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PubMed1996

Crystal structure of human trypsin 1: unexpected phosphorylation of Tyr151.

The X-ray structure of human trypsin 1 has been determined in the presence of diisopropyl-phosphofluoridate by the molecular replacement method and refined at a resolution of 2.2 A to an R-factor of 18%. Crystals belong to the space group P4, with two independent molecules in the asymmetric unit packing as crystallographic tetramers. This study was performed in order to seek possible structural peculiarities of human trypsin 1, suggested by some striking differences in its biochemical behavior as compared to other trypsins of mammalian species. Its fold is, in fact, very similar to those of the bovine, rat and porcine trypsins, with root-mean-square differences in the 0.4 to 0.6 A range for all 223 C alpha positions. The most unexpected feature of the human trypsin 1 structure is in the phosphorylated state of tyrosine residue 151 in the present X-ray study. This feature was confirmed by mass spectrometry on the same inhibited sample and also on the native enzyme. This phosphorylation strengthens the outstanding clustering of highly negative or highly positive electrostatic surface potentials. The peculiar inhibitory behaviour of pancreatic secretory trypsin inhibitors of the Kazal type on this enzyme is discussed as a possible consequence of these properties. A charged surface loop has also been interpreted as an epitope site recognised by a monoclonal antibody specific to human trypsin 1.

Amino Acid Sequence↗
PubMed1993

Modification of K-ATP channels in pancreatic beta-cells by trypsin.

The inside-out configuration of the patch-clamp method was used to study the effects of trypsin on the activity of ATP-sensitive potassium (K-ATP) channels from isolated mouse pancreatic beta-cells. Trypsin (20 micrograms/ml) irreversibly enhanced channel activity around twofold by reducing the interburst intervals without altering the burst kinetics. No effect on the single channel conductance or the inward rectification produced by internal Mg2+ was observed: however, the protease did reduce the inhibitory effect of Mg2+ on channel activity. Trypsin both prevented rundown of K-ATP channel activity and reactivated the channels after complete rundown. These effects of trypsin were absent in the presence of trypsin inhibitor. The protease also reduced the inhibitory effect of ATP on channel activity, increasing the dissociation constant from 7 to 49 microM. Trypsin removed the activating effect of ADP (0.1 mmol/l) on channel activity and reduced the inhibitory effect of tolbutamide (0.5 mmol/l). Carboxypeptidase A did not activate K-ATP channels in excised patches, although it was able to slightly reactivate channels after complete rundown, whereas chymotrypsin increased K-ATP channel activity but it did not produce reactivation. The effects of papain were similar to those of trypsin.

Adenosine Diphosphate↗
PubMed1986

Variations in dried blood spot immunoreactive trypsin in relation to gestational age and during the first week of life.

Dry blood spot immunoreactive trypsin was measured by radioimmunoassay in 84 preterm babies and in 65 full-term newborns studied daily from the first to the fifth day of life. In a control group of 3858 full-term newborns, trypsin concentrations at days 4-6 of life exhibited a log-normal pattern distribution, the geometric mean being 18 ng/ml serum. Immunoreactive trypsin concentrations did not change significantly between days 1, 2, 3, 4 and 5 after birth. Immunoreactive trypsin was found to be significantly lower (geometric mean 9 ng/ml, P less than 0.01) in preterm newborns before 32 weeks of gestation. In hypotrophic newborns of 34 weeks gestational age, immunoreactive trypsin values were higher than those observed at 31 weeks of gestation in eutrophic newborns, the mean birth weight not being different between both groups. These data suggest that trypsin production by the pancreas is dependent on maturity but does not seem related to intrauterine nutritional status. Immunoreactive trypsin concentrations do not change after 32 weeks gestational age and during the first postnatal week.

Blood Stains↗
PubMed1981

Circulating trypsin-like immunoreactivity in chronic pancreatitis.

