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PubMed1978

Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism.

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of beta-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.

Journal Article↗
PubMed1979

Preparation and properties of polyethylene glycol-trypsin adducts.

The covalent attachment of polyethylene glycol of 5000 daltons to non-essential groups on trypsin produces an adduct that no longer precipitates with anti-trypsin antibody. In comparison with trypsin, polyethylene glycol-trypsin preparations show equal or greater activity against N-alpha-benzoyl-L-arginine ethyl ester, about one-fourth activity against angiotensin II, and little activity against bovine liver catalase. The polyethylene glycol-trypsin adduct dissolves soft blood clots at one-fourth the rate of trypsin. Soybean trypsin inhibitor produces two-thirds inhibition of the adduct under conditions that cause complete inhibition of trypsin.

Angiotensin II↗
PubMed1981

Problems associated with the radioimmunoassay of serum trypsin.

A commercial trypsin radioimmunoassay (RIA) kit was used for its ability to measure trypsin bound to the serum protease inhibitors, alpha 2 macroglobulin (alpha 2 M) and alpha 1 anti-trypsin (alpha 1 AT). Only 20% of trypsin bound to alpha 2 M and 70% bound to alpha 1 AT was detected by the assay system. Recovery of trypsin added to human serum varied from 0 to 20%. Standard curves prepared from purified human cationic trypsin did not exhibit parallelism with the kit standard curves. Inclusion of horse serum in the standard solutions improved the parallelism observed. Immunoreactive trypsin (IRT) levels obtained for serum samples were found to vary considerably depending on the standard curve used to calculate the assay results. Lower IRT levels were observed when trypsin standards prepared in the absence of horse serum were used as reference.

Humans↗
PubMed1987

Similarity and dissimilarity in the stereogeometry of the active sites of thrombin, trypsin, plasmin and glandular kallikrein.

The relationship between chemical modifications of arginine derivatives and inhibitory activity to trypsin, plasmin and glandular kallikrein was investigated comparing with that of thrombin and concluded as follows: The hydrophobic binding pocket, which has been reported previously to be stereogeometrically very similar in trypsin and thrombin, corresponded to the length of ethylpiperidine. Concerning the site (termed the P site) next to the hydrophobic binding pocket, there were large differences in stereogeometry between trypsin and thrombin; the binding site of trypsin extended further to allow propyl and phenyl group attached to piperidine, while that of thrombin would be much narrower and unable to allow them. The P sites of plasmin and glandular kallikrein resembled that of trypsin in being able to allow phenyl group. To substantialize the hydrophobic binding pocket and the P site, a (2R, 4R)-MQPA-trypsin complex model was generated using the results of X-ray crystallography of (2R, 4R)-MQPA and BPTI-trypsin complex by calculation to minimize van der Waals contacts, and it was of great use for understanding the geometry of the active sites of trypsin, thrombin, plasmin and glandular kallikrein.

Animals↗
PubMed1987

The binding and inhibition of trypsin by alpha-crystallin.

One of the major lens-structural proteins, alpha-crystallin, is a multimeric protein containing 40 subunits of approx. 20 kDa each. There are two subunit types with distinct but similar structures. This protein was capable of inhibiting trypsin, chymotrypsin and elastase, but had no effect on thrombin or kallikrein. Complete inhibition was not observed, but rather plateau levels of inhibition were obtained in each case. Maximum inhibition was observed at a ratio of 1 mol of alpha-crystallin for every 9-10 mol of trypsin. alpha-Crystallin also inhibited the labeling of the active site of trypsin by [3H]diisopropyl fluorophosphate (DFP). Greater than 90% inhibition of DFP labeling was observed at a ratio of 1 mol of alpha-crystallin for every 7-8 mol of trypsin. Both trypsin and [3H]DFP-labeled trypsin formed a complex with alpha-crystallin, as demonstrated by gel-filtration chromatography. The active site of trypsin when bound to alpha-crystallin was still capable of reacting with p-nitrophenyl p-guanidobenzoate and soybean trypsin inhibitor, but was inaccessible to alpha 1-antitrypsin. These data suggest that alpha-crystallin acts as a multivalent modified inhibitor which is consistent with the proposed quaternary structure of alpha-crystallin.

Animals↗
PubMed1998

Interaction of second and third domains of Japanese quail ovomucoid with ten mammalian trypsins.

