Acidic cysteine proteinase inhibitor in seminal plasma

A cysteine proteinase inhibitor with acidic isoelectric point (pI = 4.7-5.0) was found in human seminal plasma. Its apparent molecular mass is 16 kDa. It inhibits cysteine proteinases like ficin, cathepsin H, cathepsin B and papain. The inhibitory activity of seminal plasma against ficin is almost the same as that of human serum.     

Assay of alpha-cysteine proteinase inhibitor in serum or plasma

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.     

Characterization and amino acid sequence of a new acidic cysteine proteinase inhibitor (cystatin SA) structurally closely related to cystatin S, from human whole saliva

A cysteine proteinase inhibitor (designated as cystatin SA) was isolated from human whole saliva by procedures including chromatography on DE 32 and DEAE-Sepharose CL-6B. The amino acid sequence determined by conventional methods showed sequence homology of 90 and 87% as compared with the sequences of cystatin S and cystatin SN, respectively, both of which are salivary inhibitors characterized previously. The new inhibitor consisted of 117 residues and had a pI value of 4.3. Cystatin SA inhibited ficin and papain more strongly than cystatin S or cystatin SN did. It also exhibited inhibitory activity toward dipeptidyl peptidase I but the activity was much weaker than those toward ficin and papain.     

The preparation and properties of ficin chemically attached to carboxymethylcellulose

1. Purified ficin has been coupled to four CM-celluloses by reaction with their acid azide derivatives. Insoluble products containing 1.8-4.7mg. of ficin/100mg. of product and retaining 8.0-12.0% of the free enzyme's esterase activity have been obtained. 2. The amount of bound ficin in these preparations is dependent on the degree of carboxymethyl substitution of the CM-cellulose to which the ficin is attached. 3. A shift of the alkaline limb of the pH-activity curve of ficin when chemically attached to CM-cellulose has been shown. 4. Only a small loss has been observed in the enzymic activity of these products when stored at 2 degrees for 4 months. They are more resistant than free enzyme to heat denaturation. 5. Columns of CM-cellulose-ficin have been packed. The degree of hydrolysis of perfused substrate has been measured for different flow rates through the column. 6. The properties of these derivatives have been discussed.     

An endogenous inhibitor of thiol protease from adult lung fluke Paragonimus miyazakii

An endogenous inhibitor of thiol protease from adult Paragonimus miyazakii was found to occur during the gel filtration on Sephacryl S-300. The partially purified inhibitor was specific for thiol proteases such as ficin and papain. The inhibitor also suppressed tosyl-L-lysine alpha-naphthylester hydrolytic activity of Paragonimus thiol protease. The molecular weight of the inhibitor was found to be 430,000 by the gel filtration. This inhibitor was thermostable and resistant to trypsin and glycosidase digestions.     

Proteolysis and the diurnal decrease of asparaginase activity

The mechanism responsible for the decrease in asparaginase (EC 3.5.1.1) activity in darkened leaves of Pisum sativum L. cv. Little Marvel was investigated. Asparaginase activity, obtained from half-expanded leaves harvested at the end of the dark period, or during the light periods, was inactivated by bromelain (EC 3.4.22.4), ficin (EC 3.4.22.3), both thiol proteases, and trypsin (EC 3.4.21.4), a serine protease. Thrombin (EC 3.4.21.5), pepsin (EC 3.4.23.1), or carboxypeptidase A (EC 3.4.17.1) had no effect on dark- or light-harvested asparaginase preparations. Inactivation of asparaginase activity in crude or purified preparations by ficin was not observed in the presence of leupeptin (an inhibitor of thiol proteases). Supplying leupeptin to detached half-expanded leaves had no effect on the increase of asparaginase observed at the start of the light period, while it maintained asparaginase activity at high levels in leaves excised during or at the end of the light period. These results suggest that decreased asparaginase activity in vivo is brought about by thiol-dependent proteases.     

Serological studies with the ficin-antificin system.

Rabbit antisera prepared against ficin reacted with it in a series of serologic tests. Upon immunodiffusion analysis, ficin was found to consist of at least nine antigenic components. Ficin will adsorb spontaneously to erythrocytes which can be used in passive hemagglutination tests. The enzymatic activity of ficin was not abolished by antificin sera.     

Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki =3.3nM) and baupain (Ki=2.1x10(-8)M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.     

