Low Concentrations Of Trypsin Research Articles

Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting

Because hESC (human embryonic stem cells) are 'social cells' that require co-operative interactions and intimate physical contact with each other, it is absolutely essential to dissociate hESC colonies into cellular clumps rather than into a single-cell suspension during serial passage. The present study compared two commonly used protocols for dissociating hESC colonies. The first protocol involved mild enzymatic treatment with collagenase type IV (1 mg/ml) for approx. 5-10 min, prior to mechanical dissociation into cellular clumps through manual scraping with a plastic pipette tip. The second protocol involved a short duration of exposure (2-3 min) to low concentrations of trypsin (0.05%), followed by gentle pipetting. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to compare the recovery of viable cells after dissociating hESC colonies with these two protocols, before and after conventional freeze-thawing with 10% (v/v) DMSO. Besides undifferentiated hESC, the randomly differentiated fibroblastic progenies of hESC at various passages (P0-P4), together with an immortalized cell line (CRL-1486), were also utilized to compare the two protocols. The results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates. It is hypothesized that scratching after collagenase treatment (first protocol) somehow induces physical damage to the cells, thereby leading to a lower recovery of viable cells, both before and after freeze-thawing.

Protease Amperometric Sensor

An amperometric biosensor for the detection of trypsin was developed. The latter was based on a two-layer configuration, namely, a polymer-glucose oxidase inner layer and a gelatin outer layer. In the presence of glucose, the enzyme layer produces H2O2 and hence an amperometric signal due to H2O2 electrooxidation was generated by potentiostating the electrode at 0.6 V. The biosensor detects the change in the increase in the maximum current caused by the proteolytic digestion of gelatin, which covers the platinum electrodes, thereby facilitating a speedier access for the glucose substrate to the electrode modified with both poly(pyrrole-alkylammonium) and glucose oxidase molecules. Our biosensor detected low trypsin concentrations down to 42 pM with a response time of approximately 10 min, making it a very sensitive device in the detection of lower trypsin levels with such future putative applications as the diagnosis of pancreatic diseases.

Evidence that protease-activated receptor-2 mediates trypsin-induced reversal of stellation in cultured rat astrocytes

Serine proteases such as thrombin and trypsin play a key role in the development and repair processes in the central nervous system. Molecular actions of serine proteases include multiple cellular events like activation of protease-activated receptors (PARs). PARs belong to a family of G protein-coupled receptors that can be stimulated through their proteolytic cleavage by ligands. PAR-2 has been implicated in neurodegenerative diseases including astrogliosis. Although recent studies have shown that low concentration of trypsin activates PAR-2, its role in morphological changes in primary astrocytes has not been studied. In the present study, we investigated the effects of PAR-2 in astrocyte stellation in rat primary astrocyte culture. Both trypsin (0.1–1 U/ml) and a PAR-2-activating peptide SLIGRL-NH 2 (1–50 μM) significantly reversed the stellation induced by serum deprivation in rat astrocytes. Treatment of astrocytes with trypsin or SLIGRL-NH 2 resulted in a transient rise of the intracellular Ca 2+ level and trypsin-induced morphological changes were blocked by BAPTA, a Ca 2+ chelator. In addition, a protein kinase C (PKC) inhibitor, bisindolylmaleimide significantly inhibited the trypsin-induced morphological changes, whereas activation of PKC by phorbol-12-myristate-13-acetate acted as trypsin. Taken together, these results suggest that activation of PAR-2 by trypsin caused reversal of stellation in cultured astrocytes, in part, via the mobilization of intracellular Ca 2+ and activation of PKC.

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

Reduction and uptake of methylene blue by human erythrocytes.

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB(+)) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB(+). Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB(+) <5 microM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of d-glucose. MB(+)-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB(+)-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB(+), which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB(+), that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB(+) reduction may correspond to MB(+)-dependent NAD(P)H reductase activity in erythrocyte ghosts.

Slow internal dynamics of membrane proteins in mechanisms of protease-induced aggregation of platelets.

