Proteinaceous Trypsin Inhibitors Research Articles

Seed Protein from Artocarpus hirsutus Lam. with Trypsin Inhibitory, Micro-bicidal and Antioxidant Activities Induces Depletion of Human Skin Cancer (A431) and Colon Cancer (HT29) Cells

: Natural trypsin inhibitors are increasingly recognised as putative anticancer or antimicrobial therapeutics. In the present study, we isolated a proteinaceous trypsin inhibitor from the seeds of Artocarpus hirsutus to assess its anticancer, antioxidant and microbicidal effects. A trypsin inhibitory protein (AhTI) was isolated from the seeds of A. hirsutus using ion exchange and gel filtration chromatographic methods. The trypsin inhibitory activity of AhTI was confirmed by activity staining using Reverse Zymography. The effect of AhTI on human cancer cell lines (A431 and HT29) was studied by microscopic examination, MTT assay and LDH leakage analysis. Antioxidant activity of AhTI was analysed by ferric reducing power and DPPH radical scavenging assays. An agar well diffusion method was used to check the antimicrobial activity of AhTI. The purification protocols used in the study significantly increased the specific activity of the target protein from 72.65 ± 0.86 to 2048 ± 27.3. AhTI offers significant activity against the proliferation of the A431 and HT29 cancer cell lines. In vitro AhTI shows potent antioxidant activity and exhibits antimicrobial activity against all the bacteria and fungi isolates used in this study. Of particular note, AhTI was particularly effective against Staphylococcus aureus and Escherichia coli. This in vitro study shows that AhTIis a promising candidate for further development novel therapeutics targeting cancer, microbial infections and oxidative stress.

Antifungal and Antimicrobial Properties of a Purified Protease Inhibitor from Macrotyloma Uniflorum Seeds.

Plant protease Inhibitors (PIs) play key roles in regulation of many biological activities and being less toxic, more potent and specific in comparision to chemical inhibitors. A new proteinaceous trypsin inhibitor was isolated and purified from Macrotyloma uniflorum seeds with a molecular mass of 25 KDa was purified to homogeneity via three sequential purification steps i.e., ammonium sulphate precipitation to CNBr activativated Sepharose 4B coupled trypsin affinity chromatography. Purified protease inhibitor (PI) showed optical specific activities of 665 µmols of tyrosine released/ml/min. Overall, there was a remarkable increase in the fold of purification. MUTI is stable to denaturation by heat (upto 80°C), pH (4-10).The inhibitory activity increased to two fold in the presence of mercuric chloride and got reduced by half in the presence of ferric chloride. SDS, Dithiothreitol, β-mercaptoethanol, Hydrogen peroxide, Triton X 100 enhanced its inhibitory activity whereas activity was reduced in the presence of DMSO. Chemical modification of the inhibitor by DEPC decreased its activity but the activity was increased considerably when modified with NE and PMSF. Presence of protease inhibitor activity was confirmed by reverse zymography and Dot- blot. Also MUPI has antifungal and antimicrobial properties. MUPI inhibited Phytophthora capsici by 16.6% and Rhizoctonia solani by 27.7%. Antibacterial activity was shown against Staphylococcus aureus and Pseudomonas aeruginosa. MUPI retained 90% inhibition upon storage at 4°C for over a period of six months. Thus PI can be effectively exploited to increase the shelf life of seafood.

Potential Anticoagulant Activity of Trypsin Inhibitor Purified from an Isolated Marine Bacterium Oceanimonas Sp. BPMS22 and its Kinetics.

Protease inhibitors control major biological protease activities to maintain physiological homeostasis. Marine bacteria isolated from oligotrophic conditions could be taxonomically distinct, metabolically unique, and offers a wide variety of biochemicals. In the present investigation, marine sediments were screened for the potential bacteria that can produce trypsin inhibitors. A moderate halotolerant novel marine bacterial strain of Oceanimonas sp. BPMS22 was isolated, identified, and characterized. The effect of various process parameters like salt concentration, temperature, and pH was studied on the growth of the bacteria and production of trypsin inhibitor. Further, the trypsin inhibitor was purified to near homogeneity using anion exchange, size exclusion, and affinity chromatography. The purified trypsin inhibitor was found to competitively inhibit trypsin activity with an inhibition coefficient, Ki, of 3.44 ± 0.13μM and second-order association rate constant, kass, of 1.08 × 103M-1S-1. The proteinaceous trypsin inhibitor had a molecular weight of approximately 30kDa. The purified trypsin inhibitor showed anticoagulant activity on the human blood samples.

