sakabe, K., Sakabe, N., Higashi, T., Sasaki, K., Aibara, S., Morita, Y., Yonaha, K., Toyama, S., and Fukutani, H. (1989) J. Biochem. 105, 1-3).

Cytogenetic study of persons occupationally exposed to ethylene oxide

Ten persons occupationally exposed to ethylene oxide (EO), used in the sterilization of medical instruments, were studied at a hospital. The estimated concentration to which they were exposed was 60–69 ppm, TWA. Peripheral blood samples from 10 workers and 10 controls of the same age and sex were taken to determine the frequency of sister-chromatid exchanges (SCE) and chromosomal aberrations (CA). The mean frequencies of SCE/cell (X = S) were 13.27 for the exposed workers and 6.05 for controls. Chromosome aberration frequencies in exposed individuals were significantly increased compared with controls. A significant relationship between the frequencies of SCE and CA and EO exposure was demonstrated. Blood chemistry parameters such as urea, creatinine, uric acid, lactic dehydrogenase, glutamic oxaloacetic and pyruvic transaminases, luteinizing gonadotropin and follicle stimulating gonadotropin and thyrotropin were also measured and found to be within the normal range.

Primary hyperoxaluria type I due to a point mutation of T to C in the coding region of the serine:pyruvate aminotransferase gene

cDNA clones for serine:pyruvate aminotransferase (SPT, alternative name: alanine:glyoxylate aminotransferase) were obtained from a cDNA library constructed from the liver of a primary hyperoxaluria type I (PH1) case in which the SPT activity was approximately one-hundredth that in control liver. Six clones were isolated from 100,000 transformants and all of them contained an approximately 1.5 kbp insert which included the whole coding region for human SPT. Nucleotide sequence analysis revealed a point mutation of T to C at position 634 (relative to the 5′-end of the cDNA) encoding a Ser to Pro substitution at residue 205. The T to C conversion created a new SmaI site, which enabled us to demonstrate that the point mutation had occurred in the patient's SPT gene. SmaI digestion of genomic DNA may be useful for the diagnostic gene analysis of this type of PH1.

Enxymological characterization of a putative canine analogue of primary hyperoxaluria type 1

This paper concerns an enzymological investigation into a putative canine canalogue of the human autosomal recesive disease primary hyperoxaluria type 1 (alanine:glyoxylate / serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and seribe:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephripathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including d-glycerate dehydrogenese / glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate / serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal humanb liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is anlogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.

Dimethylarginine:pyruvate aminotransferase in rats. Purification, properties, and identity with alanine:glyoxylate aminotransferase 2.

Dimethylarginine:pyruvate aminotransferase, which plays a role in the metabolism of dimethylarginines, has been purified to homogeneity from rat kidney. The enzyme has a molecular weight of approximately 200,000 and an isoelectric point at about pH 6.3. The enzyme consists of four similar subunits having a molecular weight of about 50,000. The enzyme catalyzes the effective transaminations of guanidino-N methylated L-arginines (e.g. NG,NG-dimethyl-L-arginine, NG,N'G-dimethyl-L-arginine and NG-monomethyl-L-arginine) and the alpha-amino group of L-ornithine to pyruvate or glyoxylate. The enzyme was always accompanied by the known alanine:glyoxylate amino-transferase activity with the ratios of their specific activities remaining constant during the purification steps. The physicochemical and immunological properties of the purified enzyme were shown to be identical with those of the isozyme of alanine:glyoxylate aminotransferase (EC 2.6.1.44), designated as alanine:glyoxylate aminotransferase 2 (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The distribution profiles in tissues and the negative response to glucagon treatment further supported the identity of the two enzymes. The present data show that alanine:glyoxilate aminotransferase 2 functions in dimethylarginine metabolism in vivo in rats.

Cloning and nucleotide sequence of cDNA encoding human liver serine – pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.

Polyamine Metabolism in Ripening Tomato Fruit

The metabolism of [1,4-(14)C]putrescine and [terminal methylene-(3)H]spermidine was studied in the fruit pericarp (breaker stage) discs of tomato (Lycopersicon esculentum Mill.) cv Rutgers, and the metabolites identified by high performance liquid chromatography and gas chromatography-mass spectrometry. The metabolism of both putrescine and spermidine was relatively slow; in 24 hours about 25% of each amine was metabolized. The (14)C label from putrescine was incorporated into spermidine, gamma-aminobutyric acid (GABA), glutamic acid, and a polar fraction eluting with sugars and organic acids. In the presence of gabaculine, a specific inhibitor of GABA:pyruvate transaminase, the label going into glutamic acid, sugars and organic acids decreased by 80% while that in GABA increased about twofold, indicating that the transamination reaction is probably a major fate of GABA produced from putrescine in vivo. [(3)H]Spermidine was catabolized into putrescine and beta-alanine. The conversion of putrescine into GABA, and that of spermidine into putrescine, suggests the presence of polyamine oxidizing enzymes in tomato pericarp tissues. The possible pathways of putrescine and spermidine metabolism are discussed.

