Identification and characterization of novel broad-spectrum amino acid racemases from Escherichia coli and Bacillus subtilis.

The peptidoglycan layer of the bacterial cell wall typically contains D-alanine (D-Ala) and D-glutamic acid (D-Glu), and also various non-canonical D-amino acids that have been linked to peptidoglycan remodeling, inhibition of biofilm formation, and triggering of biofilm disassembly. Bacteria produce D-amino acids when adapting to environmental changes as a common survival strategy. In our previous study, we detected non-canonical D-amino acids in Escherichia coli grown in minimal medium. However, the biosynthetic pathways of non-canonical D-amino acids remain poorly understood. In the present study, we identified amino acid racemases in E. coli MG1655 (YgeA) and Bacillus subtilis (RacX) that produce non-canonical D-amino acids other than D-Ala and D-Glu. We characterized their enzymatic properties, and both displayed broad substrate specificity but low catalytic activity. YgeA preferentially catalyzes the racemization of homoserine, while RacX preferentially racemizes arginine, lysine, and ornithine. RacX is dimeric, and appears not to require pyridoxal 5'-phosphate (PLP) as a coenzyme as is the case with YgeA. To our knowledge, this is the first report on PLP-independent amino acid racemases possessing broad substrate specificity in E. coli and B. subtilis.

Continuous colorimetric assay that enables high-throughput screening of N-acetylamino acid racemases.

N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli L-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-D-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an L-amino acid acylase and its subsequent oxidation by an FAD-dependent L-amino acid oxidase (L-AAO). Cofactor recycling of the L-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-D-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.

Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids.

To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of D-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using (14)C-labeled D-Trp showed that D-Trp in the medium was used as a protein component in the cells, suggesting the presence of D-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with D-amino acids, especially D-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of D-amino acids for growth.

Presence of cyclophilin A in synovial fluids of patients with rheumatoid arthritis.

Cyclophilins have been suggested to act as leukocyte chemotactic factors produced in the course of inflammation. Therefore we looked for the presence of cyclophilins in the synovial fluids (SF) from patients with rheumatoid arthritis (RA). Peptidyl prolyl cis-trans isomerase activity (PPIase) was measured in SF from knee punctures of 26 patients with RA and five patients with knee osteoarthritis (OA). PPIase was detected in SF from RA patients, but not in samples from OA patients. Enzyme activity was sensitive to inhibition by cyclosporin A (IC50 = 28-50 nM). Estimated concentrations of the SF-derived cyclophilin based on the enzyme activity were in the range of 11 to 705 nM. The presence of cyclophilin in the SF showed disease correlation; its concentration correlated with the number of cells in the SF (r = 0.91, P < 0.0001) and with the percentage of neutrophils in the cellular infiltrate and was higher in more acute cases of joint swelling. In immunoblots of partially purified preparations of SF from RA patients, an approximately 18-kD protein band reacted with polyclonal antibodies that recognize cyclophilin A and B, but not with antibodies specific for cyclophilin B. Sequencing of this protein revealed identity of the NH2-terminal amino acids with those of human cyclophilin A. The finding is unexpected since cyclophilin B rather than A is generally regarded as the secreted isoform, the presence of cyclophilin A being confined to the cytoplasm. Our data support the hypothesis that cyclophilins may contribute to the pathogenesis of inflammatory diseases, possibly by acting as cytokines. This may offer a possible explanation of the effectiveness of cyclosporin A in RA, in addition to the known immunosuppressive effects of the drug.

Thyroid hormones and D-aspartic acid, D-aspartate oxidase, D-aspartate racemase, H2O2, and ROS in rats and mice.