Purification of ficin by affinity chromatography

The sulfhydryl proteinase ficin (EC 3.4.4.12) was purified by chromatography on an agarose-mercurial column. Two separate protein fractions were eluted, ficin and mercurificin, both exhibiting enzymatic activity upon activation by excess thiol.     

The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors

Native gamma-trace, a small basic protein present in high concentration in cerebrospinal fluid, semen and neuroendocrine cells, but of unknown biological function, is shown to be a potent inhibitor of the cysteine proteinases papain, ficin, and human cathepsins B, H and L. It proves to be the tightest -binding protein inhibitor of cathepsin B so far discovered. The name cystatin C is proposed for gamma-trace to reflect the many similarities in activity and structure to chicken egg-white cystatin and mammalian cystatins A and B. The inhibition constants of cystatin C, taken together with its widespread distribution in human tissues and extracellular fluids, suggest that a physiological function could well be the regulation of cysteine proteinase activity.     

Characterization of a new cysteine proteinase inhibitor of human saliva, cystatin SN, which is immunologically related to cystatin S

A new cysteine proteinase inhibitor, cystatin SN, was purified from human whole saliva by chromatography with DE32, Sephacryl S200, and CM-Sepharose CL6B. Cystatin SN is immunologically related to cystatin S and both inhibitors have a similar molecular mass of about 13 kDa. The new inhibitor, however, was clearly distinguished from cystatin S by its much higher pI value. These inhibitors showed similar inhibitory activity for ficin, but cystatin SN was a much better inhibitor for papain and dipeptidyl peptidase I. The amino acid sequence of cystatin SN deduced in the light of the known structure of cystatin S indicates that they have 10 different amino acid residues in the sequence comprising in total 113 residues.     

Cystatin S: a cysteine proteinase inhibitor of human saliva

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.     

Naturally occurring inhibitors of intracellular proteinases

The papain inhibitor isolated from chicken egg white inhibits the enzymatic activity of cathepsin B1 and cathepsin C. The inhibitor bears two nonoverlapping reactive sites: one binds cathepsin B1, papain, ficin, and bromelain, the other one cathepsin C. The inhibitor decreases the degree of an immunologic hypersensitive reaction, the so-called Arthus reaction. A statistically significant inhibition of this immunologically developed inflammation occurs only if the inhibitor is applied intradermally and simultaneously with the provoking dose of the antigen to rabbits sensitized to the same antigen. The pepsin inhibitor from the body walls of the roundworm Ascaris lumbricoides inhibits the proteolytic activity of cathepsin E. This inhibitor covalently bound to Sepharose 4B was used for affinity chromatography of cathepsin E. A cathepsin D inhibitor was isolated from potato tubers and its inhibitory and chemical characteristics were studied. The inhibitor does not inhibit either cathepsin E or pepsin yet inhibits trypsin in the alkaline pH-range. The molecular weight of the inhibitor is 21 790 and its molecule consists of 199 amino acid residues. The sequence of 17 amino acid residues was determined by Edman degradation of the inhibitor molecule.     

Resolution of non-protein amino acids via the enantioselective hydrolysis of their esters mediated by sulfhydryl proteases

Summary The esterase activity of sulfhydryl proteases, i.e., papain, ficin, and bromelain, has been utilized for the resolution of a number of non-protein amino acids.     

Protease inhibitors from Streptomyces violascens

Summary Two protease inhibitors from the culture fluid of Streptomyces violascens U 10600 have been purified with a method including freeze-drying, methanol extraction, dialysis, and ultrafiltration. By gel filtration on Sephadex G-15 a separation in two active inhibitors, one of trypsin and one of chymotrypsin, was made.The inhibitors were stable at 100°C, pH 5, for 20 min and at 24°C between pH 1.8 to 9.7. Both inhibitors were dialysable. They had no bacteriostatic or fungistatic effects. The trypsin inhibitor inhibited also papain and proteases from Aspergillus oryzae, Alternaria tenuissima, Entomophthora coronata, and to some extent Gibberella fujikuroi, but not chymotrypsin, kallikrein, ficin, or pepsin. The chymotrypsin inhibitor inhibited also papain and proteases from Aspergillus oryzae, Alternaria tenuissima, and Gibberella fujikuroi, but not trypsin, kallikrein, ficin, pepsin, or protease from Entomophthora coronata.     

