Proteolytic enzymes of Neurospora crassa. Purification and some properties of five intracellular proteinases.

Five intracellular proteolytic enzymes from Neurospora crassa were isolated and partially characterized: an acidic and an alkaline endopeptidase, one carboxypeptidase and two aminopeptidases. All these proteinases were purified from the same crude extract to homogenity by heat treatment, precipitation with ammonium sulfate, chromatography on DEAE-cellulose, CM-cellulose, DEAE-Sephadex, hydroxyapatite and by gel filtration. The acid proteinase hydrolysed acid-denatured haemoglobin at pH 3.0. The alkaline proteinase and the carboxypeptidase are serine proteinases that require a sulfhydryl group for activity. The aminopeptidases are both metallo-proteinases; one posseses broad specifity to the B-chain of oxidized insulin, the other posseses only narrow specifity and can only split the N-terminal basic amino acids of peptides.     

Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutinin-encoding gene of Porphyromonas gingivalis

The obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black-pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N-terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine-specific cysteine proteinase (Arg-gingipain, RGP)-encoding gene ( rgp1 ) and was also present in genes for lysine-specific cysteine proteinases ( prtP and kgp ) and a haemagglutinin ( hagA ) of P. gingivalis . The HGP15 domain protein was purified from an HGP15-overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa haemoglobin-binding protein in the envelope of P. gingivalis. P. gingivalis wild-type strain showed pH-dependent haemoglobin adsorption, whereas its non-pigmented mutants that produced no HGP15-related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis .     

Acid proteinases in various species of cellular slime mold

The proteolytic activities of the cellular slime moldsDictyostelium mucoroides, Dictyostelium purpureum, Polysphondylium pallidum, andPolysphondylium violaceum have been examined. Myxamoebae possessed activity against Hide Powder Azure at pH 2–5 which was enhanced by dithiothreitol: this enhancement was small inDictyostelium species but three- to four-fold in thePolysphondylium species. Following electrophoresis on polyacrylamide gels containing denatured haemoglobin five or more proteinases could be detected in each species. Activity against Hide Powder Azure was inhibited severely by HgCl₂ and to a lesser extent by other thiol proteinase inhibitors such asN-α-p-tosyl-l-lysine chloromethyl ketone-HCl, antipain, and leupeptin. Inhibitors of aspartyl and serine proteinases had no effect. All proteinases visualized on gels were inhibited by HgCl₂, and some, but not the major one of each species, were sensitive to the other thiol proteinase inhibitors. Extracts of fruiting bodies retained acid proteolytic activity. New proteinases were detected inD. mucoroides; there was a relative increase in one proteinase inP. violaceum but three proteinases were lost during fruiting body formation inP. pallidum. During microcyst formation inP. pallidum there was a decrease in proteolytic activity but most of the myxamoebal proteinases could be detected. Overall the results demonstrate that the cellular slime molds possess similar types of proteinase although there were significant differences between the actual proteinases observed in individual species.     

Characterization of the Proteinases Present in Germinating Seeds of Scots Pine, Pinus sylvestris

Methods were developed to determine proteinase activity in germinating seeds of Scots pine. The assays were based on the liberation of TCA-soluble peptides from haemoglobin at pH 3.7 and from casein at pH 5.4 and pH 7.0; the reaction products were determined by the Lowry method. — Endosperms separated from seeds at the time of rapid storage protein mobilization (seedling length between 20 and 50 mm) showed high proteinase activities in all three assays. Experiments with different inhibitors suggested that at least four enzymes were involved. One of the enzymes resembled mammalian and microbial pepsin-like acid proteinases: the pH optimum was 3.7 and the enzyme was inhibited by pepstatin.—The proteinase activities in the endosperms were high enough to account for the mobilization of the reserve proteins during germination. Moreover the activities at pH 3.7.5.4. and 7.0 in the endosperms were 10-, 25-, and 50-fold the corresponding activities in the growing seedlings (a “reference” tissue). Consequently, it seems that both the acid and neutral proteinases take part in the mobilization of storage proteins in the germinating seed.     