A Comparison of Pyrimidinone Analogue Immunomodulators for Treatment ofPhlebovirusInfections in Mice

The substituted pyrimidinone immunomodulators 5-bromo-2,3-dihydro-2-imino-6-phenyl-4(1H)-pyrimidinone (ABPP), 5-iodo-2,3-dihydro-2-imino-6-phenyl-4(1H)-pyrimidinone (AIPP), 5-bromo-2,3-dihydro-2-imino-6-methyl-4(1H)-pyrimidinone (ABMP), 5-chloro-2,3-dihydro-2-imino-6-phenyl-4(1H)-pyrimidinone (ACPP), 5-bromo-2,3-dihydro-2-iminp-6-(3-fluorophenyl)-4(1H)-pyrimidinone (ABmFPP) and 5-chloro-2,3-dihydro-2-imino-6-(2,3-difluorophenyl)-4(1H)-pyrimidinone (AComF2PP) were evaluated against the hepatotropic and immunosuppressive infection of mice induced by Punta Toro virus, a Phlebovirus related to Rift Valley fever virus. Using intraperitoneal (i.p.) treatments once daily for 3 days beginning 1 day prior to virus exposure, the order of efficacy was ABPP > ABmFPP > AComF2PP > AIPP > ABMP > ACPP, with prevention of death as end-point. Using single i.p. treatment 1 day prior to virus exposure, antiviral activity was seen in the order ABPP > ABmFPP > ABMP > AIPP > ACPP > AComF2PP. Three active pyrimidonones, ABPP, ABMP and AIPP, were given by gavage (p.o.) using the above 3-day treatment schedule. In this experiment, expanded parameters were used, with prevention of death seen as well as decreased hepatic icterus, as evidenced by lowered liver scores and serum glutamic oxalacetic and pyruvic transaminases. In addition, recoverable virus titres were reduced in the livers and sera of the mice. Greatest activity was seen using ABPP, followed by ABMP and then by AIPP. ABPP in doses of 100–800 mg kg−1was markedly effective when administered p.o. in single treatments given as late as 24 hr after virus inoculation; a single dose, 400 mg kg−1, prevented death when given as late as 48 hr after virus exposure. In comparison of three once-daily, every-other-day, or every 2-day p.o. treatments, ABPP was most effective using the every-other-day regimen.

Effects of some insecticides on several enzymes of tryptophan metabolism in rats

The activities of tryptophan 2,3-dioxygenase (EC 1.13.11.11), indoleamine 2,3-dioxygenase (EC 1.13.11.17), kynurenine 3-hydroxylase (EC 1.14.13.9), kynureninase (EC 3.7.1.3), kynurenine transaminases, and pyridoxal phosphokinase (EC 2.7.1.35) in the liver, kidney and lung rats were measured after administration of a single dose and repeated doses of dimethoate, carbaryl and fenvalerate, respectively. Ten percent LD50 of each insecticide was orally administered to a rat for a single dose, while 5% LD50 was orally given for five consecutive days as repeated doses. The control animals received the same volume of vehicle (polyethylene glycol 300). Body weight and organs weight losses were recognized only after repeated doses of dimethoate, while protein content remained constant compared to control animals. Repeated administration of dimethoate caused significant decrease in the activity of kynurenine 3-hydroxylase (28.3% decrease in liver, and 32.5% in kidney), kynurenine-pyruvate transaminase (EC 2.6.1.7) (40% in liver, and 24.2% in kidney), kynurenine- pyruvate transaminase (EC 2.6.1-) (24.5% in kidney) and pyridoxal phosphokinase (36.1% in liver). Repeated doses of carbaryl resulted in a significant decrease in the activity of apo-tryptophan 2,3-dioxygenase (42.8%), kynurenine-2-oxoglutarate transaminase (40% in liver), kynurenine-pyruvate transaminase (30.6% in liver), and serine-glyoxylate transaminase (EC 2.6.1.51) (47.9% in liver). Externally added insecticides at different concentrations to the incubation mixture resulted in an inhibition to tryptophan 2,3-dioxygenase, while the other enzymes examined showed no change in their activities.