Second and third domains were prepared from Japanese quail ovomucoid and association equilibrium constants, Kas, were measured at 25 degreesC and pH 8 for these domains with trypsins from ten mammalian species: cat, cow, dog, guinea pig, hog, horse, man, rabbit, rat, and sheep. The values ranged from 108 M-1 to 1010 M-1 for the second domain-trypsin associations and from 106 M-1 to 108 M-1 for the third domain-trypsin associations. Changes in Ka values for the interactions between the trypsins and each domain are attributed to slight changes in surface conformation caused by the residue changes in the inhibitor-binding region other than the S1 pocket of the trypsin species. The representative of such residue changes is assumed to be the one observed at residue 217 of trypsin molecule. Concerning each trypsin, the Ka value with the second domain was always higher than that with the third domain. However, the ratios between the two equilibrium constants varied from 3 to 60 depending upon trypsin species. This means that amino acid changes in enzyme-contact residues other than the P1 site of the Kazal-type inhibitor can make it possible to recognize even a slight difference in inhibitor-binding surface among the enzymes with the same S1 pocket and highly similar overall three-dimensional structure.

Amino Acid Sequence↗
PubMed1998

Highly selective mechanism-based thrombin inhibitors: structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes.

The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined and refined to R values 0.152-0. 170 at 1.8-2.1 A resolution. Since the selectivity of two of the inhibitors was >1600 with respect to trypsin, the structures of trypsin-inhibited complexes of these inhibitors were also determined (R = 0.142-0.157 at 1.9-2.1 A resolution). The selectivity appears to reside in the inability of a benzenesulfonamide group to bind at the equivalent of the D-enantiomorphic S3 site of thrombin, which may be related to the lack of a 60-insertion loop in trypsin. All the inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. Differences in the binding constants of these inhibitors are correlated with their interactions with thrombin and trypsin. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism. The structures of both thrombin and trypsin complexes show similar well-defined transition states in the S1 site and at the electrophilic carbon atom and Ser195OG. The trypsin structures, however, suggest that the first step in a two-step kinetic mechanism may involve formation of a weak transition-state complex, rather than binding dominated by the P2-P4 positions.

Aldehydes↗
PubMed1955

The inactivation of dilute solutions of crystalline trypsin by x-radiation. II. Effects of enzyme concentration, medium, pH, and temperature.

The proteolytic activity of dilute solutions of clystalline trypsin is destroyed by x-rays, the amount of inactivation being an exponential function of the radiation dose. The reaction yield increases steadily with increasing concentration of trypsin, varying, as the concentration of enzyme is increased from 1 to 300 microM, from 0.068 to 0.958 micromole of trypsin per liter inactivated per 1000 r with 0.005 N hydrochloric acid as the solvent, from 0.273 to 0.866 with 0.005 N sulfuric acid as the solvent, and from 0.343 to 0.844 with 0.005 N nitric acid as the solvent. When the reaction yields are plotted as a function of the initial concentration of trypsin, they fall on a curve given by the expression Y alpha X(K), in which Y is the reaction yield, X is the concentration of trypsin, and K is a constant equal to 0.46, 0.20, and 0.16, respectively, with 0.005 N hydrochloric, sulfuric, and nitric acids as solvents. The differences between the reaction yields found with chloride and sulfate ions in I to 10 microM trypsin solutions are significant only in the pH range from 2 to 4. The amount of inactivation obtained with a given dose of x-rays depends on the pH of the solution being irradiated and the nature of the solvent. The reaction yield-pH curve is a symmetrical one, with minimum yields at about pH 7. Buffers such as acetate, citrate, borate and barbiturate, and other organic molecules such as ethanol and glucose, in concentrations as low as 20 microM, inhibit the inactivation of trypsin by x-radiation. Sigmoid inactivation-dose curves instead of exponential ones are obtained in the presence of ethanol. The reaction yields for the inactivation of trypsin solutions by x-rays are approximately 1.5 times greater when the irradiation is done at 26 degrees C. than when it is done at 5 degrees C., when 0.005 N hydrochloric acid is the solvent. The dependence on temperature is less when 0.005 N sulfuric acid is used, and is negligible with 0.005 N nitric acid. The difficulties involved in interpreting radiation effects in aqueous systems, and in comparing the results obtained under different experimental conditions, are discussed.

Buffers↗
PubMed2000

L-Isoaspartate 115 of porcine beta-trypsin promotes crystallization of its complex with bdellastasin.

