Gene, 2000 Aug 22, 254(1-2), 67 - 76 Expression of a TGF-beta superfamily protein, macrophage inhibitory cytokine-1, in the yeast Pichia pastoris; Fairlie WD et al.; The methylotrophic yeast, Pichia pastoris, has been used to express both human and murine macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor beta (TGF-beta) superfamily cytokine . This is the first report of the expression of a correctly folded TGF-beta superfamily protein in a microbial organism . The protein is secreted in its correctly folded dimeric form at milligram per litre quantities, which are significantly higher than we have been able to achieve using mammalian expression systems . Purification schemes are described, and the purified protein is immunologically identical to protein produced in a mammalian expression system . Protein expression was influenced by a number of factors, most significantly by the concentration of methanol used during the induction phase . However, with very high levels of MIC-1 induction, substantial amounts of MIC-1 monomer were also secreted. Plant J, 2000 Aug, 23(4), 481 - 8 Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana; Seo M et al.; Abscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses . Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 1.2.3.1) . We have reported the occurrence of three isoforms of aldehyde oxidase, AOalpha, AObeta and AOgamma, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde . Here we report a new isoform, AOdelta, found in rosette leaf extracts, which efficiently oxidizes abscisic aldehyde . AO delta was specifically recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4) . Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOdelta . These results indicate that AOdelta is encoded by AAO3 . AOdelta produced in P . pastoris exhibited a very low Km value for abscisic aldehyde (0.51 microM), and the oxidation product was determined by gas chromatography-mass spectrometry to be ABA . Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves . When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment . These results suggest that AOdelta, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves. J Biol Chem, 2000 Dec 8, 275(49), 38410 - 6 Identification of positive and negative determinants of malonyl-CoA sensitivity and carnitine affinity within the amino termini of rat liver- and muscle-type carnitine palmitoyltransferase I; Jackson VN et al.; The extreme amino terminus and, in particular, residue Glu-3 in rat liver (L) carnitine palmitoyltransferase I (CPT I) have previously been shown to be essential for the sensitivity of the enzyme to inhibition by malonyl-CoA . Using the Pichia pastoris expression system, we now observe that, although mutants E3A (Glu-3 --> Ala) or Delta(3-18) of L-CPT I have markedly lowered sensitivity to malonyl-CoA compared with the wild-type protein, the mutant Delta(1-82) generated an enzyme that had regained much of the sensitivity of wild-type CPT I . This suggests that a region antagonistic to malonyl-CoA sensitivity is present within residues 19-82 of the enzyme . This was confirmed in the construct Delta(19-30), which was found to be 50-fold more sensitive than wild-type L-CPT I . Indeed, this mutant was >4-fold more sensitive than even the native muscle (M)-CPT I isoform expressed and assayed under identical conditions . This behavior was dependent on the presence of Glu-3, with the mutant E3A-Delta(19-30) having kinetic characteristics similar to those of the E3A mutant . The increase in the sensitivity of the L-CPT I-Delta(19-30) mutant was not due to a change in the mechanism of inhibition with respect to palmitoyl-CoA, nor to any marked change of the K(0.5) for this substrate . Conversely, for M-CPT I, a decrease in malonyl-CoA sensitivity was invariably observed with increasing deletions from Delta(3-18) to Delta(1-80) . However, deletion of residues 3-18 from M-CPT I affected the K(m) for carnitine of this isoform, but not of L-CPT I . These observations (i) provide the first evidence for negative determinants of malonyl-CoA sensitivity within the amino-terminal segment of L-CPT I and (ii) suggest a mechanism for the inverse relationship between affinity for malonyl-CoA and for carnitine of the two isoforms of the enzyme. Biochem Biophys Res Commun, 2000 Aug 28, 275(2), 279 - 85 Biochemical characterization of cloned Aspergillus fumigatus phytase (phyA); Ullah AH et al.; The gene for Aspergillus fumigatus phytase (phyA) was cloned and expressed in Pichia pastoris . The enzyme expressed was purified to near homogeneity using sequential ion-exchange chromatography and was characterized biochemically . Although A . fumigatus phytase shows 66.2% sequence homology with A . ficuum phytase, the most widely studied enzyme, the cloned phytase showed identical molecular weight and temperature optima profile to the benchmark phytase . The pH profile of activity and kinetic parameters, however, differed from A . ficuum phytase . The cloned enzyme contains the septapeptide RHGARYP motif, which is also identical to the active site motif of A . ficuum phytase . Chemical probing of the active site Arg residues using both cyclohexanedione and phenylglyoxal resulted in the inactivation of phytase . The cloned A . fumigatus phytase, however, was more resistant to phenylglyoxal-induced inactivation . Both cloned A . fumigatus and A . ficuum phytases were identically affected by cyclohexanedione . Both the thermal characterization data and kinetic parameters of cloned and expressed A . fumigatus phytase indicate that this biocatalyst is not superior to the benchmark enzyme . The sequence difference between A . fumigatus and A . ficuum phytase may explain why the former enzyme catalyzes poorly compared to the benchmark enzyme . In addition, differential sensitivity toward the Arg modifier, phenylglyoxal, indicates a different chemical environment at the active site for each of the phytases . Biochem Soc Trans, 2000, 28(4), 353 - 7 Expression of recombinant human type I-III collagens in the yeast pichia pastoris; Myllyharju J et al.; An efficient expression system for recombinant human collagens will have numerous scientific and medical applications . However, most recombinant systems are unsuitable for this purpose, as they do not have sufficient prolyl 4-hydroxylase activity . We have developed methods for producing the three major fibril-forming human collagens, types I, II and III, in the methylotrophic yeast Pichia pastoris . These methods are based on co-expression of procollagen polypeptide chains with the alpha- and beta-subunits of prolyl 4-hydroxylase . The triple-helical type-I, -II and-III procollagens were found to accumulate predominantly within the endoplasmic reticulum of the yeast cells and could be purified from the cell lysates by a procedure that included a pepsin treatment to convert the procollagens into collagens and to digest most of the non-collagenous proteins . All the purified recombinant collagens were identical in 4-hydroxyproline content with the corresponding non-recombinant human proteins, and all the recombinant collagens formed native-type fibrils . The expression levels using single-copy integrants and a 2 litre bioreactor ranged from 0.2 to 0.6 g/l depending on the collagen type. J Interferon Cytokine Res, 2000 Aug, 20(8), 677 - 83 Antiviral and antiluteolytic activity of recombinant bovine IFN-omega1 obtained from Pichia pastoris; Boue O et al.; The gene coding for bovine interferon-omega1 (BoIFN-omega1) was recently cloned and expressed at high levels in the yeast Pichia pastoris . The recombinant BoIFN-omega1 protein shows antiviral activity in different cell lines and has an antiluteolytic effect in cyclic ewes . In this article, we describe a method for purification of BoIFN-omega1 expressed in the methylotrophic yeast P . pastoris and characterization of its activity in vivo . The recombinant protein secreted to the culture medium had low activity because of self-aggregation . BoIFN-omega1 was solubilized using urea and desalting and finally purified by ion exchange chromatography on Q-Sepharose Fast Flow . The yield of purified product was approximately 300 mg/L of fermentation culture, with a specific antiviral activity of 10(8) IU/mg . Its purity was at least 80% . The biologic characterization of purified BoIFN-omega1 was determined by induction of an antiviral state on ewes challenged with 100 lethal doses (LD) of Aujeszky virus and by the extension of the corpus luteum life span and interestrous interval in cyclic cows . Ewes treated with 2 x 106 IU/kg BoIFN-omega1 were protected from Aujeszky virus infection . In cows receiving an intrauterine infusion of 1 mg BoIFN-omega1, equally distributed between the two uterine horns, twice daily from day 14 to day 22 of the experimental estrous cycle, the lifespan of the corpus luteum (25 vs . 19 days) and the interestrous intervals (26 vs . 21 days) were extended when compared with a control group (p < 0.05) . We show that recombinant BoIFN-omega1 purified from P . pastoris has high antiviral activity and is an effective antiluteolytic agent in cattle. Receptors Channels, 2000, 7(2), 93 - 107 The human ET(B) endothelin receptor heterologously produced in the methylotrophic yeast Pichia pastoris shows high-affinity binding and induction of stacked membranes; Schiller H et al.; The human endothelin B receptor (ET(B) receptor) was produced in the methylotrophic yeast Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 (AOX1) gene promoter . In the expression plasmids pPIC9KFlagET(B)Bio and pPIC9KFlag deltaGPET(B)Bio the ET(B) receptor coding region was fused in frame to the Saccharomyces cerevisiae alpha-factor prepropeptide and the FLAG-tag . In both constructs, the receptor was also fused to a biotinylation domain . Additionally, in pPIC9KFlag deltaGPET(B)Bio the putative N-glycosylation site and a protease site have been deleted by site directed mutagenesis . Crude membranes prepared from recombinant P . pastoris revealed specific and saturable binding of {125I}ET-1 with a K(D) of about 42 pM . Receptor levels of 60 pmol/mg and 35 pmol/mg for the Flag deltaGPET(B)Bio and the FlagET(B)Bio construct, respectively, were determined . The pharmacological profile for ET-1, ET-2 and ET-3 were as expected for a subtype B endothelin (ET) receptor . Immunoblot analysis showed an apparent molecular mass of 55 kDa for the Kex2-processed and about 74 kDa for the Kex2-unprocessed receptor . Contrary to the Flag deltaGPET(B)Bio construct, the FlagET(B)Bio construct was not correctly processed by the internal Kex2 endopeptidase . As was detected by ultrastructural analysis of recombinant yeast cells, high-level production of the receptor resulted in the formation of stacked membranes. Eur J Biochem, 2000 Sep, 267(17), 5493 - 501 Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii; Costello CA et al.; A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance . The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield . The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM . The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones . Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis . The 1.0-kb ketoreductase gene was cloned and sequenced from a Z . rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein . Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active . Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class. Int J Food Microbiol, 2000 Jul 25, 59(1-2), 29 - 36 Typing of non-Saccharomyces yeasts with enzymatic activities of interest in wine-making; Fernandez M et al.; Enzymatic activity of potential interest in wine-making was studied for 182 non-Saccharomyces yeasts isolated from musts before and at the onset of fermentation at wine cellars operating under the La Mancha Appellation of Origin in Spain . Tests were carried out on plates containing differential substrates appropriate for each case (casein, gelatin, polygalacturonic acid, and arbutin) to determine whether each of the isolates exhibited proteolytic, polygalacturonase, and beta-glucosidase activities . Nearly 80% of the wild yeasts possessed one or more enzymes of biotechnological interest . Once the enzymatic activities of the isolates had been established, 69 of the isolates that exhibited pronounced enzymatic activity and 11 randomly selected isolates that were devoid of any activity were typed using PCR/RFLP, which gave 13 different molecular profiles . The isolates for each of the profiles were then identified by classical methods . The enzyme beta-glucosidase was linked to the species Metschnikowia pulcherrima, and polygalacturonase activity was common in most of the species identified . Proteolytic activity was observed in Pichia membranifaciens and in Metschnikowia pulcherrima . Typing revealed the possibility of intraspecific differences in Pichia membranifaciens, because six different molecular profiles with one or more shared restriction bands were recorded for that species. Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1683 - 6 Pichia hawaiiensis sp . nov., occurring in decaying bark of Charpentiera trees in the Hawaiian archipelago; Phaff HJ et al.; A description is given for Pichia hawaiiensis sp . nov., a nitrate-utilizing member of the genus Pichia E . C . Hansen emend . Kurtzman . Seven strains of the new species were isolated during the years 1972, 1973 and 1978 from rotting bark of the Hawaiian tree genera Charpentiera, Pisonia and Cheirodendron . P . hawaiiensis is heterothallic but appears to occur in nature mainly in the diploid state . Asci are deliquescent and produce up to four hat-shaped spores per ascus . Phylogenetic analysis of the 600 nucleotide D1/D2 domain of the 26S rDNA showed that P . hawaiiensis is most closely related to Pichia populi and Williopsis californica (syn . Hansenula californica) . The type strain of P . hawaiiensis, isolated on the island of Hawaii from the rotting bark of Charpentiera sp . containing insect larvae, is strain UCD-FST 72-181T (= ATCC MYA-137T = CBS 8760T = NRRL Y-27270T). J Biotechnol, 2000 Jul 28, 81(1), 55 - 61 Expression of a mammalian alpha 2,6-sialyltransferase gene in Pichia pastoris; Chotigeat W et al.; Terminal sialic acid on oligosaccharides of glycoproteins shows several biological functions of the glycoproteins . The yeast Pichia pastoris normally does not contain sialic acid on the oligosaccharides of glycoproteins . A sialyltransferase (ST) gene was transfected into P . pastoris to assess the possibility of using yeast cells as a host to produce sialoglycoproteins . The expression vectors pPIC3.5 and pPIC9 were used as carriers . The recombinant P . pastoris harbouring ST-pPIC3.5 and ST-pPIC9 had sialyltransferase activity of 1.1 and 10.2 mU l(-1) respectively . The ability of the recombinant ST-pPIC3.5 and ST-pPIC9 to transfer the fluoresceinyl-NeuAc into the cell glycoproteins was 36.9 and 20.9 pmol mg -1 protein respectively. Eur J Biochem, 2000 Aug, 267(16), 5209 - 16 Biochemical characterization of recombinant subunits of type 2A protein phosphatase overexpressed in Pichia pastoris; Swiatek W et al.; Methylotrophic yeast Pichia pastoris was used for a medium-scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A protein phosphatase (PP2A) . Constructs encoding these subunits, which were designed to introduce eight histidines at their N-termini, were introduced into the KM71 Pichia strain by homologous recombination . Recombinant proteins overproduced after methanol induction were purified from cell-free extracts by anion-exchange chromatography on DEAE-Sepharose, and Ni2+/nitrilotriacetate/agarose . In addition, chromatography on omega-aminohexyl-Sepharose was applied to purify recombinant (r)PR65/A . This purification scheme yielded approximately 5 mg and 100 microg of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture . The specific activity of rPP2Ac measured with {32P}phosphorylase a {1.7 micromol.min-1.(mg protein)-1} and its inhibition by okadaic acid (IC50 = 0.66 nM) were similar to PP2A isolated from rabbit skeletal muscle . As demonstrated by immunodetection with methylation state-specific antibodies, recombinant PP2Ac was carboxymethylated at the last C-terminal leucine residue . Recombinant PP2A subunits were able to form a complex as demonstrated both by activity assays in the presence of protamine and by chromatography on protamine-agarose . In summary, P . pastoris provides a convenient heterologous system for the production of recombinant subunits of PP2A. Acta Crystallogr D Biol Crystallogr, 2000 Jul, 56 ( Pt 7), 894 - 7 Purification and crystallization of the extracellular domain of human neutral endopeptidase (neprilysin) expressed in Pichia pastoris; Dale GE et al.; Neutral endopeptidase (NEP) is a mammalian zinc metalloprotease involved in the inactivation of a wide variety of regulatory peptides such as enkephalins and atrial natiuretic factor . The soluble extracellular domain of NEP (sNEP) was expressed in the methylotrophic yeast Pichia pastoris . The protein was purified to homogeneity and single crystals have been obtained . Enzymatic deglycosylation of the enzyme was essential for the production of crystals suitable for X-ray analysis for both the NEP-phosphoramidon binary complex and the apo enzyme. FEMS Microbiol Lett, 2000 Aug 15, 189(2), 233 - 7 Can the grey mould disease of the grape-vine be controlled by yeast? Masih EI, Alie I, Paul B. Botrytis cinerea has been found to be highly pathogenic to 'Chardonnay' and 'Pinot noir' cultivars of the grape-vine producing the characteristic grey mould symptoms within 7 days of inoculation to the vitro-plants . The yeast Pichia anomala (strain FY-102), isolated from apple skin, was found to be antagonistic to B . cinerea as it completely inhibited the appearance of the grey mould symptoms when grown together . The yeast was responsible for morphological changes such as coagulation and leakage of the cytoplasm of B . cinerea . The pathogen, when applied together with P . anomala, failed to bring about the grey mould symptoms on the grape-vine, suggesting that the yeast could control the expression of this disease . An account of the interaction between B . cinerea and P . anomala, as well as the sequences of the complete ITS region of the ribosomal DNA of the yeast are described here. FEMS Microbiol Lett, 2000 Aug 15, 189(2), 165 - 9 Ribonuclease U2: cloning, production in Pichia pastoris and affinity chromatography purification of the active recombinant protein; Martinez-Ruiz A et al.; RNase U2 is an endoribonuclease secreted by the fungus Ustilago sphaerogena . Its genomic DNA (rnu2), containing an intron of 116 bp, has been isolated and cloned . The corresponding cDNA has also been synthesized . The recombinant RNase U2 was successfully produced in Pichia pastoris, fused to the yeast alkaline phosphatase signal peptide . The recombinant RNase U2, purified by affinity chromatography, contains three extra amino acids at its amino-terminal end and retains the enzymatic and spectroscopic properties of the natural fungal protein. Appl Environ Microbiol, 2000 Aug, 66(8), 3381 - 6 Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures; Eliasson A et al.; For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity . Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway . However, the high ethanol yield and productivity seen with glucose have not yet been achieved . To quantitatively analyze metabolic fluxes in recombinant S . cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001 . The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S . cerevisiae CEN.PK 113-7A . The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations . Anaerobic ethanol formation from xylose by recombinant S . cerevisiae was demonstrated for the first time . However, the strain grew on xylose only in the presence of oxygen . Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h(-1) g (dry weight) of cells(-1) (0.24 to 0.30 g h(-1) g {dry weight} of cells(-1)) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h(-1) . The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose . The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h(-1) g (dry weight) of cells(-1) when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h(-1) g (dry weight) of cells(-1) when the feed ratio was 3:1 . With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux. RNA, 2000 Jul, 6(7), 1004 - 18 dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing; Palladino MJ et al.; We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR . The dADAR locus has been localized to the 2B6-7 region of the X chromosome and the complete genomic sequence organization is reported here . dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B . Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates . Transcripts of the dADAR locus originate from two regulated promoters . In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs . dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system . Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain . Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated. Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1181 - 8 Purification and characterization of a novel alpha-L-arabinofuranosidase from Pichia capsulata X91; Yanai T et al.; An intracellular alpha-L-arabinofuranosidase from Pichia capsulata X91 was purified and characterized . The enzyme was purified to homogeneity from a cell-free extract by ammonium sulfate treatment, Concanavalin A-Sepharose, ion-exchange chromatography with DEAE Bio-Gel A agarose, arabinose-Sepharose 6B affinity chromatography, and hydroxyapatite column chromatography . The apparent molecular mass of the enzyme was estimated to be 250 kDa by native-PAGE . The enzyme molecule was suggested to be a tetramer with a subunit molecular mass of 72 kDa by SDS-PAGE . The enzyme had an isoelectric point at 5.1, and was most active at pH 6.0 and at around 50 degrees C . The alpha-L-arabinofuranosidase was active at ethanol concentrations of wine . The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate . The enzyme hydrolyzed beet arabinan and arabinogalactan, and efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice . A considerable amount of monoterpenols was produced in the Muscat wine coupled with the enzyme addition. Bioorg Khim, 2000 Jun, 26(6), 433 - 41 {Deletion mutants of the natriuretic peptide receptor type B: expression of cDNA, purification and characteristics of proteins}; Solov'eva OV et al.; The C type natriuretic peptide (CNP) is a peptide hormone stimulating vasorelaxation and inhibiting cell proliferation . CNP activates the type B natriuretic peptide receptor (NPR-B), known as the guanylate cyclase B membrane enzyme, which results in the cGMP release . To study functional properties of NPR-B, its gene fragments were expressed in methylotrophic yeasts Pichia pastoris . Conditions were found providing for secretion of functionally active recombinant proteins NPR-Bs and NPR-B1 into the cultural medium in a yield of 25 mg/ml culture . Their specific activity was 0.97 and 0.93 mumol cGMP min-1 mg-1 protein, respectively . It was shown that NPR-B belongs to the family of Ser/Thr protein kinases and can be autophosphorylated at the serine residues. Yeast, 2000 Aug, 16(11), 979 - 93 Isolation of Pichia pastoris genes involved in ER-to-Golgi transport; Payne WE et al.; Pichia pastoris has discrete transitional ER sites and coherent Golgi stacks, making this yeast an ideal system for studying the organization of the early secretory pathway . To provide molecular tools for this endeavour, we isolated P . pastoris homologues of the SEC12, SEC13, SEC17, SEC18 and SAR1 genes . The P . pastoris SEC12, SEC13, SEC17 and SEC18 genes were shown to complement the corresponding S . cerevisiae mutants . The SEC17 and SAR1 genes contain introns at the same relative positions in both P . pastoris and S . cerevisiae, whereas the SEC13 gene contains an intron in P . pastoris but not in S . cerevisiae . Intron structure is similar in the two yeasts, although the favoured 5' splice sequence appears to be GTAAGT in P . pastoris vs . GTATGT in S . cerevisiae . The predicted amino acid sequences of Sec13p, Sec17p, Sec18p and Sar1p show strong conservation in the two yeasts . By contrast, the predicted lumenal domain of Sec12p is much larger in P . pastoris, suggesting that this domain may help localize Sec12p to transitional ER sites . A comparison of the SEC12 loci in various budding yeasts indicates that the SEC12-related gene SED4 is probably unique to the Saccharomyces lineage. J Biol Chem, 2000 Oct 13, 275(41), 31655 - 60 Characterization of a cDNA encoding a novel human Golgi alpha 1, 2-mannosidase (IC) involved in N-glycan biosynthesis; Tremblay LO et al.; A human cDNA encoding a 70.9-kDa type II membrane protein with