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.     

Protease (PrA and PrB) and prolyl and arginyl aminopeptidase activities from Debaryomyces hansenii as a function of growth phase and nutrient sources

The effects of nutrient sources and growth phase of Debaryomyces hansenii on the protease (PrA and PrB) and aminopeptidase (prolyl-[PAP] and arginyl-[AAP] aminopeptidases) activities were investigated. These activities were also monitored during growth on a whole sarcoplasmic muscle protein extract (WSPE) and on an equivalent medium but free of compounds under 10 kDa (SPE>10 kDa). The levels of specific protease and aminopeptidase activities were higher when cells were grown in urea and dipeptides than when grown in either ammonium or free amino acids as nitrogen sources. The level of each aminopeptidase (PAP or AAP) activity was preferentially induced by its own substrate (ProLeu or LysAla), suggesting a role in the utilization of exogenous peptides. Higher specific activities for all proteolytic enzymes were detected when using acetate as carbon source. The time course experiments carried out on urea or sarcoplasmic protein-containing media revealed an increase in all activities during transition and advanced stages of stationary phase of growth. In muscle protein extracts, the absence of low molecular mass nutrients (SPE>10 kDa) initially induced the production of PrA, PrB, and AAP activities, possibly involved in the breakdown of muscle oligopeptides.     

The shock of vacuolar PrA on glycolytic flux,oxidative phosphorylation,and cell morphology by industrial <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> WZ65

Proteinase A (PrA) is one of the most significant vacuolar proteinase in S. cerevisiae, and it plays an important role in S. cerevisiae physiology and metabolism, especially under unfavorable environment. In this study, the differences in pyruvate kinase (PYK) level under fructose-1,6-diphosphate (FDP) induction and ATP synthesis block among SC1 (the wild-type yeast that was industrial Saccharomyces cerevisiae WZ65), SC2 (PEP4 partial deletion) and SC3 (PEP4 complete deletion) were examined. Results showed that the induction caused by FDP clearly increased PYK expression no matter for which strain, but the increasing effect is more significant for SC2 (P < 0.05). The comparative results of intracellular ATP accumulation showed that the induction by FDP may be affected at the presence of PrA. The block experiment of ATP synthesis showed that PYK activities in PEP4-modified strains are lower than that of the wild type, but the intracellular ATP levels in the wild-type one are generally higher than the PEP4-modified strains after rotenone treatment (P < 0.01). This implies that the effect of PrA deficiency on intracellular ATP accumulation was much more pronounced than the effect of rotenone on oxidative phosphorylation. The cell morphology of three strains was comparatively examined by means of transmission electron microscopy (TEM). The PEP4-modified strains possessed more vacuoles, and cell structure were more integrated than the wild-type strain. Current data preliminarily indicated that the deletion of PEP4 gene in industrial S. cerevisiae WZ65 may not only affected PYK expression but also modulated the oxidative phosphorylation flux.     

Effect of PrA encoding gene-<Emphasis Type="Italic">PEP4</Emphasis> deletion in industrial <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">cerevisiae</Emphasis> WZ65 on key enzymes in relation to the glycolytic pathway

S. cerevisiae proteinase A (PrA) is a member of the aspartic proteinase superfamily. The roles of PrA in S. cerevisiae physiology and cell metabolism are controversial. The objective of the current study was to elucidate the effects of the absence of PrA on key enzymes with regard to the glycolytic flux in industrial S. cerevisiae. The observed activities of hexokinase (HK), phosphofructokinase (Pfk) and pyruvate kinase (PYKi) in PrA-modified S. cerevisiae strains (SC2 and SC3) were lower than that of the wild-type strain (p < 0.01). Compared to other strains, SC3 revealed the lowest activity of three key glycolytic enzymes. Current results imply that PrA in industrial S. cerevisiae may control the glycolytic enzymes expression (HK, Pfk and PYKi) in the direct or indirect manner and thus lead to the delay of cell metabolism. The observed intracellular ATP levels between the wild-type strain and PrA-modified strains (SC1 and SC2) were significantly different (p < 0.01). The pronounced differences of key glycolytic enzymes between the wild-type and PEP4-modified strains were as well characterized by SDS–PAGE. The intracellular protein concentration in the presence of PrA is higher than those of the PrA-modified strains. As a result, the interaction mode of PrA and the glycolytic enzymes was postulated in this work. The findings herein suggest that the glycolytic flux direction and rate may be regulated by vacuolar PrA in industrial S. cerevisiae. The present data obtained provide insights into understanding the roles of PrA in industrial S. cerevisiae.     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

The proteolytic system of the yeast Kluyveromyces lactis.

