A peptidyl derivative of [3H]aniline as a sensitive, stable, protease substrate.

A peptidyl derivative of [³H]aniline, Gly-Gly-Arg-[³H]anilide, can be used as a substrate in a convenient and sensitive assay procedure for trypsin, urokinase, and plasminogen activator from transformed cells. The extent of hydrolysis can be determined simply by selective extraction of the product [³H]aniline into an organic phase containing a scintillant. (The uncleaved peptide is not appreciably soluble in this phase and is not counted.) The reaction is of comparable sensitivity to fluorimetric assays, but has the advantage that no cleanup of the biological sample is required, since it is far less subject to interference from fluorescence quenching. Other peptidyl anilides should be useful for assaying proteolytic enzymes with widely varying specificities.     

Liposome Aggregation: Promoted by Trypsin and Papain in the Absence of Cations

Abstract

Phospholipid vesicle aggregation is usually mediated by phospholipid-binding proteins such as the annexins in a Ca²⁺-dependent manner. Here, we describe aggregation of unilamellar liposomes by trypsin and papain in the absence of cations. Cations including Ca²⁺ inhibited the aggregation. While both trypsin and papain promoted aggregation of liposomes made of phosphatidylcholine and phosphatidylglycerol, only papain elicited aggregation of liposomes made of exclusively phosphatidylcholine. Incubation of trypsin for 30 min at 37°C destroyed its liposome aggregating activity, similar treatment had no effect on papain's. Chymotrypsin and pepsin had no liposome aggregating activity.     

Epidermal growth factor-urogastrone receptor: selective alteration in simian virus 40 transformed mouse fibroblasts

Comparative data on the properties of four thiol proteinase inhibitors, and of four serine proteinase inhibitors (two subtilisin and two trypsin inhibitors) isolated from seeds of Vigna are presented. They were similar in their molecular weights (5000–15,000) and dissociation constants (10^?8–10^?9m). The range of isoelectric points of the thiol proteinase inhibitors was 6.5 to 10.6, and of the serine proteinase inhibitors was 5.0 to 5.9. The amino acid compositions of one papain isoinhibitor, one of subtilisin, and one of trypsin are presented. Papain inhibitor A1 and subtilisin inhibitor 2a were low in cystine. All of the inhibitors were stable upon heating to 80 °C for 5 min at low pH. The subtilisin inhibitor did not bind to catalytically inactive subtilisin derivatives, whereas the papain inhibitor was stoichiometrically bound to the Hg or thioacetamide derivatives of papain. Incubation of the subtilisin inhibitor with catalytic amounts of subtilisin led to the formation of a modified form with the same inhibitor activity as the native inhibitor but with a different electrophoretic mobility. There was no indication of a similar modification of the papain inhibitor by papain. Separate sites are present on the trypsin-chymotrypsin inhibitors for trypsin and chymotrypsin. The papain inhibitors have the same binding sites for papain and ficin.     

Macrophage membrane proteins: Possible role in the regulation of priming for enhanced respiratory burst activity

Brief exposure of macrophages to the proteolytic enzymes papain, elastase, or trypsin primed them for enhanced production of superoxide anion (O₂⁻) in response to stimulation by phorbol myristate acetate (PMA). Priming by trypsin was achieved at 0 °C, at which temperature trypsin functions as a protease but is not internalized, supporting the concept that protease priming depends on modification of the plasma membrane. Analysis of external membrane proteins after radioiodination of intact cells and separation by gel electrophoresis indicated that papain treatment of macrophages resulted in the cleavage of a membrane protein with a molecular weight of approximately 305K. Membranes from macrophages primed by elicitation with Corynebacterium parvum also demonstrated a reduced amount of the membrane protein at approximately 305 kDa, as well as a reduction of a protein at about 270 kDa. Lipopolysaccharideelicited macrophages showed a reduced amount of a protein at about 175 kDa. Continuous spectrophotometric assays of O₂⁻ release from adherent macrophages indicated that after exposure to a stimulus, protease-treated cells produced O₂⁻ more quickly than did control cells (reduced lag time). Inhibitors of protein synthesis augmented the priming effect of papain when added with the protease. These results suggest that protease-induced priming results from inactivation of a membrane protein (or proteins) that exerts a down-regulating effect on the respiratory burst.     