sequence similarity to class I alpha1,2-mannosidases was isolated . The enzymatic properties of the novel alpha1,2-mannosidase IC were studied by expressing its catalytic domain in Pichia pastoris as a secreted glycoprotein . alpha1,2-Mannosidase IC sequentially hydrolyzes the alpha1,2-linked mannose residues of {(3)H}mannose-labeled Man(9)GlcNAc to form {(3)H}Man(6)GlcNAc and a small amount of {(3)H}Man(5)GlcNAc . The enzyme requires calcium for activity and is inhibited by both 1-deoxymannojirimycin and kifunensine . The order of mannose removal was determined by separating oligosaccharide isomers formed from pyridylaminated Man(9)GlcNAc(2) by high performance liquid chromatography . The terminal alpha1,2-linked mannose residue from the middle branch is the last mannose removed by the enzyme . This residue is the mannose cleaved from Man(9)GlcNAc(2) by the endoplasmic reticulum alpha1, 2-mannosidase I to form Man(8)GlcNAc(2) isomer B . The order of mannose hydrolysis from either pyridylaminated Man(9)GlcNAc(2) or Man(8)GlcNAc(2) isomer B differs from that previously reported for mammalian Golgi alpha1,2-mannosidases IA and IB . The full-length alpha1,2-mannosidase IC was localized to the Golgi of MDBK and MDCK cells by indirect immunofluorescence . Northern blot analysis showed tissue-specific expression of a major transcript of 3.8 kilobase pairs . The expression pattern is different from that of human Golgi alpha1,2-mannosidases IA and IB . Therefore, the human genome contains at least three differentially regulated Golgi alpha1, 2-mannosidase genes encoding enzymes with similar, but not identical specificities. Can J Microbiol, 2000 Jun, 46(6), 495 - 505 Nutrition and phylogeny of predacious yeasts; Lachance MA et al.; Yeast predation was studied with respect to the range of its distribution among ascomycetous yeasts, the range of yeast species that can be affected, and nutritional aspects of the phenomenon . The yeasts identified as predators belong to the Saccharomycopsis clade as defined on the basis of rDNA sequence relatedness . The 11 recognized species in the clade, plus three undescribed but related Candida species, were shown to be incapable of utilizing sulfate as sole source of sulfur, and all but two (Saccharomycopsis capsularis and Saccharomycopsis vini) were observed to penetrate and kill other yeasts under some conditions . Other unrelated sulfate transport-deficient yeasts (strains in the genera Pichia and Candida and the two known species of Starmera) are not predacious . The predacious species vary considerably as to the optimal environmental conditions that favour predation . Some are inhibited by the presence of rich nitrogenous nutrients, organic sulfur compounds, or higher concentrations of ammonium nitrogen, whereas other species may be stimulated under the same conditions . An attempt was made to correlate prey susceptibility to the excretion of substances that stimulate the growth of predators, but no correlation was detected between the two phenomena . The range of susceptible prey covers both ascomycetes and basidiomycetes, and includes Schizosaccharomyces pombe, which was previously thought to be immune . The achlorophyllous alga Prototheca zopfii is not killed by predacious yeasts, but the initial steps of penetration have been observed in some cases . Predacious species attack other predacious species, and in some cases, young cultures may penetrate older cultures of the same strain. FEBS Lett, 2000 Jul 7, 476(3), 179 - 85 Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac protein; Briand L et al.; Aphrodisin is a soluble glycoprotein of hamster vaginal discharges, which stimulates male copulatory behavior . Natural aphrodisin was purified and its post-translational modifications characterized by MALDI-MS peptide mapping . To evaluate its ability to bind small volatile ligands, the aphrodisiac protein was expressed in the yeast Pichia pastoris as two major isoforms differing in their glycosylation degree, but close in conformation to the natural protein . Dimeric recombinant aphrodisins were equally able to efficiently bind odors (2-isobutyl-3-methoxypyrazine and methyl thiobutyrate) and a pheromone (dimethyl disulfide), suggesting that they could act as pheromone carriers instead of, or in addition to, direct vomeronasal neuron receptor activators. Protein Expr Purif, 2000 Aug, 19(3), 369 - 74 Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris; Roderfeld M et al.; A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71 . High levels of secreted protein were detected . Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated . This active enzyme was highly purified and characterized with respect to its biochemical properties . The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low . The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP . The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)) . Protein Expr Purif, 2000 Aug, 19(3), 329 - 34 Expression, purification, and characterization of equistatin in Pichia pastoris; Rogelj B et al.; Equistatin (EI) is a cysteine protease inhibitor that was isolated from the sea anemone Actinia equina . It belongs to a recently discovered group of thyroglobulin type-I domain inhibitors called thyropins . Since native EI is found only in low amounts in the body of sea anemone and expression of recombinant EI in Escherichia coli yielded only 1 mg/liter of protein, we used the Pichia pastoris expression system to obtain higher yields . A cDNA encoding EI was inserted into pPIC9 vector and transformed into the P . pastoris, strain GS115 . Clones expressing high levels of EI were selected from 48 transformants . Recombinant EI was produced in 2-liter shake flasks and recovered from the fermentation broth by affinity chromatography using CM-papain-Sepharose . SDS-PAGE and N-terminal sequence analysis revealed that EI was N-terminally intact and running at the expected molecular weight of 22 kDa . The equilibrium dissociation constants of EI with papain and bovine cathepsin D were determined and were found to be similar to the results for the native inhibitor . EI production was scaled up to a bench top fermentor with a 25 mg/liter yield of active EI . Biol Reprod, 2000 Aug, 63(2), 619 - 28 Production, purification, and carboxy-terminal sequencing of bioactive recombinant bovine interferon-stimulated gene product 17; Pru JK et al.; An interferon (IFN)-stimulated gene (ISG) encodes a bovine 17-kDa protein (bISG17) that is released from endometrial cells but also conjugates to intracellular proteins through a ubiquitinlike mechanism . During early pregnancy in ruminants, conceptus-derived IFN-tau induces endometrial ISG17 . The present experiments were designed to generate bioactive recombinant (r) bISG17 . The Pichia pastoris yeast expression system was used because previous experiments expressing the human ISG15 ortholog in bacteria were confounded by inherent carboxypeptidase activity that cleaved C-terminal residues resulting in an inactive protein . In a series of extensive yeast culture experiments using shaker-bath and fermentation approaches, optimal conditions were determined for a transformant containing a multi-ISG17 gene insertion . Recombinant bISG17 was purified . Carboxy-terminal sequencing revealed that rbISG17 retained the C-terminal Gly that is potentially critical for the first step in covalent attachment to targeted intracellular proteins . The rISG17 induced (P < 0.0001) IFN-gamma mRNA (reverse transcription-polymerase chain reaction) and release of IFN-gamma protein (ELISA) by bovine peripheral blood mononuclear cells . The IFN-gamma mRNA also was upregulated (P < 0.0001) in endometrium from pregnant (Day 18) when compared with nonpregnant (Days 14 and 18) cows . It is concluded that rbISG17 generated in a yeast expression system retains cytokine/hormonal activity . This is the first description coupling the biology of two distinct IFNs (gamma and tau) through the intermediary ubiquitin homolog ISG17. Biotechnol Bioeng, 2000 Sep 5, 69(5), 486 - 94 Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters; Boer H et al.; Heterologous expression of T . reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P . pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermentor . Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded . Cel7A expressed in P . pastoris is overglycosylated at its N-glycosylation sites as compared to the native T . reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae . The k(cat) and K(m) values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced . The measured pH optimum also closely resembles that of purified T . reesei Cel7A . Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements . On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T . reesei Cel7A . The constitutive GAP promoter expression system of P . pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates . With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T . reesei expression system . J Agric Food Chem, 2000 Jun, 48(6), 2401 - 8 Effects of hexanal, trans-2-hexenal, and storage temperature on shelf life of fresh sliced apples; Corbo MR et al.; In this paper, the effects of hexanal and trans-2-hexenal, which are both natural molecules characterizing apple aroma, on the microbial population and on color retention of fresh sliced apples were evaluated . In particular, a central composite design (CCD) was developed to assess the individual and interactive effects of the chosen volatile molecules and storage temperatures on (i) the growth of the naturally occurring microflora, (ii) the evolution over time of an inoculated spoilage yeast (Pichia subpelliculosa), and (iii) the enzymatic browning reaction in minimally processed apples . The inclusion of hexanal and trans-2-hexenal in the storage atmosphere of apple slices determined a significant extension of shelf life also when P . subpelliculosa was inoculated at levels of 10(3) colony-forming units/g and abusive storage temperatures were used . In fact, the presence of these molecules in the packaging atmospheres considerably prolonged the lag phases of the inoculated yeast and reduced the growth potential of naturally occurring bacteria . Moreover, the addition to the modified atmosphere of low levels of the hexanal increased the color stability of the products up to 16 days. Syst Appl Microbiol, 2000 Apr, 23(1), 156 - 64 Polyphasic identification of wild yeast strains isolated from Greek sourdoughs; Paramithiotis S et al.; A total of forty-five wild yeast strains were isolated from five traditional Greek wheat sourdoughs . Strains were identified using the classical identification technique along with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell proteins (SDS-PAGE), Fourier transform-infrared spectroscopy (FT-IR) and the randomly amplified polymorphic DNA-polymerase chain reaction analysis (RAPD-PCR) . The latter methods confirmed the classical identification . According to the results obtained, fourteen strains were identified as Saccharomyces cerevisiae strains, twenty-five as Pichia membranaefaciens strains and six as Yarrowia lipolytica. Int Arch Allergy Immunol, 2000 Jun, 122(2), 101 - 7 Immunological activity of recombinant Ole e 1 in patients with Olea europaea pollinosis; Quiralte J et al.; BACKGROUND: Recombinant allergens have potential advantages over conventional allergenic extracts . However, these recombinant allergens should be evaluated for their antigenic activity and compared with their natural counterparts before being used for clinical purposes . METHODS: We studied 33 patients with seasonal rhinitis and/or bronchial asthma and a positive skin prick test to Olea europaea pollen extract, 10 atopic patients with no history of pollinosis and a negative skin prick test to O . europaea extract and 10 healthy controls . Skin prick tests and determination by ELISA of specific IgE to natural Ole e 1 (nOle e 1) and recombinant Ole e 1 (rOle e 1) expressed in Pichia pastoris were performed in all patients and controls . Inhibition assays were performed between nOle e 1 and rOle e 1 by ELISA . RESULTS: All patients with O . europaea pollinosis had positive skin test responses to both commercial O . europaea extract and nOle e 1 allergen, and all reacted to rOle e 1 on the skin prick test . The nonatopic and atopic control subjects with negative olive pollen skin test results did not react to