Major proteolytic activities were characterized in the yeast K. lactis NRRL 1118, grown in chemostat cultures. This yeast expressed proteolytic activities similar to those found in S. cerevisiae. This fact was particularly evident in the case of proteases such as PrA, PrB and CpY with regard to substrate specificity, activation at pH 5. 0 and inhibition patterns. The presence of a CpS activity could not be detected in either fresh or activated cell-free extracts by using the dipeptide N-Cbz-Gly-Leu, even in the presence of Zn(+2). On the other hand, K. lactis exhibits at least two major intracellular Ap activities different from those reported in other yeasts, and these seem to be carried out by closely related proteins. These activities corresponded to molecular masses of about 60 kDa, close pI values, and a similar behaviour in non-denaturing polyacrylamide electrophoresis. Both activities were enhanced by Co(+2) and inhibited by EDTA. Among different aminoacyl-p-NAs, they preferentially hydrolysed Lys-p-NA. No increase of Ap activity was obtained by incubation of extracts at acid pH. The maximum PrA and PrB activities detected in N-limited cultures were six-fold higher than those expressed under C- or P-limitation. The effect of culture conditions on the Cp and Ap expression was much less pronounced in comparison with PrA and PrB activities, Ap levels even being slightly higher in C-limited cells. This fact suggests that hydrolysis of protein to peptides might be the limiting step in the pathway of general protein degradation in the vacuole.     

The structure and function of Saccharomyces cerevisiae proteinase A

Saccharomyces cerevisiae proteinase A (saccharopepsin; EC 3.4.23.25) is a member of the aspartic proteinase superfamily (InterPro IPR001969), which are proteolytic enzymes distributed among a variety of organisms. Targeted to the vacuole as a zymogen, its activation at acidic pH can occur by two different pathways, a one-step process to release mature proteinase A, involving the intervention of proteinase B, or a step-wise pathway via the autoactivation product known as pseudo-proteinase A. Once active, S. cerevisiae proteinase A is essential to the activities of other yeast vacuolar hydrolases, including proteinase B and carboxypeptidase Y. The mature enzyme is bilobal, with each lobe providing one of the two catalytically essential aspartic acid residues in the active site. The crystal structure of free proteinase A reveals that the flap loop assumes an atypical position, pointing directly into the S(1) pocket of the enzyme. With regard to hydrolysis, proteinase A has a preference for hydrophobic residues with Phe, Leu or Glu at the P1 position and Phe, Ile, Leu or Ala at P1', and is inhibited by IA(3), a natural and highly specific inhibitor produced by S. cerevisiae. This review is the first comprehensive review of S. cerevisiae PrA.     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Construction of recombinant industrial <Emphasis Type="Italic">S. cerevisiae</Emphasis> strain with barley lipid-transfer protein 1 secretion capability and lower PrA activity

Beer barley LTP1 in beer is an important component of beer foam, and it participates in the formation of beer foam. The digestion of beer barley LTP1 by proteinase A from brewing yeast leads to the decline of beer foam stability, especially for the unpasteurized beer. The objective of this study was to construct an industrial brewing yeast strain to secrete recombinant barley LTP1 into fermenting wort during beer fermentation for the foam stability improvement. We constructed barley LTP1 expression cassette and transformed into the host industrial yeast cells to replace partial PEP4 alleles using homologous recombination method. The expression of b-LTP1 was under control of the constitutive yeast ADH1 promoter, and the concentration of recombinant barley LTP1 secreted by recombinants reached 26.23 mg/L after incubation in YEPD medium for 120 h. The PrA activity of the recombinant strain declined compared with the host strain. The head retention of beer brewed with the recombinant industrial strain (326 ± 12 s) was improved when the host strain WZ65 (238 ± 7 s) and the constructed strain S.c-P-1 (273 ± 10 s) with partial PEP4 gene deficiency were used as control. The present study may provide reference for brewing industries and researches on beer foam stability.     

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.     