A study of the nascent polypeptides synthesized on the free polyribosomes of rat brain in vivo

The free polyribosomes of the cerebral cortex of the immature rat (12-14 days old) were exposed to very low concentrations of trypsin at 0°C and for very brief periods of time and the conditions under which their breakdown to smaller aggregates occurs were determined. Trypsin also caused the release of nascent, radioactive polypeptides from polyribosomes prelabelled with [¹⁴C]amino acids in vivo. An examination of the kinetics of release of the nascent chains by trypsin revealed that it was dependent on the concentration of trypsin as well as on the duration of incubation in the presence of trypsin. The influence of the nature of the [¹⁴C]amino acid used as precursor of the nascent polypeptides and of the duration of the radioactive pulse in vivo was also determined. The radioactivity associated with polyribosomes as a result of the brief radioactive pulses administered (2 to 10 minutes) was incompletely removed even after the ribosomes were dissociated into subunits by EDTA. These findings suggest that the assembly of the cerebral ribosome in vivo must be a very rapid process, particularly in the immature animal. The nature of the nascent, radioactive polypeptides was studied by disc gel and high voltage electrophoresis and by thin-layer and column chromatography. Evidence was obtained that a rather limited number of qualitatively different molecules resides on the polyribosomes at any given moment.     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.     

Trypsin-induced masking of tetrodotoxin receptor of the sodium channels in mollusc neurones

At the early stage of trypsin treatment of mollusc neurones tetrodotoxin cannot block the Na⁺ current. In the course of further exposure of neurones to trypsin, tetrodotoxin-sensitivity is restored completely, so its temporal loss results from shielding rather than destruction of the tetrodotoxin-binding site. Pronase and papain do not affect the tetrodotoxin action on the Na⁺ current.     

Completion of the amino acid sequence of papain

Papain was inhibited with bromo[2-(14)C]acetic acid, the tertiary structure of the inhibited enzyme was unfolded and the disulphide bridges were reduced with mercaptoethanol and aminoethylated. Digestion with trypsin gave a radioactive peptide consisting of residues 18-58 inclusive and containing therefore the sequence of the thirteen unknown residues 29-41 in the primary sequence of papain. This peptide was digested with pepsin to give a radioactive peptide consisting of residues 18-47, which after digestion with 0.4m-hydrochloric acid gave a radioactive peptide consisting of residues 24-43 inclusive. Further digestion with 6m-hydrochloric acid gave peptides that were used to determine the sequence: Ser-Ala-Val-Val-Thr-Ile-Glx-Gly-Ile-Ile-Lys-Ile-Arg for the residues 29-41, so completing the amino acid sequence of papain.     

Interaction of proflavine with 2-hydroxy-5-nitrobenzylated papain

Interaction of proflavine with papain, modified by hydroxynitrobenzylation of Trp-177 (HNB-papain), is characterized by a dissociation constant about twice that of proflavine's binding to native papain. Kinetic analyses revealed that proflavine's activation of papain-catalyzed hydrolysis of benzoyl-L-arginine ethyl ester persists with the HNB-enzyme; however, hydroxynitrobenzylation of papain precludes proflavine's inhibition of benzoyl-L-arginine p-nitroanilide (BzArgNan) hydrolysis. Yet, proflavine noncompetitively inhibits hydrolysis of BzPheValArgNan by both native and HNB-papain to about the same extent. Thus, proflavine appears to reduce nonproductive binding of smaller substrates, but may also interfere with conformational repositioning necessary for anilide hydrolysis.     

Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling

Crotoxin is a neurotoxic phospholipase A₂ capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin withN-hydroxysuccinimidyl-4-azidobenzoate and with Na[¹²⁵I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin,Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC₅₀ of about 10^–8 M. Mojave toxin, caudoxin, notexin,Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while -bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.On leave from Department of Biochemistry and Biophysics, University of Hawaii School of Medicine, Honolulu, Hawaii.     

A study of the surface proteins of Entomophthora egressa protoplasts and of larval spruce budworm hemocytes

Entomophthora egressa protoplasts either exposed to or not exposed to trypsin were not attacked by either trypsinized or non-trypsinized larval spruce budworm granulocytes. Granulocytes adhered to protoplasts exposed to papain, and this adhesion could be prevented by papainizing the hemocytes. Differences were observed in the responses of two E. egressa isolates when exposed to papain or to the papain-control solutions. Exposure of hemocytes to trypsin did not reduce either the number of Absidia repens sporangiospores per granulocyte or the percentage of granulocytes with spores, whereas, exposure to papain did. The role of surface proteins, particularly glycoproteins, in hemocyte-fungal cell interactions is briefly discussed.     