Higher Activities of Defense-Associated Enzymes may Contribute to Greater Resistance of Manchurian Ash to Emerald Ash Borer Than A closely Related and Susceptible Congener.

Emerald ash borer (EAB) is an invasive beetle native to Asia that infests and kills ash (Fraxinus spp.) in North America. Previous experiments indicated that larvae feeding on co-evolved, resistant Manchurian ash (F. mandshurica) have increased antioxidant and quinone-protective enzyme activities compared to larvae feeding on susceptible North American species. Here, we examined mechanisms of host-generated oxidative and quinone-based stress and other putative defenses in Manchurian ash and the closely related and chemically similar, but susceptible, black ash (F. nigra), with and without exogenous application of methyl jasmonate (MeJA) to induce resistance mechanisms. Peroxidase activities were 4.6-13.3 times higher in Manchurian than black ash, although both species appeared to express the same three peroxidase isozymes. Additionally, peroxidase-mediated protein cross-linking activity was stronger in Manchurian ash. Polyphenol oxidase, β-glucosidase, chitinase, and lipoxygenase activities also were greater in Manchurian ash, but only lipoxygenase activity increased with MeJA application. Phloem H2O2 levels were similar and were increased by MeJA application in both species. Lastly, trypsin inhibitor activity was detected in methanol and water extracts that were not allowed to oxidize, indicating the presence of phenolic-based trypsin inhibitors. However, no proteinaceous trypsin inhibitor activity was detected in either species. In response to MeJA application, Manchurian ash had higher trypsin inhibitor activity than black ash using the unoxidized water extracts, but no treatment effects were detected using methanol extracts. Based on these results we hypothesize that peroxidases, lignin polymerization, and quinone generation contribute to the greater resistance to EAB displayed by Manchurian ash.

Purification, characterization, and antimicrobial activity of nontoxic trypsin inhibitor from Albizia amara Boiv.

The present investigation describes the purification, and characterization of a novel, thermostable, nontoxic, proteinaceous trypsin inhibitor extracted from seeds of Albizia amara Boiv. The proteinaceous protease inhibitor (API) was purified via acetone fractionation, ion-exchange chromatography (diethylaminoethyl (DEAE) cellulose), and gel permeation chromatography (GPC; Sephadex G-100). The apparent molecular weight of the API is 49kDa and is identified as a serine protease inhibitor. The API remains active in a wide pH range (3.0–8.0), and showed thermal stability at 60°C. The API inhibits trypsin by a mixed inhibitory mechanism with an inhibition constant (Ki) of 1.24×10−8M using BApNA (Nα-benzoyl-dl-arginine-p-nitroanilide hydrochloride) as the substrate. The API also showed substantial activity in the presence of several metal ions, surfactant (Triton X-100), oxidizing agent (dimethyl sulfoxide; DMSO), and NaCl (5%). Moreover, API retained 85% trypsin inhibition activity upon storage at 4°C over a period of 6 months. The antimicrobial effectiveness of the API against Pseudomonas aeruginosa, Bacillus subtilis, Alternaria alternata, Alternaria tenuissima, and Candida albicans reported in this study suggests its utility as a potential antimicrobial component.

EFFECT OF Moringa oleifera FLOWER EXTRACT ON LARVAL TRYPSIN AND ACETHYLCHOLINESTERASE ACTIVITIES IN Aedes aegypti

Aedes aegypti control is crucial to reducing dengue fever. Aedes aegypti larvae have developed resistance to organophosporous insecticides and the use of natural larvicides may help manage larval resistance by increasing elements in insecticide rotation programs. Here, we report on larvicidal activity of Moringa oleifera flower extract against A. aegypti L(1), L(2), L(3), and L(4) as well as the effect of flower extract on gut trypsin and whole-larval acetylcholinesterase from L(4.) In addition, the heated flower extract was investigated for larvicidal activity against L(4) and effect on larval gut trypsin. Moringa oleifera flower extract contains a proteinaceous trypsin inhibitor (M. oleifera flower trypsin inhibitor, MoFTI), triterpene (β-amyrin), sterol (β-sitosterol) as well as flavonoids (kaempferol and quercetin). Larvicidal activity was detected against L(2), L(3), and L(4) (LC(50) of 1.72%, 1.67%, and 0.92%, respectively). Flower extract inhibited L(4) gut trypsin (MoFTI K(i) = 0.6 nM) and did not affect acetylcholinesterase activity. In vivo assay showed that gut trypsin activity from L(4) treated with M. oleifera flower extract decreased over time (0-1,440 min) and was strongly inhibited (98.6%) after 310 min incubation; acetylcholinesterase activity was not affected. Thermal treatment resulted in a loss of trypsin inhibitor and larvicidal activities, supporting the hypothesis that flower extract contains a proteinaceous trypsin inhibitor that may be responsible for the deleterious effects on larval mortality.

