Electrochemical formation and investigation of a self-assembled [60]fullerene monolayer.

The formation and characterisation of a C(60) monolayer at the electrode|electrolyte interface has been studied by cyclic voltammetry, potential step chronoamperometry and ac voltammetry. The presence of the monolayer is evidenced by the presence of a very sharp peak P in the voltammogram, attributed to the faradaic phase formation of an ordered monolayer, and of a reduction post peak Q associated with the reduction of adsorbed species. The chronoamperograms exhibit a well-defined maximum, characteristic of a nucleation and growth mechanism. By comparison with existing models of phase transitions, a progressive polynucleation and growth mechanism is demonstrated. The monolayer is proposed to consist of a 2D fulleride salt. It is suggested that the formation of the monolayer can take place for a broad range of solution compositions, but requires an atomically smooth substrate such as mercury.

The open reading frame YAL048c affects the secretion of proteinase A in S. cerevisiae.

Over-expression of the yeast PEP4 gene encoding the vacuolar aspartic protease proteinase A (PrA) leads to saturation of the vacuolar targeting system of the cell and missorting of PrA to the growth medium. In a screen for genes affecting the secretion of over-expressed PrA we found that multiple copies of the open reading frame (ORF) YAL048c enhanced PrA secretion. Since no function has hitherto been ascribed to YAL048c, we undertook further studies of this ORF. Deletion of YAL048c resulted in slightly reduced secretion of over-produced PrA. Furthermore, strains deleted for YAL048c showed a growth inhibition phenotype resulting in wrinkled colony morphology when grown on rich medium containing high concentrations of calcium. YAL048c is predicted to encode a polypeptide of 662 amino acid residues containing two consensus ATP/GTP-binding site motifs and a putative carboxy-terminal transmembrane region. In addition, the amino acid sequence contains two putative calcium-binding domains. The YAL048c protein may be evolutionarily conserved, as homologues exist in humans and Caenorhabditis elegans. We suggest that the YAL048c protein is involved in vesicle transport in the secretory pathway.

Heterologous expression of human cholecystokinin in Saccharomyces cerevisiae. Evidence for a lysine-specific endopeptidase in the yeast secretory pathway.

Precursors of the human regulatory peptide cholecystokinin (CCK) have been expressed in Saccharomyces cerevisiae, and the post-translational processing of secreted CCK-related products analyzed. Recombinant plasmids expressing native human prepro-CCK and a hybrid molecule encompassing the prepro leader of the yeast alpha-mating pheromone fused to pro-CCK were examined. The latter construct resulted in considerably higher levels of pro-CCK secretion and was therefore analyzed in more detail. Two of the protein modifications essential for CCK bioactivity, C-terminal alpha-amidation and tyrosyl sulfation, were not detected in S. cerevisiae. Proteolytic cleavage of pro-CCK occurred C-terminally of three basic sites; (i) Arg105-Arg106 which, upon exposure to carboxypeptidase activity, leads to the production of glycine-extended CCK; (ii) Arg95 to produce CCK-8 related processing intermediates; and (iii) Lys81 resulting in CCK-22 related products. To elucidate which protease(s) are involved in these endoproteolytic cleavage events, pro-CCK was expressed in yeast mutants lacking various combinations of the Mkc7, Yap3, and Kex2 proteases. Only in S. cerevisiae strains deficient in Kex2 function was any of the above mentioned pro-CCK cleavages abolished, namely processing at the Arg105-Arg106 and Arg95 sites. This suggests that mammalian Kex2-like serine proteases may process pro-CCK at single arginine residues. Our data suggests that an as yet uncharacterized endopeptidase(s) in the S. cerevisiae secretory pathway is responsible for the lysine-specific cleavage of pro-CCK.

Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A.

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.

Antithrombotic effect of recombinant truncated tissue factor pathway inhibitor (TFPI1-161) in experimental venous thrombosis--a comparison with low molecular weight heparin.