Total concentrations of thyroid hormones T(3) and T(4), and of their free forms, FT(3) and FT(4), D-aspartic acid (D-Asp), D-aspartate oxidase (D-AspO), D-aspartate racemase, H(2)O(2), and ROS (reactive oxygen species) were determined in rats and mice. T(3) and T(4) were 1 and 50 ng/ml, respectively, in serum, and 750 and 40000 ng/g, respectively, in thyroid. Concentrations of the free forms FT(3) and FT(4) were ca. 250 times lower than their respective total concentrations. The endogenous content of D-Asp in thyroid gland was ca. 100 nmol/g tissue, whereas the activity of D-AspO was ca. 80 units/mg thyroid, and that of D-aspartate racemase was ca. 15 units/mg thyroid. H(2)O(2) Concentration in rat and mouse thyroid gland was ca. 290 pmol/g thyroid, and the concentration of ROS was ca. 10 pmol/DCF/min/mg protein. H(2)O(2) is essential for the iodination of the tyrosyl residues to produce mono- and diiodotyrosine that are the precursors for the synthesis of T(3) and T(4). Production of H(2)O(2) in thyroid glands occurs by oxidation of endogenous D-Asp by D-AspO (D-Asp+O(2)+H(2)O-->alpha-oxaloacetate+NH(3)+H(2)O(2)). D-Aspartate racemase catalyzes the in vivo production of D-Asp from L-Asp. Thus, interaction of endogenous D-Asp, D-AspO, and D-aspartate racemase in thyroid gland constitutes an additional biochemical pathway for the production of H(2)O(2) and consequently for the synthesis of thyroid hormones.

s-cyclophilin is retained intracellularly via a unique COOH-terminal sequence and colocalizes with the calcium storage protein calreticulin.

Cyclophilins (cyclosporin A-binding proteins) are conserved, ubiquitous, and abundant proteins that accelerate the isomerization of XaaPro peptide bonds and the refolding of proteins in vitro. s-Cyclophilin is a member of the cyclophilin family with unique NH2- and COOH-terminal extensions, and with a signal sequence. We now report that s-cyclophilin is retained in the cell, and that the conserved s-cyclophilin-specific COOH-terminal extension VEKPFAIAKE is sufficient to direct a secretory protein to s-cyclophilin containing structures. Antibodies to s-cyclophilin-specific peptides were produced and the location of the protein was determined by an immunocytochemical study at the light microscopic level. s-Cyclophilin colocalized with the Ca(2+)-binding protein calreticulin and, to a lesser extent, with the microsomal Ca(2+)-ATPase in the myogenic cell line L6, and with the Ca(2+)-binding protein calsequestrin in skeletal muscle. In activated platelets, s-cyclophilin immunoreactivity was detected in a ring-like structure that might correspond to the Ca(2+)-storing and -releasing dense tubular network. In spreading cells, s-cyclophilin containing vesicular structures accumulated at actin-rich protrusion sites. While s-cyclophilin consistently codistributed with Ca2+ storage site markers, the distribution of s-cyclophilin immunoreactivity was not identical to that of ER markers. To determine whether the COOH-terminal extension of s-cyclophilin was involved in its intracellular transport we added this sequence to the COOH-terminus of the secretory protein glia-derived nexin. Appropriate constructs were expressed transiently in cultured cells and proteins were detected with specific antibodies. We found that glia-derived nexin with the COOH-terminal sequence VEKPFAIAKE (but not with the control sequence GLVVMNIT) colocalized with endogenous s-cyclophilin, indicating that the sequence contained retention information. These results indicate that s-cyclophilin is a retained component of an intracellular organelle and that it may accumulate in specialized portions of the ER, and possibly in calciosomes. Because of its conserved structure, widespread distribution, and abundance s-cyclophilin may be a useful marker to study the biogenesis and distribution of ER subcompartments.

Direct dating of human fossils.

The methods that can be used for the direct dating of human remains comprise of radiocarbon, U-series, electron spin resonance (ESR), and amino acid racemization (AAR). This review gives an introduction to these methods in the context of dating human bones and teeth. Recent advances in ultrafiltration techniques have expanded the dating range of radiocarbon. It now seems feasible to reliably date bones up to 55,000 years. New developments in laser ablation mass spectrometry permit the in situ analysis of U-series isotopes, thus providing a rapid and virtually non-destructive dating method back to about 300,000 years. This is of particular importance when used in conjunction with non-destructive ESR analysis. New approaches in AAR analysis may lead to a renaissance of this method. The potential and present limitations of these direct dating techniques are discussed for sites relevant to the reconstruction of modern human evolution, including Florisbad, Border Cave, Tabun, Skhul, Qafzeh, Vindija, Banyoles, and Lake Mungo.

Leaf Extract from Lithocarpus polystachyus Rehd. Promote Glycogen Synthesis in T2DM Mice.

