Igor Kregar

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.

Acid sulphydryl protease from calf lymph nodes

Abstract A new sulphydryl acid protease has been isolated from calf lymph nodes. Having a molecular weight of 14 000 ± 1500 , it was separated from cathepsin D (EC 3.4.23.5) on a column of Sephadex G-100. Hemoglobin hydrolysis was optimal at pH 3.0. It was not inhibited by pepstatin. Sulphydryl effectors characteristically influence this activity.

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.

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.

Cellulolytic complex of Aspergillus niger under conditions for citric acid production. Isolation and characterization of two β-(1→4)-glucan hydrolases

SummaryTwo β-(1→4)-endoglucanases have been purified from industrial waste broth of Aspergillus niger grown under conditions which produce citric acid. Molecular weights for endoglucanase A were 43,000 and 25,000 for endoglucanase B. Both enzymes exhibited very similar properties: a rather broad pH optimum between pH 2 and 7 for CM-cellulose hydrolysis and an inability to degrade crystalline cellulose. The endoglucanases have a higher thermal stability at acid pH (up to 60°C) than at alkaline pH. They are inhibited by iodine, HgCl2 and N-bromosuccinimide.

Rapid isolation of cathepsin D by affinity chromatography on the immobilized synthetic inhibitor.

In previous work we have shown, that a rapid isolation of lysosomal proteinase cathepsin D (EC 3.4.4.23) is a prerequisite for obtaining this enzyme in undegraded form [ 11. An immobilized substrate, such as hemoglobin is a suitable affinity ligand for this purpose, although immobilized inhibitors are more commonly used for the isolation of enzymes by affinity chromatography. Renin [2,3] and cathepsin D [4,5] were purified on the affinity column packed with immobilized pentapeptide pepstatin, a strong inhibitor of acid proteinases. Synthetic enanthiomeric substrate analogues are known to be competitive inhibitors of various proteinases [6-81 and they can serve as convenient ligands in affinity chromatography. Bound to Sepharose, they were used for the affinity chromatography of pepsin and acid proteinase from Aspergillus awamori [7]. We have found that synthetic octapeptide Gly-Glu-Gly-Phe-Leu-Gly-DPhe-I_eu is a competitive inhibitor of cathepsin D [9]. This report describes its use as an affinity ligand for the simple and rapid isolation of cathepsin D from tissue extracts in a single or double step procedure.

Amino Acid Sequence of Lamb Preprochymosin and its Comparison to Other Chymosins

Chymosin (EC 3.4.23.4) is one of the most important aspartic proteinases used as a milk-clotting enzyme in cheese production. The primary structures of two calf chymosin forms (A and B) are known.1,2 The only difference in their structures is at position 302 (preprochymosin numbering). More recently, other forms of calf chymosin were also found and sequenced.3–8 Calf chymosin has been cloned in many laboratories using different expression vector/host systems. Recombinant chymosin is comparable to the wild type enzyme in cheesemaking properties.9,10 Like traditionally used calf rennet, lamb rennet has also been utilized in cheese making because it has similar high milk-clotting and low proteolytic activities.11 Recently, we briefly reported on the deduced amino acid sequence of lamb preprochymosin.12

Molecular Cloning and Immunocytochemical Localization of Jasmonic Acid Inducible Cathepsin D Inhibitors from Potato

Proteins which have the ability to inhibit proteolytic activity of certain enzymes are found throughout the plant kingdom. They are usually accumulated as storage proteins in seeds and tubers (1). Many inhibitors of serine (2–4), cysteine (5–8) but only a few inhibitors of aspartic (9) proteinases have been isolated from potato tubers. The protein sequences of two closely related isoinhibitors, PDI (10) and NID (11), isolated from potato tubers, were determined.

Isolation of Cathepsin D by Affinty Chromatography on Immobilized Pepstatin

Affinity chromatography with an immobilized substrate proved to be a successful tool in the preparation of pure and undegraded cathepsin D (1). Immobilized reversible inhibitors are even more suitable for the isolation because the enzyme remains inactive during the affinity chromatography procedure. Pepstatin is known as a strong inhibitor of carboxyl proteases (2) and we wanted to make use of its inhibitory property for the isolation of cathepsin D. In this report, a method for isolation of cathepsin D is described using affinity chromatography on pepstatin-agarose.