Vacuole Segregation in the Saccharomyces cerevisiae vac2-1 Mutant: Structural and Biochemical Quantification of the Segregation Defect and Formation of New Vacuoles

The conditional vacuolar segregation mutant vac2-1 [Shaw and Wickner (1991) EMBO J. 10, 1741–1748] shifted to non-permissive temperature (37°C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole. Some cells still contain normal segregation structures. Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37°C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion. Thus, 10–15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell. To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping. The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37°C. Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles. Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min. Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate. The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding. © 1997 by John Wiley & Sons, Ltd.     

Construction of self‐cloning industrial brewer's yeast with SOD1 gene insertion into PEP4 prosequence locus by homologous recombination

Superoxide dismutase (SOD, encoded by SOD1), which can scavenge active oxygen free radicals, is an ideal endogenous antioxidase in beer. In this study, the SOD1 expression cassette was constructed, and this cassette contained the PGK1 promoter, the PGK1 terminator and the SOD1 gene fused to the signal sequence of the yeast mating pheromone α‐factor (MFα1_s). One of the prosequences of the PEP4 gene (encoding proteinase A, PrA) in Saccharomyces cerevisiae strain S‐6 was replaced by the SOD1 expression cassette via homologous recombination and the self‐cloning strain S54PS, which could improve the antioxidant capability and foam stability of beer, was successfully obtained. Fermentation results showed that the SOD activity of the final beer brewed with S54PS was increased by 21.06%. Accordingly, the DPPH‐radical scavenging activity of S54PS increased by 30.6% compared with that yielded by the parental strain S‐6. Furthermore, the PrA activity of S54PS was always lower than that of the parental strain at all stages of beer fermentation. The head retention of the beer (255 ± 4 s) was better than that of the parental strain (224 ± 1 s). Hence, this research implies that S54PS exhibits good brewing performance and can be applied to improve the industrial brewing process. Copyright © 2016 The Institute of Brewing & Distilling     

Purification and characterisation of proteases A and D from Debaryomyces hansenii

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.     

Construction of the mutant strain in Aspergillus oryzae 3.042 for abundant proteinase production by the N+ ion implantation mutagenesis

Because of secreting large amounts of enzymes, Aspergillus oryzae was widely used in the fermentation of soy sauce in many Asian countries. Here, N⁺ ion implantation mutagenesis was applied to improve the ability of A. oryzae to secrete acid protease. Several mutants were screened using three kinds of plates (Casein medium, Czapek’s medium and carboxymethyl cellulose medium agar plates). Compared with the other mutants, the mutant A100‐8 could secrete the most protease. Acid protease activity of A100‐8 was enhanced about 44.1% at 36 h by koji fermentation test. In addition, A100‐8 was stable by periodically subculturing and maintaining on the agar slant. The mRNA expression levels of two kinds of acid protease (serine carboxypeptidase and aspartyl protease) were measured by RT‐PCR at different periods. Serine carboxypeptidase and some kinds of aspartyl protease of A100‐8 were significantly (P < 0.01) expressed higher than the control at all times.     

Vacuolar functions are involved in stress-protective effect of intracellular proline in Saccharomyces cerevisiae

Proline protects yeast cells from damage caused by various stresses. A yeast Saccharomyces cerevisiae mutant with high levels of intracellular proline grown in a minimal medium accumulated proline in its vacuole, but when grown in a nutrient medium, accumulated proline mainly in the cytosol. To understand the role of the proline pool in the vacuole, we examined the stress-protective effect of proline in proline-accumulating yeast cells deficient in vacuolar functions. The disruption of PEP3 encoding a vacuolar membrane protein required for vacuolar biogenesis caused hypersensitivity to heat shock and ethanol stresses, probably due to disappearance of normal vacuoles. The vph1-disrupted cells lacking vacuolar-ATPase activity showed resistance to heat shock without any change in proline localization, but showed severe growth defects in an ethanol-containing medium. These results indicate that vacuolar functions are involved in the stress-protective effect of proline in S. cerevisiae. Also, it appears that excess proline is transported to the vacuole in an ATP-independent manner.     

Saccharomyces cerevisiae mutants altered in vacuole function are defective in copper detoxification and iron-responsive gene transcription

The metal ions, Cu^2+/+ and Fe^3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu²⁺ for high affinity transport of Fe²⁺ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly or acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe²⁺ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution or redox states of Fe^3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions. © 1997 John Wiley & Sons, Ltd.     