Generation from a single gene of two mRNAs that encode the mitochondrial and peroxisomal serine:pyruvate aminotransferase of rat liver.

In rat liver there are two types of serine:pyruvate aminotransferase (SPT) whose natures are indistinguishable but whose subcellular localization are different. One is a mitochondrial enzyme (SPTm) and the other a peroxisomal enzyme (SPTp). We compared, in this study, the structure of mRNAs encoding SPTm and SPTp by comparison of the sizes after removal of poly(A) tail by ribonuclease H and by means of RNA blot analysis and S1 nuclease protection assay. No differences were detected between these two mRNAs other than that about 100 nucleotides of the 5'-terminal sequence of SPTm mRNA are lacking in SPTp mRNA, and the length of the poly(A) tail is different. Southern blot analysis of rat genomic DNA showed that the SPT gene is single. Primer extension and S1 nuclease mapping analyses, using a DNA fragment of a genomic clone, revealed that the SPTm and SPTp mRNAs are transcribed from different initiation sites, about 70 nucleotides apart, in the same exon, exon 1. Ribonuclease protection assay performed with RNA hybridization probe corresponding to 5'-terminal portion of SPTm mRNA also showed that the 5'-terminal sequence of SPTp mRNA is about 70 nucleotides shorter than that of hormone-responsive SPTm mRNA. These results indicate that the different organelle distribution of SPTm and SPTp, the products of the same SPT gene, arises from transcription from different initiation sites, conferring N-terminal extension peptide, the mitochondrial targeting signal, only on the translation product of SPTm mRNA.

4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Laboratory cultures of cowpea Rhizobium MNF2030 grew on 4-aminobutyrate (GABA) as sole source of carbon and nitrogen. GABA transport was active since it was inhibited by carbonyl cyanide mchlorophenyl hydrazone and 2,4-dinitrophenol and cells developed a 400-fold concentration gradient across the cell membrane. Arsenite treatment of GABA-grown cells revealed stoichiometric conversion of GABA to pyruvate, indicating that 2-oxoglutarate is not an intermediate in GABA catabolism. GABA catabolism by cells of strain MNF2030 grown on GABA appreared to involve GABA transaminase, succinic semialdehyde dehydrogenase and malic enzyme; the first two enzymes were specifically induced by growth on GABA. The deamination process and removal of NH3 in cells catabolizing GABA involved GABA: 2-oxoglutarate transaminase; glutamate: oxaloacetate aminotransferase; asparate: pyruvate aminotransferase and alanine dehydrogenase.

Purification and characterization of d-3-Aminoisobutyrate—pyruvate aminotransferase from rat liver

d-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40) was purified 1900-fold from rat liver extract. The purified enzyme showed a molecular mass of 180 kDa by gel-permeation HPLC analysis using a TSK gel G3000SW column. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate resulted in identification of a single band of approx. 50 kDa, indicating that the native enzyme is probably a tetrametric protein. The specific activity of the purified enzyme was 1.14 μmol/min per mg protein. d-3-Aminoisobutyrate and β-alanine were good amino donors. The K m value for l-3-aminoisobutyrate was 100-times larger than that for the d-isomer. The apparent K m values for d-3-aminoisobutyrate and β-alanine were 35 and 282 μM, respectively. Pyruvate, glyoxylate, oxalacetate 2-oxo- n-valerate, and 2-oxo- n-butyrate were good amino acceptors. The apparent K m values for pyruvate and glyoxylate were 32 and 44 μM, respectively.

Effects of applied nitrogen upon aspects of nitrogen metabolism and transport in developing nodulated winged bean plants

Nodulated winged bean [Psophocarpus tetragonolobus (L.) DC., cv. UPS 122] were grown under constant environmental conditions and supplied with mineral nutrient solution in which nitrogen was absent or was present as nitrate (12 mg N week‐1 plant‐1).Nitrate treatment dramatically promoted plant growth, increased fruit weight 1.6 fold, was necessary for tuberisation and enhanced nodulation. The in vitro accumulation of 14C into asparagine and aspartate components of excised nodules supplied with exogenous 14CO2 and [14C]‐D‐glucose was greater for nitrate‐treated plants, whilst accumulation into ureides was reduced by nitrate treatment. Levels of amino acids in xylem sap were greater for plants supplied with a complete nutrient solution, than those grown without applied nitrate, particularly for asparagine, glutamine and proline. Xylem ureide levels were greater for plants grown in the absence of supplementary nitrate.Nitrogen accumulated in leaf, stem and petiole, and root nodule tissues for utilisation during fruit development; peak nitrogen levels and time of anthesis were retarded for plants grown without applied nitrate. The shoot ureide content increased during fruiting, coincident with decreases in the total nitrogen content, indicating that ureide pools are not utilised during the early reproductive phase. However ureide reserves, particularly allantoin, were utilised during the later stages of pod fill.Enzyme activity which metabolised asparagine was found throughout the plant and was identified as K+‐dependent asparaginase (EC 3.5.1.1) and an aminotransferase. Apart from temporal differences in developmental profiles of enzyme activity, the activity of these enzymes and of allantoinase (EC 3.5.2.5) in developing tissues were similar for both treatments. The main differences were greater asparaginase and asparagine:pyruvate aminotransferase activities in root tissues and fruit of nitrate‐supplied plants; allantoinase activity in the primary roots of plants grown without nitrate decreased during development, whilst activity in developing tubers (nitrate‐supplied plants) increased.