The purpose of this study was to investigate the effects of leaf extract from Lithocarpus polystachyus Rehd. on type II diabetes mellitus (T2DM) and the active ingredients of this effect. In addition, this study determined, for the first time, the underlying molecular and pharmacological mechanisms of the extracts on hyperglycemia using long-term double high diet-fed and streptozotocin (STZ) induced type II diabetic mice. In the present study, leaf extract, phloridzin and trilobatin were assessed in vivo (gavage) and in vitro (non-invasive micro-test technique, NMT) in experimental T2DM mice. The biochemical parameters were measured including blood glucose and blood lipid level, liver biochemical indexes, and hepatic glycogen. The relative expression of glycometabolism-related genes was detected. The effect of leaf extracts on physiological glucose flux in liver tissue from control and T2DM mice was also investigated. Body weight of experimental T2DM mice increased significantly after the first week, but stabilized over the subsequent three weeks; body weight of all other groups did not change during the four weeks' study. After four weeks, all treatment groups decreased blood glucose, and treatment with leaf extract had numerous positive effects: a) promoted in glucose uptake in liver, b) increased synthesis of liver glycogen, c) reduced oxidative stress, d) up-regulation of glucokinase (GK), glucose transporter 2 (GLUT2), insulin receptor (IR) and insulin receptor substrate (IRS) expression in liver, e) down-regulation of glucose-6-phosphatase (G-6-P) expression, and f) ameliorated blood lipid levels. Both treatment with trilobatin or phloridzin accelerated liver glycogen synthesis, decreased oxidative stress and increased expression of GK. IRS and phosphoenolpyruvate carboxykinase (PEPCK) were both up-regulated after treatment with trilobatin. Expression of GLUT2, PEPCK and G-6-P were also increased in liver tissue after treatment with phloridzin. Our data indicate that leaf extract from L. polystachyus Rehd. has a preferable hypoglycemic effects than trilobatin or phloridzin alone. Leaf extract significantly increased glucose uptake and hepatic glycogen synthesis while also inducing a decline of hepatic gluconeogenesis and oxidative stress in T2DM mice. From this study, we draw conclusions that L. polystachyus promoted glycogen synthesis in T2DM mice, and that the active compounds were not only the trilobatin or phloridzin.

Intact disulfide bonds decelerate the folding of ribonuclease T1.

Disulfide bonds in a folding protein chain are equivalent to prematurely formed native-like tertiary interactions. We investigated whether the mechanism of protein folding is changed by the presence of disulfide bonds. As a model we used the S54G/P55N-variant of ribonuclease T1, a protein with two disulfide bonds and a single cis proline (Pro39), and we measured both the direct and the proline-limited folding reactions before and after breaking of the disulfide bonds. The folding kinetics were compared under refolding conditions, in the regions of the urea-induced unfolding transitions of the two forms, and under unfolding conditions. The kinetics in the transition regions were analyzed on the basis of a three-species mechanism and all microscopic rate constants of folding and of prolyl isomerization could be determined as a function of the urea concentration from the measured rates and amplitudes. These kinetic analyses indicated that the disulfide bonds can be rather unfavorable for the folding of S54G/P55N-ribonuclease T1. Under strongly native conditions they retard the rate-limiting trans-->cis isomerization of Pro39 because they allow the rapid formation of partially ordered structure prior to the proline-limited refolding reaction. Under unfolding conditions the isomerization of Pro39 is not affected. The direct unfolding and refolding reactions in the transition region of polypeptide chains with correct prolyl isomers are also decelerated when the disulfide bonds are present. These changes in the folding kinetics are possibly related to the decrease in chain flexibility that is caused by the disulfide bonds. A high flexibility is probably important throughout folding, and in the case of ribonuclease T1 a premature locking of tertiary contacts by intact disulfide bonds can interfere unfavorably with both the direct and the proline-limited folding reactions.

A sensitive assay for measuring aspartate-specific amino acid racemase activity.