SECONDARY STRUCTURE OF SOME ASPARTYL PROTEINASES

A structural study of some aspartyl proteinases was undertaken. Secondary structure prediction methods indicate that chymosin, pepsin, penicillopepsin and Mucor miehei proteinase have relatively high proportions of β-sheet with active site aspartic acid residues located in β-turn regions. Secondary structure determination from far-UV CD spectral data support the above finding that the aspartyl proteinases have a high proportion of β-sheet. The proportion of β-sheet generally decreased at pH values greater than 6.3. More extensive unfolding occurred with pepsin and penicillopepsin than chymosin, Mucor miehei proteinase, Mucor pusillus proteinase and Endothia parasitica proteinase in the neutral to alkaline pH range. Results obtained from the near-UV CD spectra of the aspartyl proteinases indicate a change in spectra in the neutral to alkaline pH range which suggests the importance of aromatic groups to tertiary structure stability.     

Protease B from Debaryomyces hansenii: purification and biochemical properties

The protease B (PrB; EC. 3.4.21.48) of Debaryomyces hansenii CECT 12487 was purified by selective fractionation with protamine sulfate followed by three chromatographic separations. The whole procedure resulted in 324-fold purification with a recovery yield of 1.0%. PrB was active at neutral-basic pH ranging from 6.0 to 12.0 with an optimum at pH 8.0. The molecular mass of the denatured enzyme was 30 kDa. Polyclonal-antibodies raised against PrB from Saccharomyces cerevisiae cross-reacted with the corresponding 30-kDa protein from D. hansenii. The serine protease inhibitor 3,4-DCI and sulphydryl group reagents markedly reduced the enzyme activity. The Km against N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was 1.79 mM. The presence of endogenous inhibitor for PrB was detected in cell-free extracts of D. hansenii although their inhibitory effect was lost after incubation at 25 degrees C for 20 h. PrB was able to hydrolyze muscle sarcoplasmic proteins by in vitro assays. This is the first endopeptidase purified and characterized from the yeast D. hansenii, whose possible contributions to meat fermentation processes are discussed.     

Construction of a Single PEP4 Allele Deletion in Saccharomyces carlsbergensis and a Preliminary Evaluation of Its Brewing Performance

The secretion of proteinase A (encoded by PEP4) from brewer's yeast is detrimental to the foam stability of unpasteurized beer. The aim of this study was to construct mutants of the allopolyploid Saccharomyces carlsbergensis strain TT, which were partially or completely deficient in proteinase A activity. Allelic PEP4 genes were consecutively disrupted by using the Cre‐loxP recombination system combined with PCR‐mediated gene disruption. A single PEP4 deletion mutant TT‐M was successfully constructed. However, no viable mutant could be obtained when the second allelic PEP4 gene was deleted. The brewing performances of the parent strain and the modified strain were compared on a 100 L pilot fermenter scale. Proteinase A activity in fermented wort brewed with mutant strain TT‐M was significantly lower (p<0.05) than that of the parent strain TT, whereas no significant difference on either maltose or maltotriose assimilation (p>0.05) was found. The mutant TT‐M remained genetically stable, as shown by diagnostic PCR, after re‐streaking for 20 generations. The flavor and taste of the final fermented wort, brewed with the mutant strain TT‐M, was evaluated by the Tsingtao expert sensory panel, and found to be comparable to that of the parent strain and exhibited no distinct defects. The flavor component profiles of these two finished products were also comparable. The study demonstrated allelic genes in polyploid industrial yeasts could be efficiently and consecutively deleted by the retractive primer disruption strategy, and the mutant of Saccharomyces carlsbergensis partially deficient in proteinase A contributed to an improvement in foam stability.     

Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have been characterized using antiserum raised against this enzyme. A 42 kDa pro-enzyme form of AXP was identified from lysates of radiolabelled Y. lipolytica cells and found to contain no N-linked carbohydrate moieties. Using pulse-chase immune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where, under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of the AXP pro-form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly-catalysed reaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalytic processing came from the side-chain specificity of mature AXP towards the oxidized B-chain of insulin. © 1998 John Wiley & Sons, Ltd.