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Acute intoxication of selected embryo-larval stages of M. galloprovincialis within successive 8-hr exposure intervals by elevated zinc and cadmium seawater concentrations, indicated stage- and metal-specific variations appearing in different proportions in normally developed straight-hinge veliger larvae. Zinc was found to be markedly more toxic than cadmium, particularly within the period of early cleavage and blastula stages. Embryos undergoing tissue differentiation and formation of the trochophora stage were substantially more sensitive regarding both metals tested in comparison with later veliger stages. Combined cadmium-zinc toxicity within each exposure interval tested appears to be less than additive, possibly associated with a markedly higher level of induced MT. The ability of developing embryos to concentrate seawater zinc (CF65Zn = 399) was approximately 2.5 times higher than for cadmium (CF109Cd = 160), which may also contribute to the observed difference in their tolerance levels

Developing eggs of Mytilus galloprovincialis were exposed to elevated concentrations of sea water cadmium, zinc and mercury, 48 hr post-fertilization, in order to study the toxic effects at the morphological and biochemical level comparatively. Decreasing order of metal toxicity (Hg > Zn > Cd) was based on the proportion of normally formed straight-hinge veliger larvae and upon the growth inhibition of the primary larval shell. Metal-induced proteins isolated from homogenate extract of the straight-hinge veliger larvae by Sephadex G-75 chromatography were considered to belong to the metallothionein class due to their high metal content as well as according to their chromatographic and spectral properties. The inverse relationship between relative toxicity level and amounts of total and induced metal bound to MT has been found to be described by the function of a potency type. Combined exposure to zinc and cadmium simultaneously, resulted in an antagonistic effect, showing a larger proportion of normally formed veliger larvae and a smaller growth inhibition in comparison with the sum of single metal effects. The induction of MT in larvae subjected to cadmium and zinc mixture was markedly larger than the addition of individual metal effects.

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.

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.

Adult mussels ( Mytilus galloprovincialis ) (shell length 5–7 cm) exposed to elevated concentrations of Cd (0.1–1.3 mg l −1 ), in a flowing seawater aquarium under laboratory conditions, responded by elevated synthesis of metal-binding proteins in a digestive gland. Response depended on the Cd concentration level as well as on duration of the exposure time. Results obtained show that inducible proteins (isolated by gel-filtration column chromatography and characterized by electrochemical and spectrophotometric methods) should be considered metallothionein-like proteins. The induced metal-binding proteins of mussels in specific organs or tissues are considered to be a potential biological indicator of metal pollution, taking into account specific qualities of the mussel response to different metals. The advantages of the analytical methods applied (gel-chromatography and polarography) and the relevance of the laboratory data when transferred to environmental conditions are discussed. Previously reported results dealing with metal-binding proteins of Mytilus species are also summarized.

Affinity chromatography has been successfully applied as a purification step in the procedure for isolation of Aspergillus niger and Claviceps purpurea carboxyl proteinases. The purification procedure includes ultrafiltration, affinity chromatography on pepstatin-aminohexyl-Sepharose 4B and gel filtration. Some enzymatic properties of both enzymes were determined.

Cathepsin B, H and S were purified simultaneously from bovine spleen by a method involving acid extraction, acetone fractionation, gel chromatography on Sephadex G-50, covalent chromatography on thiol Sepharose 4B and chromatography on CM - cellulose. All three enzymes were isolated in electrophoretically pure forms. The enzymes exist in multiple forms. Molecular weight, substrate specificity, effect of inhibitors and some other enzymatic properties are presented.

Some properties of cathepsin B and a-N-benzoylarginine-~- naphthylamide (BANA) hydrolase from bovine lymph nodes have been studies. a-N-benzoylarginine-~-naphthylamide was a sensitive substrate for both enzymes. Leucine-2-naphthylamide was cleaved only by BANA hydrolase. Degradation of low molecular weight substrates was optimal at pH = 6.0. At this pH value, the enzymes were most stable. Cathepsin B inactivated aldolase, was inhibited by 1 μM leupeptin and by thiol blocking compounds. BANA hydrolase was not inhibited by 1 μM leupeptin but showed that it required thiol compounds and EDTA for full activation. It was concluded that BANA hydrolase is very similar or identical to cathepsin H from rat liver lysosomes.