D-Aspartate (D-Asp), a free D-amino acid found in mammals, plays crucial roles in the central nervous, neuroendocrine, and endocrine systems. In mammalian tissues, D-Asp oxidase (DDO) is a degradative enzyme that stereospecifically acts on D-Asp. Asp racemase, a synthetic enzyme that produces D-Asp from L-Asp, has been identified in several lower organisms; however, the biosynthetic pathway of D-Asp in mammals remains to be fully clarified. The aim of this study was to establish a simple, accurate, and sensitive enzymatic method for the determination of Asp racemase activity. Using recombinant Streptococcus thermophilus Asp racemase as a model enzyme, two enzymatic methods for the determination of Asp racemase activity were optimized. In these methods, recombinant human DDO was used to degrade D-Asp formed from L-Asp by the Asp racemase reaction to 2-oxo acid, the amounts of which were then determined using a colorimetric assay. In one method, designated the coupling method, DDO was concomitantly included in the Asp racemase reaction mixture, and the Asp racemase reaction was readily coupled to the D-Asp degradative reaction by DDO during the incubation. In the other method, designated the separating method, an aliquot of the Asp racemase reaction mixture was mixed with DDO after the reaction to determine the amounts of D-Asp produced by Asp racemase. Under optimized conditions, the accuracy and sensitivity of these two methods were examined and compared, both to one another and conventional high-performance liquid chromatography (HPLC). The results presented here suggest that the coupling method is more accurate and sensitive than the other two methods and can be used for the determination of Asp racemase activity. The coupling method may help to advance our current understanding of the biosynthetic pathway of D-Asp in mammals.

Cyclophilin-A is a zinc-dependent DNA binding protein in macrophages.

The association of cyclosporin A (CsA) immunosuppression with inhibition of transcription factor-dependent lymphokine gene activation formed the basis of our decision to investigate nuclear-associated Cyp isoforms. Immunofluorescence microscopy of mouse macrophages cell line with a monoclonal antibody mAb7F1 raised against CypA shows a co-localisation of CypA in the nucleus and in the cytosol. Nuclear CypA binds to DNA in a zinc ion-dependent manner, in contrast to recombinant CypB. Peptidyl-prolyl cisltrans isomerase (PPIase) activity of nuclear CypA is inhibited by zinc ions. The zinc inhibited CypA does not bind cyclosporin A (CsA). We suggest that nuclear Cyp in complex with zinc ions recognizes DNA sequences and is involved in transcription modulating processes.

Characterization of a Schistosoma mansoni cDNA encoding a B-like cyclophilin and its expression in Escherichia coli.

A cDNA encoding a Schistosoma mansoni cyclophilin (SmCyP) has been cloned by polymerase chain reaction amplification using degenerate oligonucleotides based on known conserved cyclophilin (CyP) sequences and by screening an expression cDNA library. The cDNA sequence encodes a 21.5-kDa protein, which shares 59% sequence identity with human CyP B. The SmCyP protein was expressed in Escherichia coli with a hexahistidine affinity tag at its amino terminus and antibodies to the purified (His6)-SmCyP fusion protein were raised in a rabbit. Fractionation of parasite material followed by immunoblot analysis revealed that schistosome CyP is a soluble protein. The N-terminus of the predicted protein contains a hydrophobic region, suggestive of a signal sequence. Accordingly, a recombinant SmCyP protein, lacking the first 23 amino acids was found to share the same gel electrophoretic mobility as the parasite-derived CyP protein, suggesting cleavage of a leader sequence. Hybridization of genomic DNA to a full-length cDNA probe indicates that the SmCyP gene is present as a single copy. Immunohistological experiments in conjunction with confocal scanning laser microscopy and immune electron microscopy show that SmCyP is present in abundance in the adult worm as well as in the schistosomula. The function of CyP in the schistosome is presently unclear, but since its ligand, cyclosporin A, has antischistosomal activity, its function is expected to be a vital one.

Immunofluorescent localization and immunochemical determination of cyclophilin-A with specific rabbit antisera.

We raised rabbit antisera against homogeneous bovine cyclophilin A (CypA) and we report their use for its immunofluorescent and immunochemical detection without resorting to cyclosporine binding. Indirect immunofluorescence demonstrated that in tissue culture cells CypA is present in the cytoplasm diffusely and also associated with vesicles and the Golgi apparatus. In mitotic cells CypA is increased in amount and redistributed away from cytoplasmic organelles. High levels of CypA were demonstrated in murine splenic erythroblasts and myeloblasts, but they became undetectable during differentiation to mature erythrocytes and granulocytes. Large, often granular, lymphocytes stained very intensely, but small lymphocytes demonstrated variable staining. Dot blot immunoassays demonstrated that murine tissues contain similar amounts of CypA. During CsA treatment murine liver can increase its CypA content much more than spleen. In summary, we demonstrated that cells known to be resistant to the effects of CsA have high levels of CypA. Also tissues that are resistant to CsA can increase their levels more than sensitive tissues upon CsA exposure. Taken together these results suggest that CypA plays a role in cell cycle progression and that sensitivity to CsA may not be simply a reflection of the baseline CypA levels, but may also be affected by the regulation of these levels. Further work is needed in order to delineate the role of CypA in the cell cycle and its relation to the action of CsA.