Crithidia guilhermei: gelatin- and haemoglobin-degrading extracellular metalloproteinases

The extracellular metalloproteinases of the insect trypanosomatid Crithidia guilhermei were characterized through the incorporation of different protein substrates (gelatin, casein, haemoglobin, and bovine serum albumin) into SDS-PAGE. Two gelatinases (60 and 80 kDa) showed ability to degrade casein as well and a 67-kDa enzyme presented the broadest specificity since it was also able to degrade casein and haemoglobin. Besides the 67-kDa extracellular proteinases detected on haemoglobin-SDS-PAGE, a 43-kDa haemoglobinase was only observed with this substrate. All C. guilhermei proteinases were incapable of using bovine serum albumin. C. guilhermei was also grown in four different culture media and the best proteinase production was reached using yeast extract-peptone medium containing glucose as the major carbon source. The results point to the importance of the use of distinct culture media and proteinaceous substrates on the characterization of extracellular proteolytic activities in trypanosomatids, since alterations in growth conditions and methods of detection could lead to distinct proteolytic profiles.     

Proteolysis in the axis of the germinating pea seed

Autoproteolytic, caseolytic and haemoglobin degrading activities, carboxypeptidase and aminopeptidase activities have all been measured in extracts prepared from the radicle of germinating pea seeds (Pisum sativum L.). With increasing time from the beginning of imbibition, the spectrum of protein degrading enzyme activities changed in a complex manner. As a proportion of total autoproteolytic activity, acid proteinases declined, while sulphydryl-and serine-active site endopeptidases accounted for increased proportions of the total activity. The distribution of protein degrading enzyme activities in the root tip compared with the balance of the root was determined after 4 days, at the onset of cell division in the root apex. On a fresh weight basis the tip was enriched ca. 2-fold in protein concentration and all of the exopeptidases. Autoproteolytic activity was concentrated in the tip to a lesser degree, and haemoglobin degrading activity not at all. In contrast, the root tip was depleted in caseolytic activity.Abbreviations AP aminopeptidase - BSA bovine serum albumin - CP carboxypeptidase - DAN diazoacetyl-D, L-norleucine - ME 2-mercaptoethanol - NEM N-ethyl maleimide - PMSF phenyl methyl sulphonyl fluoride - TCA trichloroacetic acid     

Degradation and inactivation of ceruloplasmin and haptoglobin by rat liver lysosomes and some other proteinases

Lysosomal fraction was isolated from rat liver by density gradient centrifugation after pervious loading of lysosomes in vivo with Triton WR-1339. Tritosome preparations were incubated at 37 degrees C and pH 5 for 24 hr with purified human ceruloplasmin or haptoglobin. After this period approximately 20% of total alpha amino nitrogen was released from ceruloplasmin and over 40% from haptoglobin. This was accompanied by loss of peroxidase activity of haptoglobin (in complex with haemoglobin), while enzymatic activity of ceruloplasmin remained unaltered. Removal of sialic acid by neuraminidase had no effect on digestion of ceruloplasmin by rat liver tritosomes. Both glycoproteins were resistant to horse leucocyte proteinases and pancreatic eleastase but were easily inactivated by trypsin and chymotrypsin.     

Pepstatin-sensitive proteolytic activity of sorghum seeds

Two novel proteinases were isolated from resting sorghum seeds and purified 100-fold. The activity of the purified enzymes was completely inhibited by pepstatin A and was unaffected by PMSF, leupeptin, EDTA and E-64 (L-trans-epoxysuccinyl leucylamino 4 guanidino butane), which indicates that they belong to the class of aspartic proteinases. SDS-PAGE and native-PAGE revealed a monomeric 29-kDa enzyme and a heterodimeric 61-kDa enzyme with two S-S linked subunits of 49 and 12 kDa. The proteases have maximum activity at 45 °C and pH 3.5, with haemoglobin as substrate. Activity at 60 °C is higher than at 30 °C.     

Aspartic proteinases in fishes and aquatic invertebrates

1. The literature on molecular properties and physiological role of aspartic proteinases in fishes and aquatic invertebrates has been reviewed. 2. Pepsins have not been detected in invertebrates, and apparently cathepsin D, as well as other cathepsins, act both as digestive and lysosomal enzymes in many of these animals. The molecular properties of invertebrate cathepsin D correspond with cathepsin D in fishes and mammalians. 3. Fishes with a true stomach have pepsinogen secretion. Fish pepsins have higher pH optimum and are less stable in strong acid conditions than mammalian pepsins. They are very efficient at low temperatures, but less thermostable than mammalian pepsins. 4. Many fishes have two significantly different pepsins: Pepsin I and Pepsin II, which digest haemoglobin at a maximal rate in the pH ranges 3-4 and 2-3 respectively. Usually the pI of Pepsin I is in the range 6.5-7, whereas pI of Pepsin II is about 4. 5. Fish Pepsin I and cathepsin D have very similar molecular properties, and a hypothesis proposing that cathepsin D is the ancestor enzyme of aspartic proteinases in higher animals is presented.     

Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine

To elucidate the mechanism of hydrolysis of fish muscle proteins by fish proteinases in fish sauce production, each pure preparation of three alkaline proteinases and two acid proteinases from sardine was tested for its ability to hydrolyze various proteins and its stability in the presence of 0 to 25% of NaCl. Each of the alkaline proteinases hydrolyzed casein more rapidly than other proteins. A major alkaline proteinase (III) hydrolyzed sarcoplasmic protein from sardine 5-times faster than other alkaline proteinases. Each of two acid proteinases hydrolyzed hemoglobin and myoglobin more rapidly than the other proteins. After preincubation with 25% NaCl, an alkaline proteinase (III) and an acid proteinase (II) were stable although the other proteinases became unstable. The two proteinases, alkaline proteinase III and acid proteinase II, were also stable for three months after the beginning of fish sauce production. The proteolytic activity of each of alkaline and the acid proteinases was strongly inhibited by more than 15% NaCl; however, minimum inhibition was observed when sardine muscle proteins were used as the substrate.     

Activities of some peptidases and proteinases in germinating kidney bean, Phaseolus vulgaris

The activities of aminopeptidase (EC 3.4.11), dipeptidase (EC 3.4.13), carboxypeptidase (EC 3.4.16), naphthylamidase (EC 3.4.11) and proteinases (EC 3.4.21) were assayed in extracts from the cotyledons and the axial tissues of resting and germinating kidney beans ( Phaseolus vulgaris L. cv. Processor).
The activities of the alkaline peptidases (aminopeptidase hydrolyzing Leu-Tyr at pH 9.2 and dipeptidase acting on Ala-Gly at pH 8.5) and naphthylamidases (hydrolyzing Leu-β-naphthylamide at pH 6.4) were high in the cotyledons of resting seeds, but decreased during germination. This decrease was faster than the loss of the total nitrogen. On the contrary, the activities of carboxypeptidase (hydrolyzing carbobenzoxy-Phe-Ala at pH 5.9) and proteinases (acting on haemoglobin at pH 3.7 and on casein at pH 5.4 and 7.0) were low in the resting seeds, but increased during germination reaching their maximal values when the mobilization of nitrogen was highest. It has been suggested that the breakdown of storage proteins is initiated inside the protein bodies by acid proteinases and carboxypeptidases. Although the activities of the alkaline peptidases and naphthylamidases decreased during germination, these were still relatively high and enough for the completion of the proteolytic breakdown. Thus, it is suggested that, as a final step in a chain of events, the main role for the alkaline peptidases in the cotyledons of germinating seeds is to provide amino acids for the growth of the seedling.     

Studies on the interaction between Streptomyces pepsin inhibitor and several acid proteinases by means of a zinc(II)-dye complex as a probe.

The zinc(II) complex of pyridine-2-azo-p-dimethylaniline is bound to several acid proteinases, at pH 5.0, accompanied by a change is the visible absorption spectrum. Streptomyces pepsin inhibitor, which was discovered by Satoi and Murao (Satoi, S. and Murao, S. (1970) Agric. Biol. Chem. 34, 1265-1267 and Satoi, S. and Murao, S. (1971) Agric. Biol. Chem. 35, 1482-1487), is also bound to acid proteinases. Spectrophotometric studies with ten acid proteinases from different sources have revealed that in several acid proteinases, zinc(II)-pyridine-2-azo-p-dimethylaniline is released from the enzyme by the inhibitor, while some acid proteinase forms a quaternary complex, zinc(II)-pyridine-2-azo-p-dimethylaniline-inhibitor-enzyme. It is speculated that zinc(II)-pyridine-2-azo-p-dimethylaniline is bound to two catalytic carboxylate groups in the active site of the acid proteinases and the inhibitor is bound mainly to the substrate-binding site of the enzymes. The binding of the inhibitor may overlap the catalytic site completely or partially. The degree of overlapping is characteristic of the kind of acid proteinases.     