The effects of biogenic phosphates on proteinase-induced protein cleavage and functioning of plasminogen activator

We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001–0.06 M) enhances by 50–250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2–12.0 times enhances protein lysis by trypsin, α-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40–50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10^?8–10^?1 M enhanced the cleavage of a number of proteins by serine proteinases and, at concentrations of 10^?5–10^?3 M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of >10^?3 and >10^?4 M inhibited pepsinand metalloproteinase-catalyzed lysis of vritually all proteins. ATP increased casein lysis by serine proteinases, metalloproteinase, and pepsin by 20–60% at concentration of >10^?3 M and by 30–260% at 10^?2 M concentration. At concentrations of 10^?2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloproteinase by 20–100%, and, at concentrations of 10^?3 M, lysis of albumin by pepsin and other proteins (except for fibrinogen) by metalloproteinase. A GTP concentration of 10^?7–10^?2 M increased protein degradation by serine proteinases, papain, and gelatin lysis by pepsin by 20–90%, whereas albumin lysis was inhibited by 40–70%. The presence of 10^?6–10^?5 M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloproteinase, while ≥10^?3 M GTP induced a drop in the activity of the metalloproteinase by 20–50%. ADP enhanced gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloproteinase activity by 20–100% (at ≥10^?3 M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.     

小鼠抗天花粉蛋白IgE型单抗的酶解及其Fab的制备

研究了Ｐａｐａｉｎ及Ｔｒｙｐｓｉｎ裂解小鼠抗天花粉蛋白ＩｇＥ单抗的条件及Ｆａｂ的制备。Ｐａｐａｉｎ和Ｔｒｙｐｓｉｎ两者都可产生Ｆ（ａｂ′）_２,分子量在１５０～１６０ｋＤ左右;经Ｐａｐａｉｎ裂解的主要产物中还有Ｆａｂ,分子量７２ｋＤ,可通过凝胶过滤获得纯的Ｆａｂ。而Ｔｒｙｐｓｉｎ裂解物经ＤＴＴ还原、碘乙酰胺烷化虽然也可得到Ｆａｂ′（ｔ）,但不易纯化;可见,要制备Ｆａｂ以采用Ｐａｐａｉｎ裂解为好,而制备Ｆ（ａｂ′）_２则可采用Ｔｒｙｐｓｉｎ裂解。这二个酶的裂解速度是Ｔｒｙｐｓｉｎ大于Ｐａｐａｉｎ。     

Radiochemical assay for renin utilizing a synthetic insoluble substrate

In order to provide a convenient in vitro assay for renin activity, a radiolabeled renin substrate analog, N-acetyl-Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-His-[³H]-Leu-Leu-Val-Tyr-Ser-Gly-Lys-Pro-OH, was prepared by solid-phase synthesis. The substrate peptide was bound covalently to agarose through the ?-amino group of its lysine residue. Incubation of this insoluble complex with partially purified hog renin resulted in the release of biologically active tritiated peptide into the soluble phase of the incubation mixture, at a rate proportional to the quantity of renin added. The optimum pH for cleavage was 6.5. The apparent K_m of the substrate was 1 × 10⁻⁴M, and the V_max was 83 pmoles tritiated peptide released/min/mg renin preparation added. The minimum amount of renin detectable by the assay was 2 μg, a quantity that would be expected to generate 1.0 pmole angiotensin per minute from the natural plasma substrate. Chymotrypsin, trypsin, papain, and pseudorenin, were also effective in cleaving labeled peptides from the insoluble substrate, but leucine aminopeptidase did not appear to release soluble radioactivity. The assay, as described, is useful for the measurement of large numbers of renin samples because of the speed and ease with which it may be performed. It is not yet sufficiently sensitive nor specific to measure the low levels of renin found in plasma.     

Decreased formation of alpha 2-macroglobulin-protease complexes in plasma of patients with cystic fibrosis.

Alpha₂-macroglobulin from patients with cystic fibrosis is shown to have reduced binding with papain, trypsin, and thrombin. The obligate heterozygotes for cystic fibrosis revealed intermediate values between the controls and the patients. Since papain and trypsin are not plasma endopeptidases, it becomes evident that the absence of α₂-macroglobulin-protease complex in cystic fibrosis is due to a molecular defect within the macroglobulin.     

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.     