The immunophilins, FK506 binding protein and cyclophilin, are discretely localized in the brain: relationship to calcineurin.

The immunosuppressant drugs cyclosporin A and FK506 bind to small, predominantly soluble proteins cyclophilin and FK506 binding protein, respectively, to mediate their pharmacological actions. The immunosuppressant actions of these drugs occur through binding of cyclophilin-cyclosporin A and FK506 binding protein-FK506 complexes to the calcium-calmodulin-dependent protein phosphatase, calcineurin, inhibiting phosphatase activity. Utilizing immunohistochemistry, in situ hybridization and autoradiography, we have localized protein and messenger RNA for FK506 binding protein, cyclophilin and calcineurin. All three proteins and/or messages exhibit a heterogenous distribution through the brain and spinal cord, with the majority of the localizations being neuronal. We observe a striking co-localization of FK506 binding protein and calcineurin in most brain regions and a close similarity between calcineurin and cyclophilin. FK506 binding protein and cyclophilin localizations largely correspond to those of calcineurin, although cyclophilin is enriched in some brain areas that lack calcineurin. The dramatic similarities in localization of FK506 binding proteins and cyclophilins with calcineurin suggest related functions.

Analysis and localization of cyclophilin A found in the virions of human immunodeficiency virus type 1 MN strain.

Previous reports have shown that cyclophilin A (CyPA) is found to be specifically associated with human immunodeficiency virus type-1 (HIV-1) virions and is required for infectivity (Franke et al. Nature 372:359; Thali et al. Nature 372:363). We have examined CyPA associated with HIV-1MN virions. Virions from infected human lymphoid cells were analyzed by high-pressure liquid chromatography (HPLC), protein sequence, and immunoblot analysis. At least three forms of CyPA were found: an unmodified form, an N-terminally modified form, and an N-terminally modified form that migrates as a larger isoform on a reducing-SDS polyacrylamide gel. Using a protease digestion procedure, CyPA that is associated with virions was found to be located inside the viral membrane. Similar examination of SIVMne produced by HUT-78 human T cells did not detect specific incorporation of CyPA into SIV virions. Our results are consistent with the role of CyPA acting early in the infectious process of HIV-1.

Roles of peptidyl-prolyl cis-trans isomerase and calcineurin in the mechanisms of antimalarial action of cyclosporin A, FK506, and rapamycin.

The immunosuppressive peptide cyclosporin A inhibits the growth of malaria parasites in vitro and in vivo, but little is known about its mechanism of antimalarial action. The immunosuppressive action of cyclosporin A is believed to result from binding of the drug to cyclophilins (intracellular peptidyl-prolyl cis-trans isomerases), and inhibition of the protein phosphatase calcineurin by the cyclosporin A-cyclophilin complex. Two immunosuppressive macrolides, FK506 and rapamycin, bind to a distinct isomerase, FKBP12, and the FK506-FKBP complex also inhibits calcineurin. Calcineurin itself is apparently involved in signal transduction between the T-cell membrane and nucleus, and its inhibition blocks T-cell activation. Rapamycin inhibits a later step in T-cell proliferation. Peptidyl-propyl cis-trans isomerase activity was detected in extracts of Plasmodium falciparum. It was completely inhibited by concentrations of cyclosporin A above 0.1 microM, but not by FK506 or rapamycin, and probably represented one or more cyclophilins. Comparison of the antimalarial and anti-isomerase activities of a series of cyclosporin analogues failed to reveal a correlation between the two properties. Cyclosporin A and its more active 8'-oxymethyl-dihydro-derivative, in combination with the cyclophilin-containing P. falciparum extract, inhibited the protein phosphatase activity of bovine calcineurin. Therefore inhibition of a putative P. falciparum calcineurin by a complex of CsA and cyclophilin might be responsible for the antimalarial action of the drug. The most active cyclosporin, however, was a 3'-keto-derivative of cyclosporin D (SDZ PSC-833) which inhibited P. falciparum growth with a 50% inhibitory concentration (IC50) of 0.032 microM (compared with 0.30 microM for cyclosporin A), but was a poor inhibitor of the parasite isomerase. 3'-Keto-cyclosporin D has negligible immunosuppressive activity, but it strongly inhibits the P-glycoprotein of multi-drug resistant mammalian tumour cells. FK506 and rapamycin were also active antimalarials (IC50 of 1.9 and 2.6 microM, respectively) but in the absence of detectable FKBP in P. falciparum extracts, their mechanisms of antimalarial action remain unclear.