Cathepsin D (E.C. 3.4.23.5) is a carboxyl proteinase existing intracellularly in many animal tissues and is considered to be one of the major enzymes in lysosornes, responsible for protein breakdown within the cell. It has been suggested that cathepsin D plays an important role in muscular dystrophy (1), and in the pathology of connective tissue diseases, particularly rheumatoid arthritis (2). This enzyme is responsible for the major part of the breakdown of proteoglycans in cartilage degradation (3,4), as well as for the autolytic degradation of cartilage (5,6). Cathepsin D could be also responsible for the breakdown of basic myelin protein forming erctphalytogenic fragments (7). Finally, this enzyme may play a significant role in cardiac pathology (8) and tumour growth process (9).

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).

Some five years ago Keilova (1) reported on low molecular weight substrates and inhibitors of cathepsin D. In the light of the recently introduced technique of affinity chromatography, her data became interesting again. A low molecular weight peptide inhibitor containing a D-amino acid could serve as a ligand in the isolation of cathepsin D and other acid proteinases.

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.

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.

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.

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.

Acid proteinases were isolated from calf lymph nodes using acid extraction, ammonium sulphate and acetone precipitation, followed by ion exchange chromatography on CM-cellulose and _ gel chromatography on Sephadex G-100. Cathepsin D (E. C. 3.4.23.5) is present in lymph nodes. It has the molecular weight of 39 000 as determined by gel filtration on Sephadex G-100. Cathepsins Bl and B2 (E. C. 3.4.22.1) were also isolated. Molecular weights of 22 000 and 51 000 were determined for cathepsins Bl and B2, respectively. Calf lymph nodes contain also another proteinase which degrades haemoglobin at an optimum at pH = 3.0. This proteinase has a molecular weight of 14 000 as was determined by gel filtration. Its activity is not inhibited with 0,25 ~tM pepstatin which inhibits 903/o of the cathepsin D activity.

Intracellular proteinases cathepsins D, E, B and S were purified from different animal organs using procedures that involved affinity chromatography. The final preparation appeared homogeneous. Their enzymatic properties were determined. The secondary structure of these enzymes and their interactions with corresponding inhibitors were studied by circular dichroism (CD).

A method was developed for the isolation of cathepsin D by affinity chromatography on immobilized pepstatin. This inhibitor was coupled to agarose by water soluble carbodiimide. Further purification included gel filtration on Sephadex G-100. The obtained cathepsin D exists in three active forms which were resolved on DEAE cellulose. Electrophoresis in the presence of sodium. dodecyl sulphate revealed that the first form consists of only one polypeptide chain having molecular weight 42 000. The second and the third form contain also polypeptides having molecular weight 27 000 and 14 000.

Aspartic proteinases were isolated from culture filtrates of Aspergillus niqer and Claviceps purpurea using ultrafiltration, affinity chromatography on immobilized pentapeptide inhibitor pepstatin and gel filtration. Some biochemical characteristics of both enzymes were determined.

Publisher Summary This chapter focuses on the purification, characterization, and structural studies of bovine spleen cathepsins D and S. It is known that lysosomal proteases play an important role in the normal turnover of tissue proteins. The chapter presents a modified purification procedure for bovine spleen cathepsin S together with some of its basic properties. Cathepsin B activity was determined using α- N -benzoyl-DL-arginine-2-naphthyl-amide (BANA) as substrate. Cathepsin S belongs to the thiol endopeptidases. This enzyme, originally isolated from calf lymph nodes, can be found in similar quantity also in bovine spleen. The specificity of cathepsin S was studied using different protein substrates. Preliminary CD spectra of cathepsin S in the near ultraviolet region show entirely different patterns compared to cathepsin D, which is in accord with its inability to bind pepstin.

Autolysis studies of cathepsin D show that this process occurs at acid pH during an incubation period of several days. As a result the polypeptide chain of molecular weight of 42,000 was partially cleaved immediately to chains of molecular weight of 28,000 and 14,000. Prolonged incubation resulted in the disappearance of 42,000 and 14,000 polypeptide chains. The enzyme activity was also decreased. Structural and denaturation studies using circular dichroism and fluorescence show similarities with pepsin. There is an indication of the possible existence of a precursor of cathepsin D, or an endogenous inhibitor - cathepsin D complex.

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.

Calf thymus was used for the investigation of intracellular proteinases. The tissue was homogenized, centrifuged at a low speed and applied on DEAE-cellulose. Active fractions were collected and their proteolytic activity toward different protein substrates was tested. It was found that several proteinases are present in thymus; cathepsin D being the most abundant acid proteinase. The activitiy at neutral pH was ascribed to fibrinogen degrading proteinases.

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