Intracellular Autoactivation of Human Cationic Trypsinogen Mutants Causes Reduced Trypsinogen Secretion and Acinar Cell Death

Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1) inclusion of the small molecule trypsin inhibitor benzamidine in the growth medium; or (2) cotransfection with the physiological trypsin inhibitor SPINK1; or (3) by mutation of the catalytic Ser(200) residue in trypsinogen. In contrast, extracellularly added SPINK1 or other nonpermeable proteinaceous trypsin inhibitors did not restore normal secretion of the mutants, indicating that intracellular autoactivation is responsible for the observed secretion loss. Acinar cells expressing the p.D22G mutant detached from the culture plate over time, became terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive, and exhibited elevated levels of the proapoptotic transcription factor CHOP. The observations indicate that activation peptide mutants of human cationic trypsinogen undergo autoactivation intracellularly, which leads to decreased trypsinogen secretion and eventual acinar cell death. The results thus define a novel pathological pathway for parenchymal injury in hereditary chronic pancreatitis.

Analysis of protein–protein interactions with a multi-capillary electrophoresis instrument

Protein-protein interactions were analyzed by zone electrophoresis of premixed equilibrium mixtures of a fluorescence-labeled protein at a constant concentration and unlabeled protein at a variety of concentrations using a 96-CE instrument equipped with a LIF detector. The interactions between labeled-con A versus succinylated ovalbumin, labeled-trypsin versus four proteinaceous trypsin inhibitors and labeled-insulin versus seven anti-insulin monoclonal antibodies were analyzed using a dual buffer system, in which a 60 mM borate-Na buffer (pH 9.35) was used as electrophoresis buffer and 60 mM MOPS-Na (pH 7.35) containing 0.1% Tween 20 was used as a sample buffer. The dual buffer system allowed fast and reproducible analyses of interactions at a physiological pH using uncoated fused-silica capillaries. The change in the mobility moment, the first statistical moment of an electropherogram on the mobility axis (Shimura, K., Uchiyama, N., Enomoto, M., Matsumoto, H., Kasai, K., Anal. Chem. 2005, 77, 564-572), of the labeled proteins were analyzed as a function of the concentration of unlabeled proteins. The dissociation constants for seven antibodies ranging from sub nanomolar to micromolar was determined based on the results of one cycle of parallel electrophoresis runs, which completed in 30 min using 20 pmol (120 ng) of labeled insulin and 5 pmol (750 ng) each of the mAb.

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin–Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M r of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 °C and lost 40% of activity at 100 °C. CpaTI was also stable from pH 2 to 12 at 37 °C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodia interpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4 ± 15.8% and 100.0 ± 7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD 50 of 3.0 and ED 50 of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2–4% (w/w) with 15% mortality and 38% mass decrease.

Production of protease isozymes by Aphanomyces cochlioides and Aphanomyces euteiches

The production of protease activity by the sugarbeet pathogen Aphanomyces cochlioides, the legume pathogen A. euteiches, and the fish pathogen Saprolegnia parasitica was examined. Protease activity was readily detected in supernatants of water cultures of each organism using autoclaved host tissue as a nutrient source. Most of the protease isozymes extracted from sugarbeet and pea seedlings infected with A. cochlioides and A. euteiches, respectively, co-migrated with enzymes produced by the pathogens in culture. In inoculated sugarbeet seedlings, the protease activities were detected prior to or concommitent with the onset of disease symptoms and the activities were capable of digesting protein extracted from sugarbeet hypocotyls. Use of class-specific inhibitors indicated that a portion of the protease activity was of the trypsin-class. Trypsin-like isozymes that possessed a relatively fast electrophoretic migration were detected in the A. cochlioides, A. euteiches, and S. parasitica protease complements, whereas the remaining isozymes were not affected by any of the inhibitors tested. Proteinaceous trypsin inhibitors from the legumes lima bean ( Phaseolus lunatus) and soybean ( Glycine max) inhibited the trypsin-like isozymes from A. cochlioides, but not A. euteiches, whereas low molecular weight, synthetic trypsin inhibitors inhibited these isozymes from both pathogen sources. The potential role of protease inhibition in determining host range in phytopathogenic Aphanomyces species is discussed.

Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis

A screening assay for inhibitory activity against trypsin in skin mucus from 29 species of fishes reveals a wide distribution of trypsin inhibitors in skin mucus and relatively high antitryptic activity in pufferfish of the family Tetraodontidae. Two trypsin inhibitors termed TPTI 1 and 2 were purified to homogeneity from the skin mucus of Takifugu pardalis by salting out, lectin affinity, anion exchange FPLC and gel filtration HPLC. Both inhibitors are acidic glycoproteins, with an apparent molecular mass of 57 kDa in SDS-PAGE, p I below 4 and 1.9% reducing sugar for TPTI 1 and with an apparent molecular mass of 47 kDa in SDS-PAGE, p I 5.2 and 0.8% reducing sugar for TPTI 2. The inhibitors effectively repress the catalytic activity of trypsin and α-chymotrypsin, and therefore can be classified as serine protease inhibitors. The inhibitory constants against trypsin were 4.9×10 −8 M for TPTI 1 and 3.9×10 −8 M for TPTI 2. Both inhibitors react with trypsin at a molar ratio of 1:1, although TPTI 1 reversibly inactivates the proteolytic activity of trypsin non-competitively and TPTI 2, competitively. The trypsin inhibitors in the skin mucus of T. pardalis may function as defense substances to neutralize serine proteases released by invasive pathogens.

Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin, reveals its resistance to proteinaceous trypsin inhibitors

Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.

Plant density and nutrient availability constrain constitutive and wound-induced expression of trypsin inhibitors in Brassica napus.

We investigated the effects of plant density on plant size, leaf total soluble protein content, and constitutive and wound-induced levels of proteinaceous trypsin inhibitors in pot-grown Brassica napus seedlings in two greenhouse studies. We manipulated plant density by varying the number of intraspecific neighbors surrounding a target plant in the center of each pot. In general, constitutive and induced levels of trypsin inhibitors were significantly reduced by competition in a density-dependent manner, to the extent that induction was greatly reduced or abolished in target plants surrounded by six neighbors. To investigate whether the effects of plant density on inhibitor production were mediated by nutrient availability, we manipulated the concentration of a complete fertilizer applied to target plants surrounded by six neighbors in two greenhouse studies. In general, constitutive and wound-induced levels of inhibitors in plants surrounded by six neighbors were increased by nutrient addition in a dose-dependent manner, such that wound-induction was completely restored in competing plants under conditions of high nutrient availability. Leaf total soluble protein content, measured only in the second trial of each experiment, was not affected by any of the treatments. The effects of plant density, nutrient addition, and wounding on inhibitor levels in all experiments were independent of their effects on above-ground plant size at the time of wounding. Overall, our results suggest that decreasing nutrient availability mediates the density-dependent reductions in inhibitor levels in B. napus seedings.

Stimulation of exocrine pancreatic secretion by soybean trypsin inhibitor does not depend on the masking of luminal trypsin activity in rats that have bile-pancreatic juice diverted into the ileum.

We reported previously that dietary proteins stimulate pancreatic secretion by a mechanism not involved in masking trypsin activity in rats that have bile-pancreatic juice (BPJ) diverted from the proximal small intestine for 7 days. However, BPJ in the distal small intestine is possibly responsible for the stimulation of pancreatic secretion in chronic BPJ-diverted rats. To examine whether the BPJ-dependent mechanism operates in the distal small intestine of chronic BPJ-diverted rats, we investigated pancreatic responses after inhibition or removal of pancreatic trypsin activity in the distal small intestine of conscious rats. Duodenal instillation of soybean trypsin inhibitor (SBTI), which is a proteinaceous trypsin inhibitor, stimulated pancreatic secretion in the chronic BPJ-diverted rats, whereas a nonpeptidic trypsin inhibitor, FOY 305, did not. Ileal administration of both trypsin inhibitors did not enhance pancreatic secretion in the diverted rats. Exclusion of luminal BPJ from the distal small intestine was also ineffective in causing pancreatic exocrine secretion in the chronic BPJ-diverted rats. These observations reveal that ileal BPJ does not contribute to the stimulation of pancreatic secretion in rats that have BPJ chronically diverted into the ileum, as in intact rats, and that a duodenal instillation of SBTI stimulates pancreatic secretion as a protein, and not as a trypsin inhibitor.

Purification and biochemical characterization of a vacuolar serine endopeptidase induced by glucose starvation in maize roots.