The aim was to investigate whether a truncated recombinant Tissue Factor Pathway Inhibitor (TFPI1-161), which lacked the third Kunitz-type domain and the basic c-terminal region, had an antithrombotic effect comparable to LMWH in a randomised double-dummy study. The experimental thrombosis was induced in jugular veins, in a total of 40 rabbits by a combination of destruction of the endothelium and restricted blood flow. Group 1: placebo, gr 2: LMWH 60 anti-FXa IU/kg, gr 3-5: 0.1, 1.0 and 10.0 mg/kg TFPI1-161. TFPI1-161 reduced the thrombus weights in all treated groups, significantly in doses of 1.0 and 10.0 mg/kg compared to placebo. The frequency of thrombosis and occlusive thrombosis were also significantly reduced in those doses. The antithrombotic properties of TFPI1-161 (1.0-10.0 mg/kg) measured as thrombus weight, frequency of thrombosis and frequency of occlusive thrombosis was equivalent to the anti-thrombotic properties of LMWH. In the anti-FXa, APTT and PT-assays TFPI1-161 displayed a dose dependent increase of activity. Recombinant-TFPI1-161 did not influence the anti-FIIa-assay. No haemorrhagic side effects were noted.

Pharmacokinetics of full length and two-domain tissue factor pathway inhibitor in combination with heparin in rabbits.

Tissue factor pathway inhibitor (TFPI) is a feed back inhibitor of the initial activation of the extrinsic pathway of coagulation. In humans, injection of heparin results in a 2-6 fold increase in plasma TFPI and recent studies suggest that TFPI may be important for the anticoagulant activity of heparin. Full length (FL) TFPI, but not recombinant two-domain (2D) TFPI, has a poly cationic C-terminus showing very strong heparin binding. Therefore, we have investigated if heparin affects the pharmacokinetics of TFPI with and without this C-terminus. FL-TFPI (608 U/kg) and 2D-TFPI (337 U/kg) were injected intravenously in rabbits with and without simultaneous intravenous injections of low molecular weight heparin (450 anti-XaU/kg). Heparin decreased the volume of distribution and the clearance of FL-TFPI by a factor 10-15, whereas the pharmacokinetics of 2D-TFPI were unaffected by heparin. When heparin was administered 2 h following TFPI the recovery of FL-TFPI was similar to that found in the group receiving the two compounds simultaneously, suggesting that the releasable pool of FL-TFPI is removed very slowly in the absence of circulating heparin.

Characterization of human tissue factor pathway inhibitor variants expressed in Saccharomyces cerevisiae.

Human tissue factor pathway inhibitor (TFPI) and three derivatives with deletions of: 1) the complete COOH-terminal third of the polypeptide including the third Kunitz domain, 2) the third Kunitz domain alone, or 3) the penultimate basic COOH-terminal region alone were expressed in yeast as secreted products. High expression yield was obtained only with the derivative that lacked both the third Kunitz domain and the penultimate COOH tail (TFPI1-161). The purified short form was heterogeneously glycosylated with a high mannose glycan. The specific activities of the different mutant polypeptides toward FXa.tissue factor.FVIIa in a chromogenic assay were similar to that of TFPI expressed in baby hamster kidney cells, suggesting that correct folding takes place in yeast and that neither the third Kunitz domain nor the COOH-terminal region is required for this activity. However, in a clotting assay the anticoagulant activities of yeast-produced TFPI and the shortened derivative TFPI1-161 were about 5- and 50-fold lower, respectively, than for full-length TFPI from mammalian cells. Clotting assays with purified short form TFPI showed that it acted mainly via inhibition of FVIIa.tissue factor rather than FXa. The anticoagulant activity of short form TFPI was comparable with that of high affinity antibodies toward tissue factor.

Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces.