Bdellastasin is a 59-amino-acid, cysteine-rich, antistasin-type inhibitor of sperm acrosin, plasmin and trypsin, isolated from the medicinal leech Hirudo medicinalis. The complex formed between bdellastasin and porcine beta-trypsin has previously been crystallized in the presence of PEG in a tetragonal crystal form of space group P4(3)2(1)2 and has now been found to crystallize under high-salt conditions in the enantiomorphic space group P4(1)2(1)2. These structures have been solved and refined to 2.8 and 2.7 A resolution, respectively. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure. In the second new crystal form, the flexible N-terminal subdomain is rotated with respect to the C--terminal subdomain by about 90 degrees, fitting into a cavity formed by symmetry-related trypsin molecules. The canonical inhibitor-proteinase interaction is restricted to the primary binding loop comprising residues Leu31-Lys36 of bdellastasin. During the refinement, a bound sodium ion occupying the calcium-binding site of the porcine beta-trypsin component was discovered. This sodium ion is coordinated in a tetragonal-pyramidal manner, with the geometry of the enclosing loop slightly changed compared with the loop in the presence of calcium. In the crystal form of space group P4(3)2(1)2, the electron density for residue 115 of porcine beta-trypsin clearly indicates the presence of a beta-isomerized L-aspartic acid, which is placed in spatial proximity to segment Thr144--Gly148 of a symmetry-related trypsin molecule. This is the first structurally observed example of an L-isoaspartate in beta--trypsin originating from Asn. A comparison with other known crystal structures of porcine beta-trypsin-macromolecular inhibitor complexes suggests that the deamidation, isomerization and racemization of Asn115 is the key step in crystallization.

Animals↗
PubMed2004

Expression of trypsinogen-1, trypsinogen-2, and tumor-associated trypsin inhibitor in ovarian cancer: prognostic study on tissue and serum.

PURPOSE: The purpose is to study the prognostic significance of tissue expression of trypsinogen-1, trypsinogen-2, and tumor-associated trypsin inhibitor (TATI) and serum concentration of trypsinogen-2, trypsin-2-API (complex of trypsin-2 with alpha-1-proteinase inhibitor), and TATI in epithelial ovarian cancer. EXPERIMENTAL DESIGN: Expression of trypsinogen-1, trypsinogen-2, and TATI was determined by immunohistochemistry with monoclonal antibodies in tissue sections of tumors from 119 patients with untreated primary epithelial ovarian cancer. Preoperative serum concentrations of trypsinogen-2, trypsin-2-API and TATI were analyzed using specific immunoassays. RESULTS: Fifty-four percent of the tumors expressed trypsinogen-1, 45% expressed trypsinogen-2, and 30% expressed TATI. In patients with stage III and IV disease, TATI tissue expression (P = 0.002) and elevated TATI concentration in serum (P = 0.048) were associated with adverse cancer-specific and progression-free survival in univariate analysis. In multivariate analysis, TATI tissue expression (P = 0.005), tumor grade (P = 0.0001), histological type (P = 0.02), and stage (P = 0.0005) were independent prognostic factors for adverse cancer-specific survival and TATI tissue expression (P = 0.006) and grade (P = 0.0003) for progression-free survival. In multivariate analysis of all patients and those with advanced disease, serum trypsin-2-API concentration was an adverse prognostic factor for cancer-specific and progression-free survival, and it was independent of stage and histological type of the tumor (P <or= 0.01). CONCLUSIONS: Tissue expression of TATI and an elevated preoperative serum concentration of trypsin-2-API are strong independent prognostic factors in advanced epithelial ovarian cancer. These results suggest that trypsin expression plays a role in the progression of ovarian cancer. TATI and trypsin-2-API are of potential use as an aid for stratification of randomized studies and for selecting treatment strategies.

Adult↗
PubMed1978

The effect of trypsin and storage on aggregation and release of human gel-filtered platelets.

Trypsin was studied as an aggregating and release-inducing agent with gel-filtered platelets (GFP) and was compared with ADP, epinephrine and collagen. GFP aggregated irreversibly with final concentrations of 0.5-4 microgram/ml trypsin, 1.6-3.2 micrometer ADP, 2.5-5 micrometer epinephrine and 40 microlite 1/ml soluble collagen. Addition of human fibrinogen to the Tyrode-suspending buffer was required for ADP and epinephrine, but was not necessary for trypsin or collagen. Release of (14C)5HT was obtained with trypsin and collagen using the same concentrations as used in aggregation. GFP stored at room temperature for 48 h were still responsive to trypsin and collagen, whereas aggregability and (14C)5HT release induced by ADP and epinephrine were already impaired 5 h after collection of blood. CP-CPK, an ADP-removing reagent, blocked aggregation and release induced by low trypsin concentrations, suggesting that ADP plays an intermediate role in the mechanism by which trypsin activates platelets. Trypsin appears to be a valuable reagent for studying platelet physiology, particulary following storage.