rOle e 1 on the skin prick test, even at the highest concentrations, confirming the specificity of this test . We found a weak correlation between the wheal surface area produced by the prick test with nOle e 1 and the wheal surface area produced by rOle e 1 at 10 microgram/ml (r = 0.42, p < 0.05) . Comparison of specific IgE against both nOle e 1 and rOle e 1 in the patients did not reveal any significant difference . There was a strong correlation between the amount of specific IgE against nOle e 1 and rOle e 1 (r = 0.99, p < 0.01) . The two proteins displayed the same extent of binding inhibition to IgE antibodies in ELISA inhibition experiments . CONCLUSIONS: These results confirm the immunological activity of rOle e 1 expressed in P . pastoris and indicate that Ole e 1 is one of the major allergens in O . europaea pollinosis as evaluated by skin prick test and serological methods . The correlation between rOle e 1 and nOle e 1 in skin test results and serologic data indicates the potential of recombinant allergens for clinical applications and diagnosis of O . europaea pollen allergy . Protein Expr Purif, 2000 Jul, 19(2), 276 - 83 Pisum sativum mitochondrial pyruvate dehydrogenase can be assembled as a functional alpha(2)beta(2) heterotetramer in the cytoplasm of Pichia pastoris; Moreno JI et al.; Pea (Pisum sativum) mitochondrial pyruvate dehydrogenase (E1) was produced by coexpression of the mature alpha and beta subunits in the cytoplasm of the yeast Pichia pastoris . Size-exclusion chromatography of recombinant E1, using a Superose 12 column, yielded a peak at M(r) 160,000 that contained both alpha and beta subunits as well as E1 activity . This corresponds to the size of native alpha(2)beta(2) E1 . Recombinant E1 alpha (His(6))-E1 beta was purified by affinity chromatography using immobilized Ni(+), with a yield of 2.8 mg L(-1) . The pyruvate-decarboxylating activity of recombinant E1 was dependent upon added Mg(2+) and thiamin-pyrophosphate and was enhanced by the oxidant potassium ferricyanide . Native pea mitochondrial E1-kinase catalyzed phosphorylation of Ser residues in the alpha-subunit of recombinant E1, with concomitant loss of enzymatic activity . Thus, mitochondrial pyruvate dehydrogenase can be assembled in the cytoplasm of P . pastoris into an alpha(2)beta(2) heterotetramer that is both catalytically active and competent for regulatory phosphorylation . Protein Expr Purif, 2000 Jul, 19(2), 253 - 8 Expression of the Arabidopsis thaliana AtJ2 cochaperone protein in Pichia pastoris; Zhou R et al.; A vector was constructed for intracellular expression of the Arabidopsis thaliana DnaJ homologue AtJ2 in the methylotrophic yeast Pichia pastoris . The vector includes DNA encoding an amino-terminal histidine-tag, to simplify protein purification . Shake-flask cultures could be induced to produce approximately 250 mg/ L of AtJ2 . Purified recombinant AtJ2 was able to stimulate the ATPase activities of both the Escherichia coli and Zea mays cytoplasmic Stress70 chaperone proteins five- to ninefold . The carboxy terminus of AtJ2 is -CAQQ, a protein farnesylation motif . When transformed P . pastoris was induced to synthesize AtJ2 in the presence of {(3)H}mevalonolactone, radioactivity was incorporated into the protein, suggesting farnesylation . Virus Genes, 2000, 20(2), 127 - 33 Cloning and secreted expression of the extracellular domain of the mumps virus fusion protein in Pichia pastoris; McAleer B et al.; The extracellular globular domain of the mumps virus fusion (F) protein (amino-acids 28-481) has been overexpressed from GS115 his4 Pichia pastoris cells following the generation of a recombinant clone . The heterologous protein was directed for secreted expression by in-frame cloning with the S . cerevisiae alpha-factor secretion signal . The expressed protein was observed to secrete into the culture medium . An expressing clone was obtained initially by small-scale induction, metabolic labeling and immunoprecipitation . Expression analysis of the chosen clone was confirmed by western blotting with F protein specific polyclonal serum . The effects of culture volume, temperature and methanol concentration on the levels of expression, were studied . The results indicate that there is a balance required between the induction temperature and methanol concentration to achieve maximal expression . In addition, the presence of designated monomeric (47 K), dimeric (85-90 K) and trimeric (140 K) forms are dependent upon the induction conditions . Estimated secreted protein expression levels of > 1 mg/L were obtained in these studies . Further, the experiments demonstrate that the complete reconstruction of the KEX2 protease cleavage site is not necessary to facilitate secretion. Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 645 - 652 Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris; Papakonstantinou T et al.; The yeast cell factory is a potentially useful source of proteins in general . They include glutamic acid decarboxylase (GAD), which is one of the major autoantigens for Type 1 diabetes . We have created a hybrid form of GAD consisting of amino acids 1-101 of the human GAD67 protein fused to amino acids 96-585 of the human GAD65 protein, and have modified this to include a C-terminal hexa-Histidine (H6) tag sequence . This hybrid GAD67/65-H6 was expressed in two yeast hosts: constitutively under the control of the plasmid phosphoglycerate kinase promoter (PGK1) in Saccharomyces cerevisiae, and inducibly under the control of the chromosomal alcohol oxidase promoter (AOX1) in Pichia pastoris . Enzymatically active hybrid GAD was prepared from yeast lysates by purification either on an affinity column based on the GAD-1 monoclonal antibody, or by metal-affinity chromatography . The purified GAD67/65-H6 was radiolabelled with iodine-125 and tested with Type 1 diabetes sera in a radioimmunoprecipitation assay, and results were compared with those using untagged GAD67/65 and those using porcine brain GAD . The results of enzymatic and immunological assays show hybrid GAD67/65 is isolated at high specific activity and moderate yield, and the addition of the H6 tag sequences or the choice of yeast strain did not appreciably affect enzyme activity, percentage recovery of GAD, protein purification, or the utility in diagnosis of diabetes in terms of specificity and sensitivity to the various sera. Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 640 - 644 Expression and secretion of human alpha1(I) procollagen fragment by Hansenula polymorpha as compared to Pichia pastoris; de Bruin EC et al.; Secretion of a human collagen alpha1(I) chain fragment was achieved in Hansenula polymorpha using the native alpha1(I) procollagen secretory signal sequence . The N-terminal propeptide in the fragment was cleaved off during secretion, yielding the N-terminus of mature alpha1(I) collagen . In Pichia pastoris transformants, the expression of the fragment could be detected on RNA-level, but the product could not be determined extracellularly . After fusion of the fragment with a myc-HIS6 epitope, the intact product was found intracellularly . The difference in the extracellular level of the protein between the two expression hosts is most likely caused by difference in secretion efficiency. Arch Biochem Biophys, 2000 Jun 15, 378(2), 234 - 45 The cyanide-resistant alternative oxidases from the fungi Pichia stipitis and Neurospora crassa are monomeric and lack regulatory features of the plant enzyme; Umbach AL et al.; Both plant and fungal mitochondria have cyanide-resistant alternative oxidases that use reductant from the mitochondrial ubiquinone pool to reduce oxygen to water in a reaction that conserves no energy for ATP synthesis . The dimeric plant alternative oxidase is relatively inactive when its subunits are linked by a disulfide bond . When this bond is reduced, the enzyme can then be stimulated by its activators, alpha-keto acids . A Cys in the N-terminal section of the protein is responsible for both of these features . We examined the alternative oxidases in mitochondria isolated from two fungi Neurospora crassa and Pichia stipitis for dimeric structure, ability to form an intermolecular disulfide, and sensitivity to alpha-keto acids . Neither of the two fungal alternative oxidases could be covalently linked by diamide, which induces disulfide bond formation between nearby Cys residues, nor could they be cross-linked by a Lys-specific reagent or glutaraldehyde at concentrations which cross-link the plant alternative oxidase dimer completely . Alternative oxidase activity in fungal mitochondria was not stimulated by the alpha-keto acids pyruvate and glyoxylate . Pyruvate did stimulate activity when succinate was the respiratory substrate, but this was not a direct effect on the alternative oxidase . In contrast, added GMP was a strong activator of fungal alternative oxidase activity . Analysis of plant and fungal alternative oxidase protein sequences revealed a unique domain of about 40 amino acids surrounding the regulatory Cys in the plant sequences that is not present in the fungal sequences . This domain may be where dimerization of the plant enzymes occurs . In contrast to plant enzymes, the fungal alternative oxidases studied here are monomeric and their activities are independent of alpha-keto acids . Biochem J, 2000 Jul 1, 349(Pt 1), 179 - 87 Reconstitution of purified, active and malonyl-CoA-sensitive rat liver carnitine palmitoyltransferase I: relationship between membrane environment and malonyl-CoA sensitivity; McGarry JD et al.; Carnitine palmitoyltransferase I (CPT I) catalyses the initial step of fatty acid import into the mitochondrial matrix, the site of beta-oxidation, and its inhibition by malonyl-CoA is a primary control point for this process . The enzyme exists in at least two isoforms, denoted L-CPT I (liver type) and M-CPT I (skeletal-muscle type), which differ in their kinetic characteristics and tissue distributions . A property apparently unique to L-CPT I is that its sensitivity to malonyl-CoA decreases in vivo with fasting or experimentally induced diabetes . The mechanism of this important regulatory effect is unknown and has aroused much interest . CPT I is an integral outer-membrane protein and displays little activity after removal from the membrane by detergents, precluding direct purification of active protein by conventional means . Here we describe the expression of a 6 x His-tagged rat L-CPT I in Pichia pastoris and purification of the detergent-solubilized enzyme in milligram quantities . Reconstitution of the purified product into a liposomal environment yielded a 200--400-fold increase in enzymic activity and restored malonyl-CoA sensitivity . This is the first time that a CPT I protein has been available for study in a form that is both pure and active . Comparison of the kinetic properties of the reconstituted material with those of L-CPT I as it exists in mitochondria prepared from yeast over-expressing the enzyme and in livers from fed or fasted rats permitted novel insight into several aspects of the enzyme's behaviour . The malonyl-CoA response of the liposomal enzyme was found to be greater when the reconstitution procedure was carried out at 22 degrees C compared with 4 degrees C (IC(50) approximately 11 microM versus 30 microM, respectively) . When the sensitivities of L-CPT I in each of the different environments were compared, they were found to decrease in the following order: fed liver>fasted liver approximately liposomes prepared at 22 degrees C approximately P . pastoris mitochondria>liposomes prepared at 4 degrees C . In addition, pre-treatment of L-CPT I liposomes with the membrane-fluidizing reagent benzyl alcohol caused densensitization to the inhibitor . In contrast with the variable response to malonyl-CoA, the liposomal L-CPT I displayed a pH profile and kinetics with regard to the carnitine and acyl-CoA substrates similar to those of the enzyme in fed or fasted liver mitochondria . However, despite a normal sensitivity to malonyl-CoA, L-CPT I in P . pastoris mitochondria displayed aberrant behaviour with regard to each of these other parameters . The kinetic data establish several novel points . First, even after stringent purification procedures in the presence of detergent, recombinant L-CPT I could be reconstituted in active, malonyl-CoA sensitive form . Second, the kinetics of the reconstituted, 6 x His-tagged L-CPT I with regard to substrate and pH responses were similar to what is observed with rat liver mitochondria (whereas in P . pastoris mitochondria the enzyme behaved anomalously), confirming that the purified preparation is a suitable model for studying the functional properties of the enzyme . Third, wide variation in the response to the inhibitor, malonyl-CoA, was observed depending only on the enzyme's membrane environment and independent of interaction with other proteins . In particular, the fluidity of the membrane had a direct influence on this parameter . These observations may help to explain the mechanism of the physiological changes in the properties of L-CPT I that occur in vivo and are consistent with the current topographical model of the enzyme. Int Arch Allergy Immunol, 2000 May, 122(1), 20 - 32 Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion; Asero R et al.; BACKGROUND: Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability . They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple) . They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods . OBJECTIVE: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity . METHODS: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT . To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs . Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included . Pepsin digestion was used to address the role of stability in the allergenicity of LTPs . RESULTS: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi) . Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity . Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions . Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP . CONCLUSION: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin . Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen . Plant Physiol, 2000 Jun, 123(2), 743 - 56 Recombinant expression of molybdenum reductase fragments of plant nitrate reductase at high levels in Pichia pastoris; Mertens JA et al.; Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO(3) reductase (NR; EC1.6.6.1) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs . In fermenter cultures, spinach MoR was expressed at 420 mg L(-1), corn MoR at 32 mg L(-1), and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L(-1) . Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase . Methanol-induced expression yielded more target protein . All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD . MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR . MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR . Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron . Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR . P . pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies. Int J Food Microbiol, 2000 Jun 1, 56(2-3), 145 - 51 Effects of weak acid preservatives on the growth and thermal death of the yeast Pichia membranifaciens in a commercial apple juice; Veiga A et al.; Pichia membranifaciens exhibited a dissociative temperature profile (the temperature range of thermal death was distinct from the temperature range of growth) when incubation took place either in a commercial apple juice (AJ) or in a synthetic mineral medium with glucose and vitamins (MGV) . In AJ the maximum temperature for growth (Tmax) was 38.6 degrees C, which decreased to 36 degrees C in the presence of either 1 mM sorbic or 1 mM benzoic acid . The minimum temperatures of thermal death (Tmind) were, respectively, 40 and 38 degrees C with either of the acids . The yeast could grow with up to 2 mM sorbic or 3 mM benzoic acid, at 25 degrees C, which is close to the optimum temperature for growth (Top) . At temperatures slightly above Tmind, sorbic acid was an actual enhancer of death rather than benzoic, the latter conferring some protection . However, these effects were reversed at higher temperatures (above 43 degrees C), at which benzoic acid was the most operative, in contrast to sorbic which was highly protective of the yeast against thermal death . The addition of acetaldehyde to sulphur-dioxide-containing juice reduced the lag phase and increased the overall specific growth rates . Sporulated or stationary vegetative cultures were more heat-resistant than exponential cultures, particularly at temperatures above 45 degrees C. Biochemistry (Mosc), 2000 May, 65(5), 604 - 8 Constitutive biosynthesis and localization of alcohol oxidase in the ethanol-insensitive catabolite repression mutant ecr1 of the yeast Pichia methanolica; Lusta KA et al.; The activity and localization of alcohol oxidase (EC 1.1.3.13) have been studied in the Pichia methanolica mutant ecr1 defective in ethanol-induced catabolite repression of enzymes of methanol utilization . Ultrasctuctural, immunocytochemical, and biochemical analyses revealed the presence of peroxisomes containing active alcohol oxidase in the mutant grown in media with methanol, ethanol, and a mixture of both substrates . No alcohol oxidase was detected in the wild-type cells (ECR1) grown on ethanol-containing media . Mutant ecr1 growing in medium containing a mixture of different alcohols and the wild-type strain growing on methanol demonstrated similar buoyant density of peroxisomes (1.24-1.27 g/cm3)during isopicnic centrifugation of the organelles in sucrose density gradients . The integrated genetic, immunocytochemical, and biochemical data are in agreement with the model that synthesis, translocation into peroxisomes, and assembly of alcohol oxidase in P . methanolica may not require any regulatory signals induced by methanol. J Cell Biol, 2000 Jun 12, 149(6), 1171 - 8 The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane; Snyder WB et al.; Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown . We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay . Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation . The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs . By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap . Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes . These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome. Appl Biochem Biotechnol, 2000 Spring, 84-86, 201 - 16 Characterization and complementation of a Pichia stipitis mutant unable to grow on D-xylose or L-arabinose; Shi NQ et al.; Pichia stipitis CBS 6054 will grow on D-xylose, D-arabinose, and L-arabinose . D-Xylose and L-arabinose are abundant in seed hulls of maize, and their utilization is important in processing grain residues . To elucidate the degradation pathway for L-arabinose, we obtained a mutant, FPL-MY30, that was unable to grow on D-xylose and L-arabinose but that could grow on D-arabinitol . Activity assays of oxidoreductase and pentulokinase enzymes involved in D-xylose, D-arabinose, and L-arabinose pathways indicated that FPL-MY30 is deficient in D-xylitol dehydrogenase (D-XDH), D- and L-arabinitol dehydrogenases, and D-ribitol dehydrogenase . Transforming FPL-MY30 with a gene for xylitol dehydrogenase (PsXYL2), which was cloned from CBS 6054 (GenBank AF127801), restored the D-XDH activity and the capacity for FPL-MY30 to grow on L-arabinose . This suggested that FPL-MY30 is critically deficient in XYL2 and that the D-xylose and L-arabinose metabolic pathways have xylitol as a common intermediate . The capacity for FPL-MY30 to grow on D-arabinitol could proceed through D-ribulose. J Biol Chem, 2000 Sep 8, 275(36), 28254 - 60 Structure/function of the human Ga1beta1,3-glucuronosyltransferase . Dimerization and functional activity are mediated by two crucial cysteine residues; Ouzzine M et al.; Galbeta1,3-glucuronosyltransferase (GlcAT-I) that catalyzes the transfer of a glucuronic acid residue onto the trisaccharide primer of the glycosaminoglycan-protein linkage region plays an essential role in the early steps of the biosynthesis of glycosaminoglycans . In order to gain insight into the structure/function of the enzyme, the human recombinant GlcAT-I was successfully expressed in the yeast Pichia pastoris, with an apparent molecular mass of 43 kDa . Analysis of the electrophoretic mobility of the membrane-bound protein in nonreducing and reducing conditions, together with cross-linking studies, indicated that the membrane-bound GlcAT-I formed active disulfide-linked dimers . GlcAT-I expressed without the predicted N-terminal cytoplasmic tail or secreted as a polypeptide lacking the cytoplasmic tail and transmembrane domain was similarly organized as dimers, suggesting that the structural determinants for the dimerization state are localized in the luminal domain of the protein . In addition, the role of Cys(33) and Cys(301) in that process was investigated by site-directed mutagenesis combined with chemical modification of GlcAT-I by N-phenylmaleimide . Replacement of Cys(33) with alanine abolished the formation of dimers with a concomitant decrease in the catalytic efficiency mainly due to a decrease in apparent maximal velocity and in affinity for UDP-glucuronic acid . On the other hand, N-phenylmaleimide treatment or alanine substitution of the Cys(301) residue inactivated the enzyme . Our study demonstrates that GlcAT-I is organized as a homodimer as a result of disulfide bond formation mediated by Cys(33) localized in the stem region, whereas the residue Cys(301) localized in a conserved C-terminal domain is strictly required for the functional integrity of the enzyme. J Dairy Res, 2000 May, 67(2), 189 - 97 Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils; Wedlock DN et al.; The physiological effects of intramammary infusions of recombinant bovine cytokines in six lactating dairy cows on the quality and yield of milk and the bactericidal activity of milk neutrophils were investigated . Recombinant bovine interleukin-2 (rboIL-2) and interferon-gamma (rboIFN-gamma) were produced in the yeast Pichia pastoris . Two animals were given rboIL-2 (2 x 10(5) units) in two quarters, two animals were given rboIFN-gamma (6.5 x 10(5) units) in two quarters, and the other two cows received a dose of rboIL-2 in one quarter and rboIFN-gamma in a second quarter . In addition, each animal was given phosphate-buffered saline (PBS) in the other two quarters as a control . Somatic cell counts and conductivity of the fore milk were monitored before and after infusion . Neutrophils were isolated from quarter milk samples 36 h after infusion of cytokine or PBS and their bactericidal activities against Staphylococcus aureus were measured in vitro with a colorimetric assay . Quarters infused with rboIL-2 or rboIFN-gamma showed significant but transitory increases in both milk somatic cell counts and conductivity when compared with preinfusion values and with control quarters . There were minimal effects on daily milk yield . Neutrophils isolated from milk from quarters infused with rboIL-2 showed enhanced bactericidal activity against Staph . aureus . The bacterial killing from rboIL-2 treated quarters was significantly greater, with a mean of 63.5% compared with a mean of 5.4% for neutrophils taken from uninfected quarters to which PBS had been administered . The bactericidal activities for quarters treated with rboIFN-gamma and infected quarters treated with PBS were 15.0 and 30.0% respectively . The results indicate that intramammary infusions of rboIL-2 and rboIFN-gamma to lactating cows are well tolerated, and that rboIL-2 can activate milk neutrophils and augment their bactericidal activity. Protein Eng, 2000 May, 13(5), 377 - 84 Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris; Morawski B et al.; The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants . Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells . We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host . Here we examined expression of HRP in Saccharomyces cerevisiae . Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S.cerevisiae culture supernatant compared with wild-type, as measured on ABTS inverted question mark2, 2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (260 units/l/OD(600)) . Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD(600) in shake flasks . The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol. Protein Eng, 2000 May, 13(5), 353 - 60 Design and synthesis of germline-based hemi-humanized single-chain Fv against the CD18 surface antigen; Caldas C et al.; The 6.7 murine monoclonal antibody (mAb) recognizes the human CD18 antigen and is therefore of interest as an anti-inflammatory agent . The 6.7 heavy variable chain (VH) was humanized using the closest human germline sequence as the template on to which to graft the murine complementary determining regions (CDRs) . Two versions were proposed, one in which the residue proline 45 of the murine form was maintained and another in which this framework residue was changed to the leucine found in the human sequence . These VH humanized versions were expressed in the yeast Pichia pastoris as hemi-humanized single-chain Fv (scFvs), with the VL from the murine antibody . The scFv from the murine antibody was also expressed . The binding activities of the murine and both hemi-humanized scFvs were determined by flow cytometry analysis . All the constructions were able to recognize human lymphocytes harboring CD18, indicating successful humanization with transfer of the original binding capability . Some differences between the two hemi-humanized versions were observed . The method used was simple and straightforward, with no need for refined structural analyses and could be used for the humanization of other antibodies. Protein Expr Purif, 2000 Jun, 19(1), 179 - 87 High-level production of recombinant fungal endo-beta-1,4-xylanase in the methylotrophic yeast Pichia pastoris; Berrin JG et al.; Efficient production of recombinant Aspergillus niger family 11 1, 4-beta-xylanase was achieved in Pichia pastoris . The cDNA-encoding XylA fused to the Saccharomyces cerevisiae invertase signal peptide was placed under the control of the P . pastoris AOX1 promoter . Secretion yields up to 60 mg/liter were obtained in synthetic medium . The recombinant XylA was purified to homogeneity using a one-step purification protocol and found to be identical to the enzyme overexpressed in A . niger with respect to size, pI, and immunoreactivity . N-terminal sequence analysis of the recombinant protein indicated that the S . cerevisiae signal peptide was correctly processed in P . pastoris . The purified protein has a molecular weight of 19,893 Da, in excellent agreement with the calculated mass, and appears as one single band on isoelectric focusing with pI value around 3.5 . Electrospray ionization mass spectrometry confirmed the presence of one major isoform produced by P . pastoris and the absence of glycosylation . The recombinant enzyme was further characterized in terms of specific activity, pH profile, kinetic parameters, and thermostability toward birchwood xylan as substrate and compared with the xylanase purified from A . niger . Both enzymes exhibit a pH optimum at 3.5 and maximal activity at 50 degrees C . The enzyme activity follows normal Michaelis-Menten kinetics with K(m) and V(max) values similar for both enzymes . P . pastoris produced recombinant xylanase in high yields that can be obtained readily as a single form . A . niger xylanase is the first microbial xylanase efficiently secreted and correctly processed by P . pastoris . Microbiol Res, 2000 Apr, 155(1), 31 - 5 Effect of magnesium ions on fermentative and respirative functions in Pichia stipitis under oxygen-restricted growth; Mahler G et al.; Mg2+ level affected growth, xylitol and ethanol production by P . stipitis grown under microaerophilic conditions . Low Mg2+ level (1 mM) directed the C flux from ethanol to xylitol, with no effect on xylose consumption rate . The addition of pyrazole, an alcohol dehydrogenase (ADH) inhibitor, had the same effect, even in conditions of Mg2+ excess (4 mM), indicating a negative interaction between ADH and Mg2+ ions (p << 0.01) . Cells grown either with pyrazole or Mg limitation increased their intracellular NADH concentration about 3 times, but displayed no significant differences in ADH specific activities (1,000 U/mg protein, +/- 10%) . In contrast, no interaction was measured between Mg and antimycin A, excluding the possibility that Mg2+ limitation interferes with respiration. Biosci Biotechnol Biochem, 2000 Apr, 64(4), 717 - 22 Molecular cloning and heterologous expression of pea seedling copper amine oxidase; Koyanagi T et al.; The cDNA coding for copper amine oxidase has been cloned from etiolated pea seedlings (Pisum sativum) . The deduced amino acid sequence, consisting of 674 residues including the signal peptide, agreed well with those reported for the enzymes from a different cultivar of P . sativum and other plant sources, except for several evolutionary replacements located mostly on the molecular surface . A heterologous expression system for the cloned pea enzyme was constructed with the yeast Pichia pastoris, using the AOX1 promoter and the yeast alpha-factor secretion signal . Adding copper to the culture medium increased the secretion of an active, quinone-containing enzyme . Furthermore, the inactive enzyme produced in a copper-deficient medium was activated considerably by subsequent incubation with excess cupric ions . These results strongly suggest that the Tyr-derived redox cofactor, 2,4,5-trihydroxyphenylalanylquinone (topa quinone, TPQ), is produced in the plant enzyme by post-translational modification that proceeds through the copper-dependent, self-processing mechanism, as in the enzymes from bacteria and yeast. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 417 - 22 Taxonomic relationships among the taxa in the Candida guilliermondii complex, as revealed by comparative electrophoretic karyotyping; Bai FY et al.; Electrophoretic karyotypes of 15 type strains of the taxa in the Candida guilliermondii complex including Candida fukuyamaensis Nakase et al . and Candida xestobii Yarrow et S . A . Meyer were comparatively analysed by using the CHEF (contour-clamped homogeneous electric field) method of PFGE . Eighteen strains (isolated from various natural sources in China) which were originally identified as C . guilliermondii by conventional methods were also included . Six electrophoretic karyotype groups were recognized among the strains compared . The following type strains were grouped together with the type strains of C . guilliermondii (Castellani) Langeron et Guerra and Pichia guilliermondii Wickerham: Blastodendrion arztii Ota, Blastodendrion krausi Ota, Candida amidovorans Balloni et al., C . guilliermondii var . japonica Sugiyama et Goto, Candida mamillae S . Goto, Candida parapsilosis (Ashford) Langeron et Talice var . tokyoensis Suzuki et al., C . parapsilosis var . tuxtlensis Herrera et al . and six Chinese strains . The type strain of Torulopsis kestonii Scarr et Rose was classified into the group together with the type strain of Candida fermentati (Saito) Bai and seven Chinese strains . The group represented by the type strain of C . fukuyamaensis included five other strains isolated in China . The type strains of Candida xestobii, C . guilliermondii var . carpophila Phaff et M . W . Miller and Trichosporon appendiculare Batista et al . were separated into three different groups, respectively . Taxonomic relationships among the taxa studied are discussed. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 395 - 404 Four new yeasts in the Pichia anomala clade; Kurtzman CP; Four new yeasts are described that were recognized as novel from nucleotide substitutions in domain D1/D2 of 26S rDNA, a region that is sufficiently divergent to allow resolution of most ascomycetous yeast species . The new species and their type strains are as follows: Pichia maclurae NRRL Y-5377T (= CBS 8671T); Pichia misumaiensis NRRL Y-17389T (= CBS 8062T); Candida mycetangii NRRL Y-6843T (= CBS 8675T); and Candida ulmi NRRL YB-2694T (= CBS 8670T) . The two Pichia species form spherical ascospores and are heterothallic . Phylogenetic analysis of domain D1/D2 sequences placed the four new species in the Pichia anomala clade. J Virol, 2000 Jun, 74(12), 5659 - 66 Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense; Smith SA et al.; Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation . The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence . It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding . In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules . Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction . Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity . These regions were then analyzed to more specifically define their properties for binding . It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction. Biometals, 1999 Dec, 12(4), 295 - 300 Iron uptake by the yeast Pichia guilliermondii . Flavinogenesis and reductive iron assimilation are co-regulated processes; Fedorovich D et al.; Pichia guilliermondii cells overproduce riboflavin (vitamin B2) in responce to iron deprivation . The increase in ferrireductase activity in iron-starved P . guilliermondii cells correlated with the increase in flavin excretion . As in Saccharomyces cerevisiae, a typical b-type cytochrome spectrum was associated with the plasma membrane fraction of P . guilliermondii and the cell ferrireductase activity was strongly inhibited by diphenylene-iodonium, an inhibitor of flavoproteins, in both yeasts . Mutants of P . guilliermondii with increased ferrireductase activity were selected for further investigation of the relationship between iron reduction/uptake and flavin production . The obtained mutation has been called hit (high iron transport) . A hit mutant with a single recessive mutation showed the following phenotype: high ferrireductase activity, increased rate of iron uptake and elevated flavinogenic activity . Cu(II) (50 microns) strongly inhibited the growth of the hit mutant compared to the wild-type . The mutant cells grown in copper-supplemented medium (5-25 microns) showed an increase of the ferrireductase activity (up to 2-3 fold) . The copper content of the mutant cells grown under these conditions was also higher (1.5-2 fold) than that of the wild-type . The role of the HIT gene of P . guillermondii in the regulation of iron, copper and flavin metabolisms is discussed. Glycoconj J, 1999 Sep, 16(9), 487 - 97 Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin; Zeng S et al.; Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans . Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha2-3)Gal(beta1-4){Fuc(alpha1-3)}GlcNAc(beta1-6){Gal(bet a1-3)}GalNAc(alpha1-pNp, representing the O-linked form, was synthesized in an overall yield of 32% . In a first step, Gal(beta1-3)GalNAc(alpha1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta3-galactosyltransferase (EC 2.4.1.122) from rat liver . UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta1-6){Gal(beta1-3)}GalNAc(alpha1-pNp in a yield of >85% . The core 2 structure was galactosylated using UDP-Gal and purified human milk beta4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%) . Overall 1.5 micromol of product was prepared . MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure. Protein Eng, 2000 Apr, 13(4), 299 - 307 Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris; Goda S et al.; A human lysozyme expression system by Pichia pastoris was constructed with the expression vector of pPIC9, which contains the alpha-factor signal peptide known for high secretion efficiency . P . pastoris expressed the human lysozyme at about 300 mg/l broth, but four extra residues (Glu(-4)-Ala(-3)-Glu(-2)-Ala(-1)-) were added at the N-terminus of the expressed protein (EAEA-lysozyme) . To determine the effect of the four extra residues on the stability, structures and folding of the protein, calorimetry, X-ray crystal analysis and GuHCl denaturation experiments were performed . The calorimetric studies showed that the EAEA-lysozyme was destabilized by 9.6 kJ/mol at pH 2.7 compared with the wild-type protein, mainly caused by the substantial decrease in the enthalpy change (DeltaH) . On the basis of structural information on the EAEA-lysozyme, thermodynamic analyses show that (1) the addition of the four residues slightly affected the conformation in other parts far from the N-terminus, (2) the large decrease in the enthalpy change due to the conformational changes would be almost compensated by the decrease in the entropy change and (3) the decrease in the Gibbs energy change between the EAEA and wild-type human lysozymes could be explained by the summation of each Gibbs energy change contributing to the stabilizing factors concerning the extra residues. Yeast, 2000 May, 16(7), 651 - 6 The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris; Koller A et al.; We report the construction of a Pichia pastoris integrating