Human tissue factor pathway inhibitor (TFPI) is a plasma protease inhibitor that consists of three tandem Kunitz-type inhibitor domains flanked by a negatively charged NH2 terminus and a positively charged COOH-terminal tail. Previous studies have shown that the first and second Kunitz-type domains in TFPI are involved in the inhibition of factor VIIa and factor Xa activity, respectively. In the present study, we have compared the inhibitory properties of full-length recombinant TFPI and a truncated form of TFPI lacking the third Kunitz-type domain and COOH-terminal tail (TFPI1-161) with respect to inhibition of factor VIIa-tissue factor complexes on the surface of a human bladder carcinoma cell line J82. Full-length TFPI and TFPI1-161 were kinetically indistinguishable with respect to neutralization of the proteolytic activity of preformed complexes of factor VIIa-tissue factor on the J82 cell surface in the absence of factor Xa. Equimolar amounts of factor Xa augmented the anticoagulant activity of both preparations of TFPI to the same extent, and both preparations of TFPI were equally effective in inhibiting factor VIIa-tissue factor amidolytic activity in solution phase. In addition, plasma concentrations of both forms of TFPI, in stoichiometric complex with factor Xa, inhibited cell surface factor VIIa-tissue factor proteolytic activity markedly faster than plasma levels of antithrombin III, even in the presence of 1 unit/ml heparin. The results of displacement studies suggested slight differences in the affinity of the two TFPI molecules for the cell surface in that approximately 5% of a VIIa.TF.Xa.TFPI1-161 quaternary complex on J82 cells was displaceable from the cell surface by high concentrations of factor VIIa (10-100 nM), whereas only 1-2% of a VIIa.TF.Xa.TFPI complex was displaceable under comparable conditions. Pretreatment of the cells with TFPI/Xa alone or together with R152E factor VII, followed by factor VIIa treatment, revealed significant differences in the two TFPI forms with respect to the degree with which offered factor VIIa could restore factor X activation on the cell surface. These differences notwithstanding, our collective findings indicate that the third Kunitz-type domain and/or COOH-terminal tail of TFPI is not essential for the inhibition of cell surface factor VIIa-tissue factor complexes and suggests that TFPI1-161 may be a useful therapeutic agent in the treatment of thromboembolic episodes.

Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

Regulated overproduction and secretion of yeast carboxypeptidase Y.

Carboxypeptidase Y (CPY) is a glycosylated yeast vacuolar protease used commercially for synthesis of peptides. To increase the production of CPY in Saccharomyces cerevisiae we have placed its coding region (PRC1) under control of the strongly regulated yeast GAL1 promoter on multicopy plasmids and introduced the constructs into vpl1 mutant strains. Such mutants are known to secrete CPY. High levels of CPY production were obtained by induction of the GAL1 promoter when the cells had left the exponential phase, resulting in a growth-phase-dependent CPY production similar to that of cells with PRC1 under the control of its own promoter. Introduction of a high copy number 2 mu-URA3-LEU2d plasmid with GAL1p-PRC1 fusion in a vpl1 strain resulted in a 200-fold increase of secreted CPY (about 40 mg/l) as compared to a vpl1 mutant carrying a single copy of the wild-type PRC1 gene. The overproduced, secreted CPY was active and had the normal N-terminal sequence. Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed two forms of active CPY, probably due to different levels of glycosylation.

Molecular genetics of serine and threonine catabolism in Saccharomyces cerevisiae.

The catabolic L-serine (L-threonine) deaminase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. A mutant, cha1 (catabolism of hydroxyamino acids), lacking this enzyme activity has been isolated. We have cloned the CHA1 gene by complementation of a cha1 mutation. Northern analysis showed that CHA1 mRNA has a size of about 1200 ribonucleotides. CHA1 is probably the structural gene for the enzyme; it is an abundant RNA in cells grown with serine and threonine as nitrogen source, whereas it is not detected when cells are grown on ammonium or proline, i.e., the transcription of the CHA1 gene is induced by serine or threonine. Under induced growth conditions haploid ilv1 CHA1 strains do not require isoleucine, i.e., the catabolic deaminase is able to substitute for the biosynthetic threnonine deaminase encoded by the ILV1 gene. We have identified a nuclear, recessive mutation, sil1, that suppresses ilv1 mutations by increased transcription of the CHA1 gene under growth conditions leading to partial induction. The sil1 mutation could exert its effect by increasing the effective pools of the hydroxyamino acids. Alternatively SIL1 may encode a negatively acting regulatory protein for CHA1.

Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae.