Structure and metabolism of rat liver heparan sulphate

Rat liver cells grown in primary cultures in the presence of [³⁵S]sulphate synthesize a labelled heparan sulphate-like glycosaminoglycan. The characterization of the polysaccharide as heparan sulphate is based on its resistance to digestion with chondroitinase ABC or hyaluronidase and its susceptibility to HNO₂ treatment. The sulphate groups (including sulphamino and ester sulphate groups) are distributed along the polymer in the characteristic block fashion. In ³H-labelled heparan sulphate, isolated after incubation of the cells with [³H]galactose, 40% of the radioactive uronic acid units are l-iduronic acid, the remainder being d-glucuronic acid. The location of heparan sulphate at the rat liver cell surface is demonstrated; part of the labelled polysaccharide can be removed from the cells by mild treatment with trypsin or heparitinase. Further, a purified plasma-membrane fraction isolated from rats previously injected with [³⁵S]sulphate contains radioactively labelled heparan sulphate. A proteoglycan macromolecule composed of heparan sulphate chains attached to a protein core can be solubilized from the membrane fraction by extraction with 6m-guanidinium chloride. The proteoglycan structure is degraded by treatment with papain, Pronase or alkali. The production of heparan [³⁵S]sulphate by rat liver cells incubated in the presence of [³⁵S]sulphate was followed. Initially the amount of labelled polysaccharide increased with increasing incubation time. However, after 10h of incubation a steady state was reached where biosynthetic and degradative processes were in balance.     

Study of the interaction of trypsin inhibitor from the sea anemone Radianthus macrodactylus with proteases

The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: K_D = 7.38 × 10^?8 M and 9.93 × 10^?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively.     

Detachment factors for enhanced carrier to carrier transfer of CHO cell lines on macroporous microcarriers

In this publication different detachment factors were tested for enhancing carrier to carrier transfer for scale-up of macroporous microcarrier based bioprocesses. Two Chinese hamster ovary cell lines, CHO-K1 and a genetically engineered CHO-K1 derived cell line (CHO-MPS), producing recombinant human Arylsulfatase B, were examined. The cells were grown on Cytoline 1microcarriers (Amersham Biosciences, Uppsala, Sweden) in protein-free and chemically defined medium respectively. Fully colonised microcarriers were used at passage ratios of approximately 1:10 for carrier to carrier transfer experiments. To accelerate the colonisation of the non-colonised, freshly added microcarriers the detachment reagents trypsin, papain, Accutase™ (PAA, Linz, Austria), heparin and dextransulphate were used. Both cell lines showed good results with trypsin, Accutase and dextransulphate (Amersham Biosciences, Uppsala, Sweden), while papain failed to enhance carrier to carrier transfer in comparison to the non-treated reference. The maximum growth rate of cells on microcarriers with 2% dextransulphate in the medium was 0.25 ± 0.02d⁻¹ and 0.27 ± 0.03d⁻¹ for the CHO-MPS and CHO-K1, respectively. TheCHO-K1 grew best after detachment with trypsin (μ = 0.36 ± 0.03d⁻¹). This indicates, that one of the key parameters for carrier to carrier transfer is the uniform distribution of cells on the individual carriers during the initial phase. When this distribution can be improved, growth rate increases, resulting in a faster and more stable process. This revised version was published online in August 2006 with corrections to the Cover Date.     

Stereochemical aspects of forcing special substrate hydrazides to behave either as electrophiles or as nucleophiles during catalysis by crude papain

Restriction of hydrazides of N-blocked amino acids mainly to electrophilic action, in acylating crude papain, has been achieved by means of a large amount of aniline, with formation of insoluble anilides of N-acylamino acids. Similarly, nucleophilic behavior, on the part of a hydrazide, has been promoted by introducing a large proportion of an N-acylamino acid to produce an insoluble N¹,N²-diacylhydrazine. Achiral, chiral and racemic hydrazides and their corresponding N-acylamino acids were utilized in the study. Among the more informative combinations of reactants were Z-dl-alanine hydrazide with aniline and then with Z-glycine. A stereospecific response in the former situation produced Z-l-alanine anilide. In the latter case, a stereoselective interaction produced Z-Gly-NHNH-lAla-Z more rapidly than Z-Gly-NHNH-d-Ala-Z. The final incubation period yielded an optically pure D product. Differences in stereochemical control have been delineated in terms of different spatial aspects for interactions at the S and S′ subsites of sulfhydryl proteolytic enzymes. A racemic reactant encountered firm stereospecificity as an electrophile at the S subsite but only modest stereoselectivity as a nucleophile at the S′ subsite. The ready availability of crude papain allows an effective procedure for the synthesis of substantial quantities of diacylhydrazines.