vector which contains the inducible CUP1 promoter from Saccharomyces cerevisiae . We show that the promoter is indeed inducible by copper when used in P . pastoris and that the level of induction is dependent on the amount of copper in the medium . Yeast, 2000 May, 16(7), 589 - 96 Isolation and characterization of the TIM10 homologue from the yeast Pichia sorbitophila: a putative component of the mitochondrial protein import system; Kayingo G et al.; The Saccharomyces cerevisiae TIM10 gene encodes one of the few essential mitochondrial proteins that are required for the import of nuclear-encoded precursor proteins from the cytosol and their subsequent sorting into the different mitochondrial compartments . We have isolated and characterized a putative homologue of TIM10 from the halotolerant yeast Pichia sorbitophila . The Pichia TIM10 gene encodes a protein of 90 amino acids with 66% identity to S . cerevisiae Tim10p . It was capable of suppressing the temperature sensitivity of tim10-1 mutant in S . cerevisiae, suggesting that Pichia TIM10 is both a functional and structural homologue of S . cerevisiae TIM10 . The putative Pichia TIM10 gene product contains all the four conserved cysteine residues and the two CX(3)C motifs typical of the Tim family proteins in the mitochondrial intermembrane space . Using anti-Tim10p serum, Western blots detected a protein of about 10 kDa, suggesting that the Pichia Tim10p is a mitochondrial protein . The results suggest that mitochondrial import and sorting systems might be also strongly conserved in other fungi . The coding sequence of the P . sorbitophila TIM10 has been deposited in the EMBL Nucleotide Sequence Database under Accession No . AJ243940 . Eur J Biochem, 2000 May, 267(10), 3079 - 89 Ligand-binding properties and structural characterization of a novel rat odorant-binding protein variant; Briand L et al.; After characterization of a novel odorant-binding protein (OBP) variant isolated from the rat nasal mucus, the corresponding cDNA was cloned by RT-PCR . Recombinant OBP-1F, the sequence of which is close to that of previously reported rat OBP-1, has been secreted by the yeast Pichia pastoris at a concentration of 80 mg.L-1 in a form identical to the natural protein as shown by MS, N-terminal sequencing and CD . We observed that, in contrast with porcine OBP-1, purified recombinant OBP-1F is a homodimer exhibiting two disulfide bonds (C44-C48 and C63-C155), a pairing close to that of hamster aphrodisin . OBP-1F interacts with fluorescent probe 1-aminoanthracene (1-AMA) with a dissociation constant of 0.6 +/- 0 . 3 microM . Fluorescence experiments revealed that 1-AMA was displaced efficiently by molecules including usual solvents such as EtOH and dimethylsulfoxide . Owing to the large OBP-1F amounts expressed, we set up a novel biomimetic assay (volatile-odorant binding assay) to study the uptake of airborne odorants without radiolabelling and attempted to understand the odorant capture by OBP in the nasal mucus under natural conditions . The assay permitted observations on the binding of airborne odorants of different chemical structures and odors (2-isobutyl-3-methoxypyrazine, linalool, isoamyl acetate, 1-octanal, 1-octanol, dimethyl disulfide and methyl thiobutyrate) . Uptake of airborne odorants in nearly physiological conditions strengthens the role of OBP as volatile hydrophobic odorant carriers in the mucus of the olfactory epithelium through the aqueous barrier towards the chemo-sensory cells. Appl Microbiol Biotechnol, 2000 Apr, 53(4), 469 - 75 Characteristics of glycosylated streptokinase secreted from Pichia pastoris: enhanced resistance of SK to proteolysis by glycosylation; Pratap J et al.; Degradation of streptokinase (SK) has been frequently observed during large-scale protein production . An enhanced susceptibility of SK to degradation has been correlated with its existence in a partially unfolded state . The influence of the carbohydrate moiety on the stability and functional characteristics of SK has been examined by obtaining the glycoform of SK following its secretion through the methylotrophic yeast Pichia pastoris . Secretion of the protein product was achieved by replacing the native secretion signal codons of SK with those from alpha-factor leader peptide and expressing the fusion construct under the control of the methanol-inducible alcohol oxidase (ox) promoter of P . pastoris after its integration into the host chromosome . Western blot and zymographic analysis of proteins secreted from the recombinant P . pastoris indicated that SK was glycosylated by the host cells, which resulted in the appearance of a SK species migrating slowly, corresponding to a 55-kDa protein product as compared to the 47-kDa native SK . The glycosylated SK retained a plasminogen activation capability identical to that of its unglycosylated counterpart . Glycoform SK exhibited an enhanced stability profile at 25 degrees C and 37 degrees C and improved resistance towards protease treatment compared to unglycosylated SK secreted through P . pastoris after tunicamycin treatment or that secreted from the recombinant Escherichia coli . The results presented thus illustrate that N-linked glycosylation of SK results in 30-40% enhancement of the protein stability and resistance towards degradation but does not interfere with its fibrinolytic function. Carbohydr Res, 2000 Apr 20, 325(3), 216 - 21 Isolation and characterization of non-labeled and 13C-labeled mannans from Pichia pastoris yeast; Vinogradov E et al.; Mannans from genetically modified Pichia pastoris yeast, used for overproduction of neural cell adhesion molecule protein, grown on normal media or on uniformly 13C-labeled glucose and methanol, were isolated and characterized by high-field (750 MHz) NMR spectroscopy . Fully 13C-labeled oligosaccharide fragments were prepared from mannans by acetolysis . According to the data obtained, the mannan is made up of a main chain of alpha-(1-->6)-linked mannopyranosyl residues, substituted at 0-2 with alpha-mannopyranosyl or a alpha-D-Manp-(1-->2)-beta-D-Manp-(1-->2)-beta-D-Manp-( 1-->2)-alpha-D-Manp- group, and with much lower content of substitution with beta-D-Manp-(1-->2)-alpha-D-Manp- . A fraction of these oligosaccharide side chains is again substituted with alpha-D-Glcp or alpha-D-GlcpNAc through a phosphodiester linkage to the 6 position of the first mannopyranosyl residue . Improved conditions of acetolysis, cleaving all alpha-(1-->6) linkages, but not beta-mannoside linkages, are proposed. J Biochem (Tokyo), 2000 May, 127(5), 829 - 36 Divalent forms of CC49 single-chain antibody constructs in Pichia pastoris: expression, purification, and characterization; Goel A et al.; Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics . Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs . In the present study, we engineered and expressed divalent non-covalent {(scFv)(2)-His(6)} and covalent {sc(Fv)(2)-His(6)} scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris . The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA . The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively . The expression of scFvs in P . pastoris was 30 to 40-fold higher than in Escherichia coli . Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11 . 42%ID/g, respectively, at 6 h post-injection . Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05) . In conclusion, improved yields of divalent scFvs were achieved using the P . pastoris expression/secretion system . The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications. Appl Environ Microbiol, 2000 May, 66(5), 1809 - 13 (1-->6)-beta-D-glucan as cell wall receptor for Pichia membranifaciens killer toxin; Santos A et al.; The killer toxin from Pichia membranifaciens CYC 1106, a yeast isolated from fermenting olive brines, binds primarily to the (1-->6)-beta-D-glucan of the cell wall of a sensitive yeast (Candida boidinii IGC 3430) . The (1-->6)-beta-D-glucan was purified from cell walls of C . boidinii by alkali and hot-acetic acid extraction, a procedure which solubilizes glucans . The major fraction of receptor activity remained with the alkali-insoluble (1-->6)-beta- and (1-->3)-beta-D-glucans . The chemical (gas-liquid chromatography) and structural (periodate oxidation, infrared spectroscopy, and (1)H nuclear magnetic resonance) analyses of the fractions obtained showed that (1-->6)-beta-D-glucan was a receptor . Adsorption of most of the killer toxin to the (1-->6)-beta-D-glucan was complete within 2 min . Killer toxin adsorption to the linear (1-->6)-beta-D-glucan, pustulan, and a glucan from Penicillium allahabadense was observed . Other polysaccharides with different linkages failed to bind the killer toxin . The specificity of the killer toxin for its primary receptor provides an effective means to purify the killer toxin, which may have industrial applications for fermentations in which salt is present as an adjunct, such as olive brines . This toxin shows its maximum killer activity in the presence of NaCl . This report is the first to identify the (1-->6)-beta-D-glucan as a receptor for this novel toxin. Biochim Biophys Acta, 2000 Jan 31, 1490(1-2), 43 - 53 Functional expression in Pichia pastoris of human and rat intrinsic factor; Wen J et al.; Intrinsic factor (IF) has been expressed previously in Baculovirus with a yield (0.1-1 mg/l) that was inadequate for structural and metabolic studies . IF cDNAs were cloned into the shuttle vector pPIC9 of P . pastoris, and the proteins were induced and purified by cobalamin (Cbl) affinity chromatography . Expression of recombinant proteins revealed a major band of 49 kDa for both human and rat IF . Expression of human IF was achieved at 1040 mg/l, but of rat IF at only 1-2 mg/l . Reaction of human IF with a photo-activatable derivative of Cbl was demonstrated by Western blotting, and detection of IF fragments by anti-Cbl monoclonal antibody and by amino-terminal sequencing revealed at least three regions (residues 129-151, 234-254, and +294) linked to Cbl . Both recombinant human and rat {125I}IF-Cbl bound to rat and guinea pig brush border membranes with similar affinity, but the binding capacity of human IF for the rat receptor was only 10% compared with rat IF . All six amino acids within the previously identified N-terminal binding region of human IF were mutated to be identical to rat IF, but the resulting chimeric IF still bound poorly to rat membranes . Mutations of residues 26/27 (Glu26 to Asp and Asn27 to Gln) and 32/34 (Ser32 to Thr and Tyr34 to Arg) showed changes in both Ka and Vmax, with great effects on Vmax . In conclusion, P . pastoris is an expression system that produces functional human IF at a higher yield than in the baculovirus system . Cbl binding was directly demonstrated at multiple sites along the linear sequence of human IF . The receptor binding function of the amino terminal sequence 25 62 has been confirmed, but it is insufficient to reproduce all the features of IF-Cbl binding. Eur J Biochem, 2000 May, 267(9), 2632 - 9 Propeptide dependent activation of the Antarctic krill euphauserase precursor produced in yeast; Kristjansdottir S et al.; Euphauserase is a brachyurin type digestive enzyme isolated from Antarctic krill . The brachyurins belong to clan SA of the S1 family of serine endopeptidases . In this study, we demonstrate that the precursor form of recombinant euphauserase, termed pro-r-euphauserase, can be successfully expressed in Pichia pastoris . The presence of most of the 51-residue euphauserase propeptide is essential during expression, under the growth conditions of Pichia . The propeptide may be required either for correct folding or processing of the enzyme . Cod trypsin generates a fully active r-euphauserase from its precursor, which appears to be identical to the native enzyme . The mature r-euphauserase sequence contains 250 amino-acid residues including a 13-residue activation peptide, which seems to be attached to the molecule by a disulfide bond . Euphauserase shares an average sequence identity of 62% with its type I brachyurin analogue, crab collagenase I . However, the identity between these two sequences is much higher in the regions shown to be important for the broad substrate specificity and collagen binding of crab collagenase I . The type I brachyurins share only 30-40% identities with the type II brachyurins and trypsins . The low isoelectric point of euphauserase, with a calculated pI value of 3.9, is typical for the type I brachyurins. Toxicology, 2000 Apr 3, 144(1-3), 113 - 20 Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: recombinant expression in the yeast Pichia pastoris and Escherichia coli; Blum A et al.; Detoxification of aldehydes and ketones generally proceeds via reduction to their corresponding alcohols, which are then conjugated and eliminated . We focused our interest on 11beta-hydroxysteroid-dehydrogenase type 1 (11beta-HSD 1), a pluripotent enzyme which physiologically performs the interconversion of active and inactive glucocorticoid hormones, and which also participates in xenobiotic carbonyl compound detoxification . 