The threonine deaminase gene (ILV1) of Saccharomyces cerevisiae has been designated "multifunctional" since Bollon (1974) indicated its involvement both in the catalysis of the first step in isoleucine biosynthesis and in the regulation of the isoleucine-valine pathway. Its role in regulation is characterized by a decrease in the activity of the five isoleucine-valine enzymes when cells are grown in the presence of the three branched-chain amino acids, isoleucine, valine and leucine (multivalent repression). We have demonstrated that the regulation of AHA reductoisomerase (encoded by ILV5) and branched-chain amino acid transaminase is unaffected by the deletion of ILV1, subsequently revealing that the two enzymes can be regulated in the absence of threonine deaminase. Both threonine deaminase activity and ILV1 mRNA levels increase in mutants (gcd2 and gcd3) having constitutively depressed levels of enzymes under the general control of amino acid biosynthesis, as well as in response to starvation for tryptophan and branched-chain amino acid imbalance. Thus, the ILV1 gene is under general amino acid control, as is the case for both the ILV5 and the transaminase gene. Multivalent repression of reductoisomerase and transaminase can be observed in mutants defective in general control (gcn and gcd), whereas this is not the case for threonine deaminase. Our analysis suggests that repression effected by general control is not complete in minimal medium. Amino acid dependent regulation of threonine deaminase is only through general control, while the branched-chain amino acid repression of AHA reducto isomerase and the transaminase is caused both by general control and an amino acid-specific regulation.

Towards diacetyl-less brewers' yeast. Influence of ilv2 and ilv5 mutations.

During alcoholic fermentations, the off-flavour compound diacetyl is formed non-enzymatically from acetolactate leaking out from the cells. Acetolactate is an intermediate in the biosynthesis of valine. In beer fermentation, the amount of diacetyl is reduced to acceptable levels during maturation. A reduction of the time needed for maturation may be achieved by the use of a brewing yeast that produces less diacetyl. Saccharomyces cerevisiae laboratory strains with an inactive ilv2 gene can not form acetolactate, while ilv5 strains, blocked in the subsequent step, leak acetolactate in high amounts. Induction of recessive mutations in production strains of Saccharomyces carlsbergensis has not yet been achieved, as the yeast is polyploid and possibly a hybrid between S. cerevisiae and another Saccharomyces species. Thus, all chromosomes investigated so far are present in at least two genetically different versions. Genetic and molecular analysis has shown that the brewing yeast is structurally heterozygous for ILV2 and ILV5. Genetic modification of brewers' yeast to reduce diacetyl formation is being carried out by mutation of ILV2. Deletion mutations in both ILV2 alleles have been constructed in vitro to be used for gene replacement in the brewing strain. In addition, partial inactivation of the ILV2 function is carried out by selecting spontaneous dominant mutations resistant to the herbicide sulfometuron methyl. Among these mutants some produce only half the amount of diacetyl compared to the parental strain. An alternative way to reduce diacetyl production might be to increase the activity of the ILV5 gene product. Model experiments in S. cerevisiae show that the presence of the ILV5 gene on a 2-micron based multi-copy vector can reduce the diacetyl production by half.

The ILV5 gene of Saccharomyces cerevisiae is highly expressed.

The nucleotide sequence of the yeast ILV5 gene, which codes for the branched-chain amino acid biosynthesis enzyme acetohydroxyacid reductoisomerase, has been determined. The ILV5 coding region is 1,185 nucleotides, corresponding to a polypeptide with a molecular weight of 44,280. Transcription of the ILV5 mRNA initiates at position -81 upstream from the ATG translation start codon and terminates between 218 and 222 bases downstream from the stop codon. Consensus sequences have been identified for initiation and termination of transcription, and for general control of amino acid biosynthesis, as well as repression by leucine. The ILV5 gene is regulated slightly by general amino acid control. Codon usage of the ILV5 gene has the strong bias observed in yeast genes that are highly expressed. In agreement with this, the reductoisomerase monomer, with an apparent molecular weight of 40,000, has been identified in an SDS polyacrylamide gel pattern of total soluble yeast proteins as a gene dosage dependent band.