Molecular cloning and regional distribution of rat brain cyclophilin.

Cyclosporine A (CsA) is a potent immunosuppressive drug that has widespread clinical uses in organ transplantation and the treatment of autoimmune disorders. However, the drug's clinical applications are on an empiric basis with a poor understanding of the basic mechanism(s) of action. CsA may exert some of its effects by binding to a cellular receptor protein--the cyclosporine receptor (also called cyclophilin). Cyclophilin (CyP) is an ubiquitous, soluble, cytoplasmic 17 kDa protein which has recently been shown to be a peptide-prolyl isomerase. CsA specifically binds to this protein and inhibits its isomerase activity. A rat cyclophilin cDNA clone was isolated from a rat brain lambda gt11 cDNA library. Northern blot analysis shows a single 1 kb messenger RNA in rat brain. In order to determine the regional distribution of the Cyp mRNA in situ hybridization was performed. The Cyp mRNA appeared to be expressed throughout the brain but there were particularly high levels in the cerebral cortex and hippocampus compared to the relatively low levels in white matter areas and tracts. At the cellular level, the Cyp mRNA is expressed at much higher levels in neurons than in glia. The high levels of Cyp in cortical (neuronal) areas may, in part, explain the global encephalopathic symptoms clinically observed in CsA neurotoxicity.

Transfer of D-phenylalanine from gramicidin S synthetase 1 to gramicidin S synthetase 2 in gramicidin S synthesis.

The transfer of phenylalanine from gramicidin S synthetase 1 (GS 1) to gramicidin S synthetase 2 (GS 2) was studied by the use of combinations of wild-type GS 1 with various GS 2s from a wild strain and gramicidin S non-producing mutant strains of Bacillus brevis Nagano. The combinations of mutant GS 2s lacking 4'-phosphopantetheine (from BI-4, C-3, E-1, and E-2) did not transfer D-phenylalanine from GS 1, although they could activate all the constituent amino acids. Other mutant GS 2s containing 4'-phosphopantetheine, except GS 2 from BII-3 (proline-activation lacking) accepted D-phenylalanine from intact GS 1. To ascertain more directly whether 4'-phosphopantetheine is involved in the transfer of D-phenylalanine from GS 1 to GS 2, pepsin digests of GS 2 that accepted [14C]phenylalanine were analyzed by Sephadex G-50 column chromatography and thin-layer chromatography (TLC). Radioactivity of [14C]phenylalanine was always associated with a peptide containing 4'-phosphopantetheine. Furthermore, the position of radioactivity was distinct from the position of 4'-phosphopantetheine on TLC after alkaline treatment or performic acid oxidation of the digests.

Properties of glutamate racemase from Bacillus subtilis IFO 3336 producing poly-gamma-glutamate.

We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-gamma-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-gamma-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-gamma-glutamate production in B. subtilis IFO 3336.

Evidence for a single multifunctional polypeptide chain on gramicidin S synthetase 2 obtained from a wild strain and mutant strains of Bacillus brevis.

The structures of the gramicidin S synthetase 2 s (GS 2, heavy enzyme) from a wild strain and mutant strains of Bacillus brevis have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The GS 2s used were obtained from a wild strain and group IV of mutant strains (BII-3, BI-3, BI-9) which lacked one specific amino acid activating activity. SDS polyacrylamide gel electrophoresis of GS 2 bound to a radioactive substrate showed that: first, in the case of the wild enzyme, the radioactivity of the substrate amino acid was detected only in the polypeptide with a molecular weight of about 280,000, regardless of the amino acid species used as substrate; secondly, in the case of the mutant enzyme, the radioactivity of the amino acid which could be activated by the enzyme was also associated with the protein band with a molecular weight of about 280,000. Regardless of the enzyme species tested, a pantothenic acid residue was also present in the protein band with a molecular weight of about 280,000. These results suggest that GS 2 is a multifunctional one polypeptide enzyme and the mutant-type GS 2s from BII-3 (proline-lacking), BI-3 (valine-lacking), and BI-9 (leucine-lacking) are also multifunctional enzymes having molecular weights identical to that of the wild-type enzyme.