A basic residue at position 36p of the propeptide is not essential for the correct folding and subsequent autocatalytic activation of prochymosin.

Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. & Yada, R.Y. (1999) Eur. J. Biochem. 261, 746-752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptide-enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.

The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors.

High levels of protease inhibitors are induced in potato leaves by wounding. These inhibitors, when ingested by Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, induce expression of specific proteolytic activities in the gut. Induced protease activities cannot be inhibited by potato inhibitors and thus enable the insects to overcome this defence mechanism of potato plants. The induced aminopeptidase and endoproteolytic activities both have the characteristics of cysteine proteases. Twenty-one protein inhibitors of different structural types have been examined for their ability to inhibit these activities in vitro. Members of the cystatin superfamily were found to be poor inhibitors of the induced endoproteolytic activities, except for the third domain of human kininogen, which was a fairly strong inhibitor (75% inhibition). The strongest inhibition (85%) of induced endoproteolytic activity was obtained using structurally different thyroglobulin type-1 domain-like inhibitors--equistatin and MHC class II-associated p41 invariant fragment. Experiments performed using three synthetic substrates for endoproteases gave similar results and indicate the existence of at least different endoproteolytic enzymes resistant to potato inhibitors. The induced aminopeptidase activity can be inhibited only by stefin family of inhibitors in cystatin superfamily. In in vivo experiments, Colorado potato beetle larvae fed on equistatin-coated potato leaves were strongly retarded in their growth and almost 50% died after 4 days. This demonstrated the potential of equistatin to protect crops from insect attack.

Potato cysteine proteinase inhibitor gene family: molecular cloning, characterisation and immunocytochemical localisation studies.

Potato cysteine proteinase inhibitors (PCPIs) represent a distinct group of proteins as they show no homology to any other known cysteine proteinase inhibitor superfamilies, but they all belong to the Kunitz-type soybean trypsin inhibitor family. cDNA clones for five PCPIs have been isolated and sequenced. Amino acid substitutions occurring in the limited regions forming loops on the surface of these proteins suggest a further classification of PCPIs into three subgroups. Accumulation of PCPI was observed in vacuoles of stems after treatment with jasmonic acid (JA) using immunocytochemical localisation, implying that these inhibitors are part of a potato defence mechanism against insects and pathogens. Genomic DNA analysis show that PCPIs form a multigene family and suggest that their genes do not possess any introns.

Jasmonic acid inducible aspartic proteinase inhibitors from potato.

A new cDNA clone coding for an aspartic proteinase inhibitor homologue was isolated from a potato tuber cDNA library. Southern blot analysis was used to study the structural diversity of the aspartic proteinase inhibitor gene family in several species of the Solanaceae. The existence of sequence-homologous genes was confirmed in the genomic DNA of different potato cultivars (Solanum tuberosum L. cv. Désirée, Pentland Squire and Igor), tomato (Lycopersicon esculentum Mill.), aubergine (S. melongena L.) and a wild type of bittersweet (S. dulcamara L.). Northern blot hybridization of total RNA, isolated from leaves under non-stress conditions, of different solanaceous species and of potato tubers showed that the gene transcripts encoding aspartic proteinase inhibitors occur mainly in potato tubers. The presence of several cathepsin D inhibitor isoforms has been detected at the protein level. At least four isoforms were isolated by affinity chromatography on cathepsin D-Sepharose and characterized. Additionally, exogenous treatment of potato plantlets by jasmonic acid (JA) over a wide range of concentrations (0-100 microM) was performed in a stem node culture in vitro. We demonstrated that the expression of aspartic proteinase inhibitor mRNA was drastically induced in potato shoots at concentrations of 50-100 microM JA.

Interactions of mercury in rat brain.

In order to study the metabolism of mercury (Hg), its affinity to metallothionein (MT), and its influence on levels of the essential metals copper and zinc in the brain tissue of rats exposed to elemental mercury (HgO) vapor was investigated. The major findings were: 1. After long-term exposure, about 40% of mercury was found in the brain water-soluble phase (supernatant); 2. In brain supernatant, about 80% of Hg was found in the range of low-molecular-weight proteins; the MT-like protein Hg-Cu-Zn-thionein was isolated and partially characterized; 3. HgO vapor exposure resulted in increased tissue levels of essential Cu and Zn in addition to exogenous Hg; and 4. Experiments showed that HgO vapor exposure can induce the stimulation of rat brain MT synthesis.