Room temperature tryptophan phosphorescence (RTTP) of suspensions of human platelets was studied. RTTP spectra and decay kinetics of both intact platelets and those after short-term incubation with low concentrations of thrombin or trypsin (0.3-50 microg/ml) were investigated. Protease-induced changes in the RTTP lifetime of platelets were observed, and interpreted in terms of the slow internal dynamics of membrane protein modification. The functional role of membrane protein internal dynamics is discussed in the context of platelet aggregation and signal transduction processes.

Titin determines the Frank-Starling relation in early diastole.

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF–SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.

CD36-mediated nonopsonic phagocytosis of erythrocytes infected with stage I and IIA gametocytes of Plasmodium falciparum.

Gametocytes, the sexual stages of malaria parasites (Plasmodium spp.) that are transmissible to mosquitoes, have been the focus of much recent research as potential targets for novel drug and vaccine therapies. However, little is known about the host clearance of gametocyte-infected erythrocytes (GEs). Using a number of experimental strategies, we found that the scavenger receptor CD36 mediates the uptake of nonopsonized erythrocytes infected with stage I and IIA gametocytes of Plasmodium falciparum by monocytes and culture-derived macrophages (Mphis). Light microscopy and immunofluorescence assays revealed that stage I and IIA gametocytes were readily internalized by monocytes and Mphis. Pretreating monocytes and Mphis with a monoclonal antibody that blocked CD36 resulted in a significant reduction in phagocytosis, as did treating GEs with low concentrations of trypsin to remove P. falciparum erythrocyte membrane protein 1 (PfEMP-1), a parasite ligand for CD36. Pretreating monocytes and Mphis with peroxisome proliferator-activated receptor gamma-retinoid X receptor agonists, which specifically upregulate CD36, resulted in a significant increase in the phagocytosis of GEs. Murine CD36 on mouse Mphis also mediated the phagocytosis of P. falciparum stage I and IIA gametocytes, as determined by receptor blockade with anti-murine CD36 monoclonal antibodies and the lack of uptake by CD36-null Mphis. These results indicate that phagocytosis of stage I and IIA gametocytes by monocytes and Mphis appears to be mediated to a large extent by the interaction of PfEMP-1 and CD36, suggesting that CD36 may play a role in innate clearance of these early sexual stages.

Human Cationic Trypsinogen: Arg117 IS THE REACTIVE SITE OF AN INHIBITORY SURFACE LOOP THAT CONTROLS SPONTANEOUS ZYMOGEN ACTIVATION

Mutation of Arg(117), an autocatalytic cleavage site, is the most frequent amino acid change found in the cationic trypsinogen (Tg) of patients with hereditary pancreatitis. In the present study, the role of Arg(117) was investigated in wild-type cationic Tg and in the activation-resistant Lys(15) --> Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg(117) can be examined selectively. We found that trypsinolytic cleavage of the Arg(117)-Val(118) bond did not proceed to completion, but due to trypsin-catalyzed re-synthesis an equilibrium was established between intact Tg and its cleaved, two-chain form. In the absence of Ca(2+), at pH 8.0, the hydrolysis equilibrium (K(hyd) = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mm Ca(2+) reduced the rate of cleavage at Arg(117) at least 20-fold, and shifted K(hyd) to 0.7. These observations indicate that the Arg(117)-Val(118) bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. Consistent with this notion, autoactivation of cationic Tg was inhibited by the cleaved form of K15Q-Tg, with an estimated K(i) of 80 microm, while no inhibition was observed with K15Q-Tg carrying the Arg(117) --> His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg(117). Taken together, the findings suggest two independent, successively functional trypsin-mediated mechanisms against pathological Tg activation in the pancreas. At low trypsin concentrations, cleavage at Arg(117) results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg(117)-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg(117) --> His mutation.

Evidence for proteinase-activated receptor-2 (PAR-2)-mediated mitogenesis in coronary artery smooth muscle cells.