Blood Coagulation↗
PubMed2003

Pulmonary trypsin-2 in the development of bronchopulmonary dysplasia in preterm infants.

OBJECTIVES: In the preterm infant, lung injury can lead to irreversible tissue destruction and abnormal lung development. We examined whether pulmonary trypsin, a potent matrix-degrading serine proteinase and proteinase-cascade activator, is associated with the development of bronchopulmonary dysplasia (BPD) in preterm infants. METHODS: Samples of tracheal aspirate fluid were collected from 32 intubated preterm infants during their first 2 postnatal weeks. The presence and molecular forms of trypsin in tracheal aspirate fluid samples were analyzed by zymography and Western blotting. The concentrations of trypsinogen-1 and -2 and tumor-associated trypsin inhibitor were measured by immunofluorometry. For examining the expression of trypsin-2 in lung tissue, immunohistochemistry was performed on autopsy specimens of fetuses, of preterm infants who died from respiratory distress syndrome or BPD, and of term infants without lung injury. RESULTS: In infants who subsequently developed BPD (n = 18), we detected significantly higher concentrations of trypsinogen-2 during postnatal days 5 to 10 compared with those who survived without it. There was no difference in trypsinogen-1 concentrations. Tumor-associated trypsin inhibitor concentrations were significantly lower in infants who needed mechanical ventilation for >1 week. Immunohistochemistry demonstrated that trypsin-2 was predominantly expressed in bronchial and bronchiolar epithelium. In 2 preterm infants who died from prolonged respiratory distress syndrome, trypsin-2 was also expressed in vascular endothelium. CONCLUSIONS: The levels of trypsinogen-2 are higher during postnatal days 5 to 10 in infants who subsequently develop BPD. The results suggest that high levels of pulmonary trypsin-2 may be associated with the development of BPD. This raises the possibility that therapy with exogenous proteinase inhibitors might prevent the development of BPD in preterm infants with respiratory distress.

Body Fluids↗
PubMed1975

Adenylate cyclase stimulation by trypsin.

Adenylate cyclase activity of a rat embryo fibroblast cell line (F111) is markedly increased by brief treatment with 1:300 trypsin. The degree of stimulation depends upon the length of time the cells are treated and the concentration of trypsin. Crystalline trypsin produced a stimulation similar to that obtained with 1:300 trypsin. Further, the addition of soybean trypsin inhibitor blocked the stimulation of adenylate cyclase by 1:300 trypsin. Trypsin-treated adenylate cyclase responds to PGE1, but there is no increase over that of untreated enzyme. This result and the increase in fluoride-stimulated levels of activity suggest that the trypsin is acting upon the catalytic unit of the enzyme.

Adenylyl Cyclases↗
PubMed1976

Trypsin-induced increase in intracellular cyclic AMP of lymphocytes.

Trypsin increases intracellular levels of cylic AMP (cAMP) in lymphocytes. The trypsin-induced increase in cAMP is blocked by specific trypsin inhibitors and by high concentrations of different proteins. Several proteolytic enzymes from various sources, including other pancreatic proteases, do not cause an increase in cAMP under the same experimental conditions. Immobilized trypsin induced the same increase in cAMP as does free trypsin. The trypsin-induced rise in cAMP is not due to inhibition of cAMP phosphodiesterase, but consistent activation of adenylate cyclase by trypsin could not be demonstrated. The extent of the trypsin-induced increase in intracellular cAMP correlates with the type of the lymphocyte and with the state of maturity attained by the cells. Transformed lymphocytes and nonlymphoid cells do not react at all.

Animals↗
PubMed1983

[Therapeutic effects of human urinary trypsin inhibitor on acute experimental pancreatitis].