A novel cysteine protease inhibitor with lectin activity from the epidermis of the Japanese eel Anguilla japonica

A novel cysteine protease inhibitor (Eel-CPI-1) was isolated from the epidermis of the eel. Eel-CPI-1 was shown to bind strongly to both lactose- and carboxymethylated papain-affinity gels. Its molecular mass under reducing condition was determined to be 18 kDa by SDS-polyacrylamide gel electrophoresis but approximately 30.5 kDa under non-reducing-conditions. Eel-CPI-1 inhibited papain (K(i)=18 nM) and ficin (K(i)=120 nM) competitively. Combined with the data on amino acid and sequence analysis, Eel-CPI-1 is identical to the eel lectin, AJL-2. This is the first report describing a cysteine protease inhibitor with lectin activity.     

Purification and characterization of an inducible cysteine proteinase inhibitor from submandibular glands of isoproterenol-treated rats

A low-molecular-weight protein, induced in rats following prolonged isoproterenol treatment, has been purified from rat submandibular glands by chromatography on columns of Sepharose CL-6B, DEAE-Sepharose CL-6B, and Sephadex G-75. The purified protein is homogeneous based on gel electrophoresis and Ouchterlony double diffusion. The molecular weight of the purified protein was 14,000 and 15,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Superose 12 column, respectively. This protein contains 31% glutamic acid/glutamine and aspartic acid/asparagine, 3.6% cysteine, and 2.5% proline. This protein is shown to be an inhibitor of several cysteine proteinases, papain and ficin being inhibited very strongly in approximately 1:1 molar ratio of enzyme to inhibitor. The protein is not detected in normal rat tissues but is induced in submandibular and sublingual glands even after 1 day of isoproterenol treatment of rats as early as 7 days after birth. Based on cysteine proteinase inhibitor activity, molecular size, and chemical composition this protein appears to belong to the cystatin superfamily.     

Purification and characterization of a low molecular mass cysteine proteinase inhibitor from human amniotic fluid

We have purified the human low molecular mass cysteine proteinase inhibitor in good yield from amniotic fluid, using ultrafiltration through 100-kDa and 1-kDa cut-off filters, chromatography on Ultrogel AcA 54, and affinity chromatography on alkylated papain-agarose. Approximately 1-4 mg/l of this inhibitor are present in amniotic fluid. The purified inhibitor had an apparent molecular mass of 10.5-12 kDa, as judged by its electrophoretic behavior. Amino acid analysis showed it to be rich in acidic and aliphatic residues and in cysteine. No carbohydrate side-chains could be demonstrated. The purified inhibitor inhibited papain, ficin, cathepsins B, C, and H, the cathepsin B-like enzyme from B16 melanoma cells, and a bovine chromaffin granule enkephalin-converting activity. No inhibition of Ca2-dependent neutral cysteine proteinase, serine- or metallo-proteinases was seen. Analysis of the purified inhibitor by isoelectric focusing revealed 7 major bands with pI values of 7.95, 7.0, 6.7, 6.55, 6.25, 5.5, and 5.2, all of which inhibited papain.     

Purification and characterization of inhibitor against the cysteine proteinase of Rana catesbeiana

1. Inhibitors of cysteine proteinase were found in tadpole tail of metamorphosing bullfrog. 2. One of the inhibitors was purified by affinity chromatography with CM-papain agarose, gel filtration with Superose 12 and ion exchange chromatography with Mono S. 3. The molecular weight of the inhibitor was 130,000-140,000 and the isoelectric point was pH 9.6. 4. The inhibitor had inhibitory effects on ficin, papain and tadpole tail cysteine proteinase. 5. The inhibitor is possibly involved in the regulation of muscle degradation in tail regression of metamorphosing tadpole.     

Quantitative structure-activity relationships of cysteine hydrolases. Ficin hydrolysis of X-phenyl-N-methanesulfonyl glycinates

The hydrolysis of ficin of 33 X-Phenyl-N-methanesulfonyl glycinates has been studied. The resulting Km-values have been used to derive a quantitative structure-activity relationship (QSAR). The QSAR for ficin is compared with QSAR for other cysteine hydrolases. The comparisons show that although there are specific differences, overall the reaction mechanisms are very similar.