1. This study investigates, whether in addition to the thrombin receptor (PAR-1), the proteinase-activated receptor-2 (PAR-2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR-2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. 2. Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA-synthesis. A similar effect was observed with 10 nM thrombin. Trypsin-induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic effect. 3. The specific PAR-2-activating peptide SLIGRL or the PAR1-activating peptide SFFLRN did not elicit mitogenesis. 4. When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). 5. Trypsin (2 nM) as well as SLIGRL (100 microm) activated the nuclear factor KB (NFkappaB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR-2. Maximum activation of NFkappaB by thrombin (10 nM) was detected after 4-5 h. 6. These data suggest that PAR-2 is present in coronary SMC and mediates a mitogenic response. Activation of NFkappaB via either PAR-1 or PAR-2 does not predict mitogenesis.

Protease Modulation of the Activity of the Epithelial Sodium Channel Expressed in Xenopus Oocytes

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

Protease-activated receptor-2 (PAR-2) is present in the rat hippocampus and is associated with neurodegeneration.

Protease-activated receptor-2 (PAR-2) is a seven-transmembrane G protein-coupled receptor that possesses a structure and activation mechanism similar to those of the thrombin receptor. It is activated by low concentrations of trypsin (300 pM) and a synthetic hexapeptide [sequence of serine, leucine, isoleucine, glycine, arginine, leucine (SLIGRL), the rodent PAR-2 "tethered ligand"] representing the first six amino acids following the putative PAR-2 cleavage site. Previous studies have indicated that alpha-thrombin and SFLLRN (synthetic hexapeptide sequence of serine, phenylalanine, leucine, leucine, arginine, asparagine; the human thrombin receptor "tethered ligand") induce neurite retraction and neurotoxicity. Because of the strong similarities between thrombin receptor and PAR-2, we have proposed that PAR-2 may also participate in neurodegeneration. In the present study, we used reverse transcriptase polymerase chain reaction and immunocytochemistry to provide the first evidence that PAR-2 is present in the rat hippocampus. Moreover, we found SLIGRL to be toxic to hippocampal neurons in a concentration-dependent manner (> or = 100 microM). Calcium signaling studies were performed to aid in determining the mechanism by which PAR-2 activation is neurotoxic.

M-Cadherin-Mediated Cell Adhesion and Complex Formation with the Catenins in Myogenic Mouse Cells

M-cadherin is a member of the multigene family of calcium-dependent intercellular adhesion molecules, the cadherins, which are involved in morphogenetic processes. Amino acid comparisons between M-cadherin and E-, N-, and P-cadherin suggested that M-cadherin diverged phylogenetically very early from these classical cadherins. It has been shown that M-cadherin is expressed in prenatal and adult skeletal muscle. In the cerebellum, M-cadherin is present in an adherens-type junction which differs in its molecular composition from the E-cadherin-mediated adherens-type junctions. These and other findings raised the question of whether M-cadherin and the classical cadherins share basic biochemical properties, notably the calcium-dependent resistance to proteolysis, mediation of calcium-dependent intercellular adhesion, and the capability to form M-cadherin complexes with the catenins. Here we show that M-cadherin is resistant to trypsin digestion in the presence of calcium ions but at lower trypsin concentrations than E-cadherin. When ectopically expressed in LMTK −cells, M-cadherin mediated calcium-dependent cell aggregation. Finally, M-cadherin was capable of forming two distinct cytoplasmic complexes in myogenic cells, either with α-catenin/β-catenin or with α-catenin/plakoglobin, as E- and N-cadherin, for example, have previously been shown to form. The relative amount of these complexes changed during differentiation from C2C12 myoblasts to myotubes, although the molecular composition of each complex was unaffected during differentiation. These results demonstrate that M-cadherin shares important features with the classical cadherins despite its phylogenetic divergence.

Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue.

1. The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and SLIGR-NH2 (PP5-NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2. From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3. In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-1; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4. The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of cross-desensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5. In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 > > PP6-NH2 > or = PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6. The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7. In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8. In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > or = PP6 > PP5-NH2 > TP5-NH2). 9. In the rat aortic preparation, the relaxant actions of the PAR-2-derived peptides were mimicked by trypsin, at concentrations (0.5-1 u ml-1; 1-2 nM) lower than those that can activate the thrombin receptor. 10. The bioassay data obtained with the PAR-2 peptides and with trypsin, along with the molecular cloning/RT-PCR analysis, point to the presence of functional PAR-2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR-2-activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.