Therapeutic effects of human urinary trypsin inhibitor (MTI) on acute pancreatitis were examined. MTI potently inhibited not only proteases such as trypsin or alpha-chymotrypsin, but also inhibited lipase or creatine phosphokinase which are considered to be related to pancreatitis. Although gabexate mesilate (gabexate) and aprotinin also strongly inhibited trypsin, their inhibition spectra against pancreatic enzymes were narrower and aprotinin also strongly inhibited trypsin, their inhibition against pancreatic enzymes were narrower than MTI. MTI inhibited proteases released from pancreatic slice by trypsin more potently than gabexate or aprotinin. The therapeutic effects of MTI on experimental acute trypsin-induced pancreatitis in dogs or rats were stronger than those of gabexate or aprotinin. These results suggest that MTI may suppress pathogenesis and development of pancreatitis in several ways, for example, by directly inhibiting trypsin and by inhibiting tissue-damaging enzymes released from the pancreas by stimulation with trypsin.

Acute Disease↗
PubMed1996

Ability of trypsin in mimicking germ cell factors that affect Sertoli cell secretory function.

A biological factor that inhibits the in vitro secretion of testin by Sertoli cells was purified to apparent homogeneity from conditioned medium of germ cells isolated using trypsin. Partial N-terminal amino acid sequence analysis of the purified germ cell factor revealed a sequence of NH2-IVGGYTXAAN. Comparison of the sequence with the existing protein database revealed that it is homologous to trypsin. Immunoprecipitation experiments using either [35S]-labeled germ or Sertoli cell proteins and a monospecific anti-trypsin antibody failed to demonstrate the synthesis and secretion of trypsin by these testicular cells, suggesting the isolated factor is the residuary trypsin that was used for isolating germ cells from seminiferous tubules. Subsequent experiments revealed that trypsin per se can inhibit the secretion of Sertoli cell testin and clusterin dose-dependently, whose effect can be prohibited by soybean trypsin inhibitor (STI). In view of these findings, a nonenzymatic procedure was deemed necessary to prepare germ cell conditioned medium (GCCM) to assess whether an authentic biological factor(s) is indeed present. Four batches of conditioned medium of germ cells isolated by a mechanical procedure without the use of trypsin were fractionated by sequential Mono Q anion exchange and C8 reversed-phase HPLC. When these fractions were monitored for testin modulatory activity using an in vitro bioassay with primary cultures of Sertoli cells, it was shown that GCCM prepared by this procedure indeed contained testin modulatory bioactivity. Since testin is a novel component of specialized junctions between Sertoli and germ cells, the identification of a germ cell factor(s) that affects its secretion by Sertoli cells suggests a dynamic biochemical relationship between these cell types in the seminiferous epithelium.

Amino Acid Sequence↗
PubMed1994

Specificity of trypsin digestion and conformational flexibility at different sites of unfolded lysozyme.

Fourteen tryptic peptides and nine intermediates were identified as products of trypsin digestion of reduced and S-3-(trimethylated amino) propylated lysozyme. Kinetics of the appearance and disappearance of these products were observed by monitoring the peak areas on the chromatogram. In spite of the complicated reaction pathways, kinetics of the digestion of proteins and several intermediate products show simple decay curves with a single rate constant. In this paper, the trypsin susceptibility of the individual cleavage site is defined as a hydrolytic rate constant of the susceptible peptide bond in the presence of 10 nM trypsin. The cleavage sites of unfolded lysozyme are classified into two groups in terms of the trypsin susceptibility: one has a high susceptibility (10-20 h-1) and the other a low susceptibility (1.0-2.0 h-1). In the unfolded state of lysozyme, in conclusion, the region from residues 15 to 61 has a strong resistance to trypsin digestion; on the other hand, the C-terminal half of the polypeptide chain is flexible enough to fit into the active site of trypsin. In addition, six kinds of pentapeptides were synthesized as analogues of lysozyme fragments including Arg 14, Arg 21, Lys 33, Arg 45, Arg 61, and Arg 73. Kinetics of tryptic digestion of them were observed. Both kcat and KM were determined for these synthetic pentapeptides. The susceptibility of each cleavage site in pentapeptides is determined and compared with that corresponding in proteins. The susceptibility is usually higher when the susceptible peptide bond is included in proteins than in pentapeptides, so long as the conformation of peptide chain is flexible. However, susceptibilities of a few sites in proteins are lower than those in pentapeptides. This means that the peptide chains tend to fold locally to prevent trypsin from binding to the sites. It was found that the sites of Arg 21 and Arg 45 are indeed resistant to trypsin, but the site of Lys 33 is not so much, although the hydrolytic rate at Lys 33 itself is extremely slow.

Amino Acid Sequence↗
PubMed1984

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III.

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.

Alkylation↗