11beta-HSD 1 belongs to the protein superfamily of the short-chain dehydrogenases/reductases (SDR), and has been structurally and functionally characterized . 11beta-HSD 1 is a glycosylated membrane protein which is very difficult to purify in an active state . In addition, expression levels in humans differ in a wide range . In order to facilitate biochemical and molecular studies on the significance of human 11beta-HSD 1 in detoxification processes, we have successfully performed the overexpression of recombinant human 11beta-HSD 1 in the yeast Pichia pastoris and in Escherichia coli . Recombinant 11beta-HSD 1 from E . coli was purified to homogeneity and used to generate a polyclonal antibody . The enzyme had no enzymatic activity, possibly due to the lack of glycosylation and/or incorrect folding in E . coli . In contrast, 11beta-HSD 1 overexpressed in P . pastoris was enzymatically active towards its physiological glucocorticoid substrates as well as towards xenobiotic carbonyl compounds . In western blot experiments the antibody crossreacted with both recombinant 11beta-HSD 1 forms and with the native enzyme from mouse and human liver . In conclusion, recombinant 11beta-HSD 1 from P . pastoris serves as a valuable tool for future studies on carbonyl compound detoxification. Mikrobiologiia, 2000 Mar-Apr, 69(2), 180 - 4 {Synergistic effect of rib80 and hit mutations on riboflavin biosynthesis and iron transport in the yeast Pichia guilliermondii}; Fedorovich DV et al.; Mutant strains of the yeast Pichia guilliermondii, carrying both rib80 and hit mutations in a haploid genome, were derived from previously obtained strains with defective rib80 or hit genes, exerting negative control of the riboflavin biosynthesis and iron transport in Pichia guilliermondii . The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase . Iron deficiency caused an additional increase in riboflavin overproduction . These results suggest the synergistic interaction of the rib80 and hit mutations . A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of 55Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains. Biochem Biophys Res Commun, 2000 Apr 21, 270(3), 1128 - 35 Antibody binding of deletion mutants of Asp f 2, the major Aspergillus fumigatus allergen; Tang B et al.; Asp f 2, a 268 amino acid major allergen from Aspergillus fumigatus (Af) demonstrated nine linear IgE binding regions . It is not known whether any of these linear epitopes are also conformatory epitopes . Hence, we constructed deletion mutants of Asp f 2 devoid of one or more epitopes, and the IgE binding of these proteins with sera from patients with ABPA was compared with the full-length Asp f 2 expressed in E . coli and Pichia . The Pichia expressed protein reacted weakly with IgE, but strongly with IgG of ABPA sera compared to E . coli expressed Asp f 2 . Weak IgE binding only was seen when the C-terminal or N-terminal was deleted, while depletion of both ends negated all reactivity . The monoclonal antibody IL-B8 and IgE and IgG of ABPA sera bound significantly to the Asp f 2 E-4 fragment indicating that the major B-cell epitope is located at the N-terminal end of Asp f 2 . J Biol Chem, 2000 Jul 7, 275(27), 20887 - 95 In mouse alpha -methylacyl-CoA racemase, the same gene product is simultaneously located in mitochondria and peroxisomes; Kotti TJ et al.; alpha-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid degradation pathway, was studied at the protein, cDNA, and genomic levels in mouse liver . Immunoelectron microscopy and subcellular fractionation located racemase to mitochondria and peroxisomes . The enzymes were purified from both organelles with immunoaffinity chromatography . The isolated proteins were of the same size, with identical N-terminal amino acid sequences, and the existence of additional proteins with alpha-methylacyl-CoA racemase activity was excluded . A racemase gene of about 15 kilobases was isolated . Southern blot analysis and chromosomal localization showed that only one racemase gene is present, on chromosome 15, region 15B1 . The putative initial ATG in the racemase gene was preceded by a functional promotor as shown with the luciferase reporter gene assay . The corresponding cDNAs were isolated from rat and mouse liver . The recombinant rat protein was overexpressed in active form in Pichia pastoris . The presented data suggest that the polypeptide encoded by the racemase gene can alternatively be targeted to peroxisomes or mitochondria without modifications . It is concluded that the noncleavable N-terminal sequence of the polypeptide acts as a weak mitochondrial and that the C-terminal sequence acts as a peroxisomal targeting signal. Antonie Van Leeuwenhoek, 2000 Feb, 77(2), 159 - 71 Reclassification of Pichia membranifaciens sensu Kurtzman; Mikata K et al.; Forty strains which were stocked as Pichia membranifaciens sensu Kurtzman and 9 strains stocked as Candida valida, anamorphs of P . membranifaciens, in the Institute for Fermentation, Osaka (IFO) were reclassified based on the data of base composition of nuclear DNA, DNA/DNA hybridization, coenzyme Q system, electrophoretic karyotype, and base sequence of 18S rDNA . P . membranifaciens complex was assigned into 3 groups: (I) P . membranifaciens group, including 25 strains with high DNA homologies to the type strain of P . membranifaciens (72-98%); (II) P . manshurica group, including 18 strains with high DNA homology of 79-95% to the type strain of P . manshurica; and a group including the remaining 6 strains, which had low DNA homology to the above two species . GC content was 42.9-45.3 mol% for the P . membranifaciens group, 40.0-42.0 mol% for the P . manshurica group, and 27.2-44.7 mol% for the remaining group . All three groups had ubiquinone Co Q-7 . Of the 6 anomalous strains, IFO 0162 was identified as Pichia deserticola, and IFO 0839 and IFO 0840 were identified as Issatchenkia occidentalis; but IFO 0842, IFO 0843, and IFO 1788 were thought to be unknown strains. J Biol Chem, 2000 Jun 23, 275(25), 18933 - 8 Functional characterization of the Candida albicans MNT1 mannosyltransferase expressed heterologously in Pichia pastoris; Thomson LM et al.; The alpha1,2-mannosyltransferase gene MNT1 of the human fungal pathogen Candida albicans has been shown to be important for its adherence to various human surfaces and for virulence (Buurman, E . T . , Westwater, C., Hube, B., Brown, A . P . J., Odds, F . C., and Gow, N . A . R . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 7670-7675) . The CaMnt1p is a type II membrane protein, which is part of a family of proteins that are important for both O- and N-linked mannosylation in fungi and which represent a distinct subclass of glycosyltransferase enzymes . Here we use heterologous expression of CaMNT1 in the methylotrophic yeast Pichia pastoris to characterize the properties of the CaMnt1p enzyme as an example of this family of enzymes and to identify key amino acid residues required for coordination of the metal co-factor and for the retaining nucleophilic mechanism of the transferase reaction . We show that the enzyme can use both Mn(2+) and Zn(2+) as metal ion co-factors and that the reaction catalyzed is specific for alpha-methyl mannoside and alpha1,2-mannobiose acceptors . The N-terminal cytoplasmic tail, transmembrane domains, and stem regions were shown to be dispensable for activity, whereas truncations to the C-terminal catalytic domain destroyed activity without markedly affecting transcription of the truncated gene. J Biol Chem, 2000 Jun 30, 275(26), 19560 - 6 Use of six chimeric proteins to investigate the role of intramolecular interactions in determining the kinetics of carnitine palmitoyltransferase I isoforms; Jackson VN et al.; The two isoforms of carnitine palmitoyltransferase I (CPT I; muscle (M)- and liver (L)-type) of the mitochondrial outer membrane have distinct kinetic characteristics with respect to their affinity for one of the substrates (l-carnitine) and the inhibitor malonyl-CoA . Moreover, they differ markedly in their hysteretic behavior with respect to malonyl-CoA and in their response to changes in the in vivo metabolic state . However, the two proteins are 62% identical and have the same overall structure . Using liver mitochondria, we have previously shown that the protein is polytopic within the outer membrane, comprising a 46-residue cytosolic N-terminal sequence, two transmembrane segments (TM1 and TM2) separated by a 27-residue loop, and a large catalytic domain (also cytosolic) (Fraser, F., Corstorphine, C . G., and Zammit, V . A . (1997) Biochem . J . 323, 711-718) . We have now conducted a systematic study on six chimeric proteins constructed from combinations of three linear segments of rat L- and M-CPT I and on the two parental proteins to elucidate the effects of altered intramolecular interactions on the kinetics of CPT activity . The three segments were (i) the cytosolic N-terminal domain plus TM1, (ii) the loop plus TM2, and (iii) the cytosolic catalytic C-terminal domain . The kinetic properties of the chimeric proteins expressed in Pichia pastoris were studied . We found that alterations in the combinations of the N-terminal plus TM1 and C-terminal domains as well as in the N terminus plus TM1/TM2 pairings resulted in changes in the K(m) values for carnitine and palmitoyl-CoA and the sensitivity to malonyl-CoA of the L-type catalytic domain . The changes in affinity for malonyl-CoA and palmitoyl-CoA occurred independently of changes in the affinity for carnitine . The kinetic characteristics of the M-type catalytic domain and, in particular, its malonyl-CoA sensitivity were much less susceptible to influence by exchange of the other two segments of the protein . The marked difference in the response of the two catalytic domains to changes in the N-terminal domain and TM combinations explains the previously observed differences in the response of L- and M-CPT I to altered physiological state in intact mitochondria and to modulation of altered lipid molecular order of the mitochondrial outer membrane in vivo and in vitro. J Biol Chem, 2000 Jul 14, 275(28), 20985 - 95 A dynamic model for bilirubin binding to human serum albumin; Petersen CE et al.; Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding . A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution . The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin . The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species . Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species . The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined . The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform . In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin. Eur J Biochem, 2000 Apr, 267(8), 2276 - 82 Determinants of substrate specificity of a second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis; Hussein AS et al.; We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis . Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite . As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met) . A recombinant form of AChE C was highly expressed by Pichia pastoris . The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate . A double mutation (W302F/W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10-fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the 'peripheral site' inhibitor propidium iodide . In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase-specific inhibitor iso-OMPA and displaying a similar profile of excess substrate inhibition as the double mutant . These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs. J Mol Endocrinol, 2000 Apr, 24(2), 157 - 64 Biological activity of single chain chorionic gonadotropin, hCGalphabeta, is decreased