Identification of the domains of neuronal nitric oxide synthase by limited proteolysis

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.

Preferential proteolysis and activation of oxidatively modified liver microsomal glutathione S-transferase of rat.

Proteolytic activation of oxidatively modified microsomal GSH S-transferase (GSTm) was investigated. When GSTm was incubated with diamide -diazenedicarboxylic acid bis(N,N-dimethylamide)- or hydrogen peroxide in the presence or absence of glutathione, a protein-gluathione mixed-disulfide and a dimer of the enzyme were formed with a concomitant increase in transferase activity. Although control GSTm was activated 3.4-fold by 3 micrograms/ml of trypsin, the monomeric form of the transferase in which the sulfhydryl group was modified by mixed-disulfide bond formation or by covalent binding with N-ethylmaleimide was further stimulated by lower concentrations of trypsin than that used in the control. In contrast, no activation of the dimeric transferase was observed with any concentration of trypsin. In immunoblot analysis, a proteolytic product (fragment A) from the dimer transferase was detected after treatment of oxidant-modified microsomes with low concentrations of trypsin, whereas the fragment (fragment B) from the unmodified-monomeric enzyme was observed by high concentrations of trypsin. These results show that oxidatively modified GSTm is sensitive to proteolytic attack by trypsin and that only monomeric transferase is further activated by limited proteolysis.

Identification of Peptides Released from Casein Micelles by Limited Trypsinolysis

Bovine casein micelles were treated with low concentrations of trypsin before and after heating. Peptides present in the supernatant solutions of time course samples after high-speed centrifugation were separated by reversed-phase HPLC and identified principally by mass spectrometry. β-Casein was hydrolyzed rapidly, the initial cleavages occurring in the region of the plasmin-sensitive bonds. αs1-Casein was hydrolyzed more slowly, the initial cleavage points being in the N-terminal region, but some trypsin-sensitive bonds in this part of the molecule appeared to be shielded. αs2-Casein was hydrolyzed slowly from the C-terminal. Hydrolysis of κ-casein was insignificant. No peptides containing phosphoserine clusters were released. Although heating increased the rate of hydrolysis of all of the caseins, the HPLC profiles were generally similar to those obtained with unheated micelles. The results are discussed in relation to the possible structure of the surface of the casein micelle. Keywords: Caseins; micelles; milk; trypsin

Detection of functional receptors for the proteinase-activated-receptor-2-activating polypeptide, SLIGRL-NH2, in rat vascular and gastric smooth muscle.

We have studied the actions of the proteinase-activated-receptor-2 (PAR2)-activating polypeptide, SLIGRL-NH2 (SLI-NH2), in rat aorta and in gastric longitudinal muscle preparations. In the phenylephrine-precontracted aorta preparation, SLI-NH2 caused an endothelium-dependent relaxation that mimicked the action of low concentrations (0.5 U/mL) of trypsin and that was blocked by the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester. In endothelium-free aorta ring preparation, SLI-NH2 caused neither a relaxation nor a contraction. In the gastric longitudinal muscle preparation, SLI-NH2 caused a transient contraction that mimicked the action of trypsin (0.5 U/mL) and that was sensitive to inhibitors of cyclooxygenase (indomethacin) and tyrosine kinase (genistein). Further, using a reverse-transcriptase - polymerase chain reaction (RT-PCR) approach we detected, in both assay tissues, mRNA for the rat PAR2 receptor, and we ascertained, using a cloned receptor cDNA obtained from a rat intestinal cDNA library, that the putative N-terminal activating peptide sequence of the rat PAR2 receptor (SLIGRL) is identical with the one previously cloned from murine tissue. We concluded that, like the thrombin receptor, the PAR2 receptor may play a pathophysiologic role in the regulation of vascular and gastric smooth muscle contractility.

Conformational dynamics of a mobile loop in the NAD(H)-binding subunit of proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli.

Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Ths) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Ths in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Ths specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the one-dimensional 1H-NMR spectra of Ths before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Ths (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Ths with NAD+ provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Ths. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.

Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2.

We previously reported the molecular cloning of a mouse guanosine-nucleotide-binding-protein-coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase-activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northern-blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase-activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located.