Isolation and sequence analysis of the genomic DNA fragment encoding an aspartic proteinase inhibitor homologue from potato (Solanum tuberosum L.).

A genomic DNA clone encoding an aspartic proteinase inhibitor of potato was isolated from a lambda EMBL3 phage library using the aspartic proteinase inhibitor cDNA as a hybridization probe. The gene has all characteristic sequences normally found in eucaryotic genes. Typical CAAT and TATA box sequences were found in the 5'-upstream region. In this part are also two putative regulatory AGGA box sequences located. In the genomic sequence there are no intron sequences interrupting the coding region. An open reading frame of the gene encodes a precursor protein of 217 amino acids which shows high percent identity with the aspartic proteinase inhibitor cDNA.

Proteinases of various Claviceps purpurea strains.

Extra- and intracellular proteinases from various Claviceps purpurea strains grown in submerged culture have been studied. A maximum level of intracellular proteinases was observed on the 6th day of culture growth, whereas extracellular activity continued to increase throughout the culture growth. Proteinases were purified and characterized. The ergotamine strain secreted one aspartic and two serine proteinases, whereas from the disrupted mycelium only the aspartic proteinase could be isolated. The ergocornine strain secreted the aspartic proteinase in two forms and the ergocristine strain produced an aspartic and a serine proteinase.

Aspartic proteinases: their activation and structural studies.

Besides extracellular mammalian aspartic proteinases also intracellular proteinase cathepsin D is synthesized in the form of a precursor. The evidence is presented that cathepsin D zymogen (cathepsinogen D, procathepsin D) can be activated by a similar mechanism to that of pepsin, releasing an activation segment - peptide(s). The released peptide(s) show inhibitory activity towards cathepsin D and some other aspartic proteinases. The activation peptides released from bovine pepsinogen do not inhibit cathepsins D and E. The structure of different aspartic proteinases was studied by circular dichroism measurements. The binding of pepstatin causes conformational changes in the near UV CD spectrum.

Bovine intracellular cysteine proteinases.

Cathepsins B, H and S were isolated from bovine lymph nodes and bovine spleen. It was shown that the incubation of homogenate at 37 degrees C at acid pH increased the total BANA hydrolase activity and LeuNA activity, whereas it decreased the total activity of cathepsin S. All three enzymes are electrophoretically homogeneous and probably composed of a single polypeptide chain. They exist in multiple forms as shown by isoelectric focusing. Far UV CD spectra revealed a rather high percentage of unordered structure. The three cysteine proteinases were inhibited by thiol blocking reagents, leupeptin and by an inhibitor isolated from Vipera ammodytes venom. Results on the specificity toward various substrates and the influence of pH on enzymatic activity are presented.

The existence of a precursor of cathepsin D: evidence from autolysis, denaturation and activation studies.

This report evidences that the single polypeptide chain of cathepsin D undergoes in vitro autolysis resulting in heavy (Mr about 30000) and light (Mr about 15000) polypeptide chains. These two chains are held together through non-covalent interaction, thus constituting a stable active conformation. Fluorescence and circular dichroism measurements demonstrate the irreversible denaturation of cathepsin D. The existence of cathepsin D precursor, cathepsinogen D, of about 50000 molecular weight was proved. Cathepsinogen D is converted to the active enzyme by intramolecular activation, releasing activation-inhibitory peptide(s).

Studies on bovine spleen cathepsin D.

The purification procedure of cathepsin D which includes autolysis results in the destruction of the molecule to smaller polypeptide chains. Pure catepsin D obtained by the method which includes affinity chromatography, contains single polypeptide chain of 42000 daltons. The N-terminal amino acid is glycine. The specificity was studied using synthetic substrates. CD measurement of cathepsin D shows mainly unordered structure, about 26% of beta-structure and only 5% of alpha-helix. Binding of pepstatin shows pronounced changes in the CD spectrum between 250 and 300 nm; above 7.5 no interaction was observed.

Inactivation studies of cathepsin D with diazo compounds.

Cathepsin D was inactivated with various diazo compounds at very high concentration. Reaction proceeded maximally at pH 4.5 in the presence of cupric ions. With 3-diazo-indazole and triazene the inactivation was noted also in the absence of cupric ions what indicates that the mechanism is mediated through triazene. CD-spectrum of partially inactivated enzyme shows that conformational changes occurred after treatment with diazo compound.