Purification and Characterization of the Active SerineiPyruvate Aminotransferase of Rat Liver Mitochondria Expressed in Escherichia coli1

In the previous study (Oda, T., et al. (1985) Eur. J. Biochem. 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm). This specific product (SPT10) was purified to homogeneity through three different column chromatographies. The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus. The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm. Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone. The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication. In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.

Synchrotron radiation protein data collection system using the newly developed Weissenberg camera and imaging plate for crystal structure analysis (abstract)

It has been an earnest desire of protein crystallographers to collect fast, accurate, high resolution diffraction data from protein crystals, preferably with exposure time as short as possible. In order to meet this challenge, a new type of Weissenberg camera has been developed for the recording of diffraction intensity from protein crystals using synchrotron radiation. The BL6A2 line has a plane-bending mirror designed by Y. Sato. The optical bench with triangular tilt-cut Si crystal monochromator was designed by N. Kamiya and was installed in the BL6A2 hutch. The Weissenberg camera was set on the 2θ arm of the optical bench. This camera can be used with Fuji Imaging Plate (IP) as an x-ray detector, and the reading out of the image from the IP is carried out by using BA100. The characterization of this system was carried out using the native crystal of chicken gizzard G-actin DNase I complex and its Yb3+, PCMB, indium, and FMA derivatives. Since these crystals are very sensitive for x rays, the resolution limit of the diffraction was 5 Å with a 4-circle diffractometer on a rotating anode x-ray generator (N. Sakabe et al., J. Biochem. 95, 887. This complex was crystallized in space group P2,2,2, with a=42.0, b=225.3, and c=77.4 Å. The data were collected with this system with the 430-mm radius cassette when Photon Factory was operated at 2.5 GeV and 270 mA and the wavelength λ=1.004 Å was chosen. In order to avoid overlapping of diffraction spots, oscillation angle range and coupling constant (degree/mm) were settled on the basis of simulation patterns of diffraction spots up to the maximum resolution to be measured considering the direction of the crystal axes, wavelength, radius of the camera, and mosaicness of the crystal. When the oscillation axis was a axis, the oscillation angle range was selected at either 10° (1°/mm) or 5° (0.5°/mm) depending on the density of reciprocal lattice points along the incident beam, and typical exposure time in each IP was 50 and 25 s, respectively. The exposure was stopped after 10 times oscillation. The total range of 117.5° was recorded on 16 sheets of IP with an overlapping range 0.5°. The data processing was carried out using program weis coded by T. Higashi. Two complete data sets along the a and c axes were collected using two crystals, independently, and the merge R(F2) for native crystal was 0.068. In order to know the feasibility of the data (F+−F−), Patterson maps were calculated with data of each derivative, and heavy atom vectors clearly appeared as prominent peaks in the Harker sections of the Patterson maps of both Yb3+ and PCMB derivatives. The heavy atom positions were obtained from the combinations of different Patterson and different Fourier maps, and were refined by using least-squares techniques. The final figure of merit up to 2.5 Å resolution was 0.61 with 22 700 reflections. The assignment of DNase I part on the electron density maps is progressing using the Nicholson molecular model referring to its structure reported by C. Oefner and D. Suck [J. Mol. Biol. 192, 605 (1986)]. In addition to promising results of ω-aminoacid pyruvate aminotransferase [N. Watanabe et al., Book of abstracts of second Japan-China Bilateral Symposium on Biophysics, p. 83 (1988)], these results support that this data collection system consisting of a new type of Weissenberg camera using SR, Fuji Imaging Plate, BA100, and program weis is one of the fastest and most accurate systems for protein crystallography in use today. We thank the Education Ministry and the Foundations of Yamada, Naito and Toray for the financial support of this project. We are grateful to Dr. K. Namba of ERATO for our use of BA100.