Aspartyl proteinase from cucumber (Cucumis sativus) seeds. Preparation and characteristics

Aspartyl proteinase (EC 3.4.23) from cucumber seeds was purified by ammonium sulphate fractionation, chromatography on immobilized pepstatin and gel filtration on Sephacryl S-200. The preparation obtained, homogeneous on polyacrylamide-gel electrophoresis in acidic and alkaline media, has a molecular mass of 42,000, pI of 5.2, and shows the highest activity with denatured haemoglobin at pH 3.2. The proteinase is stable in slightly alkaline medium, whereas it is inactivated in acidic medium, especially in the presence of NaCl. The enzyme activity is affected neither by the inhibitors of serine proteinases, sulfhydryl-proteinases and metalloproteinases, nor by divalent metal ions, whereas the enzyme is inactivated by the inhibitors of aspartyl proteinases: 1,2,3-epoxy(p-nitrophenoxy)propane, diazoacetyl-DL-norleucine and pepstatin.     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.     

Localization and characterization of hemoglobin-degrading aspartic proteinases from the malarial parasite Plasmodium falciparum.

Three hemoglobin-degrading proteinases were partially purified from food vacuoles isolated from trophozoite-stage forms of the malarial parasite Plasmodium falciparum. Two of the proteinases (M1 and M2) were solubilized by repeated sonication. The remaining proteinase (M3) was solubilized by treatment of the particulate fraction with taurocholic acid, suggesting that proteinase M3 is a membrane-bound proteinase whereas proteinases M1 and M2 are weakly associated with parasite membrane. The location of these proteinases suggests that they may participate in the digestion of host cytosolic protein. After partial purification, but not before, proteinases M1, M2 and M3 are highly sensitive to pepstatin, supporting their designation as aspartic proteinases. These aspartic proteinases show broad specificity for protein substrates. Native hemoglobin, acid denatured hemoglobin and oxidatively damaged hemoglobin are comparable substrates. Hemoglobin within the food vacuole was shown to be primarily native hemoglobin. Chemical modification studies indicate that these three aspartic proteinases have similar properties. The peptide maps from degradation of hemoglobin, however, suggest that aspartic proteinases M1, M2 and M3 are distinct proteinases.     

Substrate specificity of proteolytic activity in the testes fluid and seminal plasma of the common carp Cyprinus carpio

Substrate specificity in the seminal plasma and testes fluids of the common carp Cyprinus carpio was determined using gelatin, casein, albumin and haemoglobin. Proteolytic profiles of the testes and seminal plasma were compared. Different ranges of pH (5·5–9·5) and temperature (4–37° C) were used during incubations of seminal plasma proteinases. Differences in proteolytic activity between testes and seminal plasma may reflect specific functions of the testes and sperm ducts in semen production. Seminal plasma metalloproteinases were characterized by higher substrate specificity than were serine proteinases. Zymography optimization for seminal plasma indicated that pH 7·5 and 22° C were the optimal conditions for gel incubations.     

Structural characterization of the papaya cysteine proteinases at low pH

Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration.     

Proteolysis of human reticulocyte membrane proteins: Evidence for a physiological role of the acid endopeptidases

Endogenous membrane proteins, labeled by incubating human reticulocytes with l-[¹⁴C]leucine, are degraded at pH 7.3 by membrane-bound acid proteinases. Solubilized membrane proteins are also degraded at neutral pH by the purified membrane acid proteinases. Exogenous proteins are not degraded by intact membranes and therefore association with the membrane seems to be an essential requirement for protein degradation in the physiological pH range. These findings provide evidence for a physiological function of the enzymes previously characterized as acid proteinases.     

Comparative biochemistry of acid proteinase from animal origins

Acid proteinases from 17 tissues of 12 animal species were compared with respect to molecular weight, inhibition by pepstatin and activation by tripolyphosphate. Gel filtration of acid proteinases from protochordates and vertebrates showed a common elution profile and three peaks with mol. wts of -20,000, -45,000 and above 150,000 were detected with acid-denatured hemoglobin as substrate at pH 3.6. The main component of vertebrate acid proteinases was identified as cathepsin D. In the invertebrate acid proteinases, the elution profiles through gel filtration were characteristic to the tissues examined, and were not so distinct as those of vertebrates. Through a biochemical survey, the animal acid proteinase was discussed from a comparative point of view.     

Aspartic proteinases in the digestive tract of marine decapod crustaceans

Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others.