An endopeptidase (designated RSIP, for root-starvation-induced protease) was purified to homogeneity from glucose-starved maize roots. The molecular mass of the enzyme was 59 kDa by SDS/PAGE under reducing conditions and 62 kDa by gel filtration on a Sephacryl S-200 column. The isoelectric point of RSIP was 4.55. The purified enzyme was stable, with no auto-proteolytic activity. The enzyme activity was strongly inhibited by proteinaceous trypsin inhibitors, di-isopropylfluorophosphate, 3,4-dichloroisocoumarin and PMSF, suggesting that the enzyme is a serine protease. The maximum proteolytic activity against different protein substrates occurred at pH 6.5. With the exception of succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin, no hydrolysis was detected with synthetic tryptic, chymotryptic or peptidylglutamate substrates. The determination of the cleavage sites in the oxidized B-Chain of insulin showed specificity for hydrophobic residues at the P2 and P3 positions, indicating that RSIP is distinct from other previously characterized maize endopeptidases. Both subcellular fractionation and immuno-detection in situ indicated that RSIP is localized in the vacuole of the root cells. RSIP is the first vacuolar serine endopeptidase to be identified. Glucose starvation induced RSIP: after 4 days of starvation, RSIP was estimated to constitute 80% of total endopeptidase activity in the root tip. These results suggest that RSIP is implicated in vacuolar autophagic processes triggered by carbon limitation.

Purification and characterization of trypsins from the digestive tract of Locusta migratoria

Two trypsin-like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE-cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p-aminobenzamidine (PABA)-Sepharose column, which also separated the two trypsins (TLEAff.1. and TLEAff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis of cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLEAff.1 and TLEAff.2, as determined by SDS-PAGE, were 17,000 and 24,000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLEAff.1 and 3.5 fold for TLEAff.2. Since most of the locust trypsin comprised TLEAff.2, the latter served as the main object of this study. TLEAff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5-9.0. The Km and kcat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLEAff.2. The locust trypsin was full inhibited by the proteinaceous trypsin inhibitors Bowman-Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site.

Degradation of proteins microinjected into HeLa cells. The role of substrate flexibility.

Increasing the flexibility of a protein enhances its susceptibility to defined proteases in vitro. To ascertain whether flexibility also affects protein stability in vivo, radioiodinated proteins with similar structures, but dissimilar flexibilities, were introduced into HeLa cells using red cell-mediated microinjection. Intracellular proteolysis was then measured as the rate of release of 125I-tyrosine into the medium. Ribonuclease A was considerably more resistant to degradation by purified proteases or in reticulocyte lysate than its flexible derivatives ribonuclease S and S-protein. In contrast, all three proteins were equally stable within HeLa cells. Like the results obtained for RNases, the rates of degradation of trypsin inhibitors, trypsin analogs, and their complexes correlated with flexibility in reticulocyte lysate. However, the intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were not affected upon formation of enzyme-inhibitor complexes. Furthermore, trypsinogen was degraded more slowly than the structurally similar anhydrotrypsin in HeLa cells, although trypsinogen has additional segmental flexibility in its activation domain. Electrophoretic analyses revealed that trypsin-inhibitor complexes remained intact following injection into HeLa cells, and that neither free inhibitors nor anhydrotrypsin formed Triton-stable complexes with soluble cytoplasmic proteins. The observation that the components of the trypsin-inhibitor complexes were degraded simultaneously indicates that neither constituent unfolded prior to the onset of proteolysis. These studies provide evidence that RNases, trypsin, and trypsin inhibitors are degraded by an intracellular proteolytic pathway(s) which recognizes surface features of the folded proteins.

Partial purification and characterization of sperm receptor hydrolase, a cortical granule proteoesterase, from eggs of the sea urchin Strongylocentrotus purpuratus

AbstractSperm receptor hydrolase, one of two classes of cortical granule proteoesterases (E.C.3.4.4.4) was purified approximately 30‐fold with 80% yield from Strongylocentrotus purpuratus cortical granule exudate. Sperm receptor hydrolase preparations were free of vitelline delaminase activity (the other class of cortical granule proteoesterase) and had less than 1% of the starting levels of cortical granule peroxidase and β‐1,3‐glucanohydro‐lase activities. Native polyacrylamide gel electrophoresis coupled with a protease activity stain showed that three proteases were present in the most highly purified preparations of sperm receptor hydrolase. Each of the three proteases has the same molecular weight of 60,000, but different isoelectric points of 2.4, 3.8, and 5.5. The Km value of the mixture of proteases for α‐N‐benzoyl‐L‐arginine ethyl ester as substrate was 263 μM at pH 8.4 and 30°C; the pH dependence of Vm showed a single prototrophic group with a pK of 6.7 and an enthalpy of ionization of 8.6 kcal‐mol−1. The values of these kinetic parameters are consistent with an enzyme‐active site containing histidine. Phenylmethyl sulfonyl fluoride, tosyl lysine chloromethyl ketone, several proteinaceous trypsin inhibitors, and p‐aminobenzamidine inhibited the esterase activity of the proteases. These data suggest that sperm receptor hydrolases are serine proteases.

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.