The present study has been designed to work out the factors regulating the fasting serum levels of trypsin-like immunoreactivity in chronic pancreatitis. One hundred patients with chronic pancreatitis have been included and studied during a painless phase of the disease. No relationships have been observed between serum trypsin-like immunoreactivity and the presence of pancreatic calcifications. Serum immunoreactive trypsin levels showed a gradual decline parallel to the progressive impairment of bicarbonate and enzyme (trypsin and chymotrypsin) outputs in duodenal aspirates during pancreatic secretory studies. Therefore, serum trypsin-like immunoreactivity levels are thought to reflect the functional capacity of the exocrine pancreas. Reduced levels of trypsin-like immunoreactivity were detected in almost all patients with diabetes and steatorrhea. However, the finding of low levels also in a minority of chronic pancreatitis patients with normal endoscopic retrograde cholangiopancreatography or pancreatic secretory tests points to other factors which, in addition to the atrophy of the pancreatic parenchyma, may influence the circulating levels of trypsin-like immunoreactivity in chronic pancreatitis.

Antigens↗
PubMed1984

Effects of trypsin and protein modification on the renal transporter of p-aminohippurate.

Basal-lateral membranous vesicles prepared from rabbit renal cortex exhibited Mg2+-stimulated, probenecid-inhibitable transport of p-aminohippurate (PAH). This uptake could be completely eliminated by incubating the membranes with trypsin at a weight ratio of 1:700 (trypsin/membrane protein). The loss of PAH uptake activity occurred in two stages. Over the first ten minutes of the vesicles' exposure to trypsin, there was a nearly linear loss, with respect to time, of about 80% of the PAH uptake activity. The remaining 20% of activity was resistant to further trypsin digestion for the next ten minutes, but by twenty-five minutes a total inactivation of the uptake activity occurred. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of normal and trypsin-treated vesicles showed very little degradation of proteins. However, two minor polypeptides (Mr - 410,000 and 388,000) were degraded during the first ten minutes of the membranes' exposure to trypsin. After twenty minutes of exposure, two other polypeptides (Mr = 94,500 and 87,500) were degraded. Chymotrypsin and clostripain also caused a loss of PAH transport activity. However, compared to the effects of trypsin, the effects of these two proteases were less complete, slower in onset, and for clostripain, a much higher concentration of enzyme was required. Other functions or properties of the vesicles including morphological appearance, degree of vesiculation, glucose space or Na+-dependent L-glutamate transport and Na+,K+-ATPase activity were not altered by the concentration of trypsin which abolished 80% of the transport of PAH. Thus, it is possible that one or more of the degraded polypeptides detected by polyacrylamide gel electrophoresis comprises the PAH transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Transport Systems↗
PubMed1985

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase.

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.

Animals↗
PubMed1987

Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion.

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.

4-Chloro-7-nitrobenzofurazan↗
PubMed1976

Studies on aspartase. III. Alteration of enzymatic properties upon trypsin-mediated activation.

Highly purified aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli, already of full activity, is further activated 3.3-fold by limited treatment with trypsin. The activation requires a few minutes to attain maximum level, and hereafter the activity gradually decreases to complete inactivation. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. Upon trypsin-mediated activation no appreciable change is detected in the molecular weight of the enzyme subunits as judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis, nor in the pH vs. activity profile in the presence of added metal ions. However, S0.5 and hill coefficient for L-aspartate considerably increase upon activation. As the trypsin-mediated activation proceeds, a marked absorbance difference spectrum of the trypsin-treated aspartase vs. untreated aspartase appears with negative absorbance maxima at 278 and 285 nm. When the trypsin-activated enzyme is denatured in 4 M guanidine-HCl, followed by removal of the denaturant by dilution, the enzyme activity is readily restored to as much as 1.5 times that of the native enzyme, indicating that the trypsin-activated enzyme is rather a stable molecule.

Ammonia-Lyases↗
PubMed1978

Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution.

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.

Animals↗
PubMed1980

Release of ribosomes from thylakoids and endoplasmic reticulum with trypsin.

Release by trypsin of chloroplast ribosomes from rough thylakoids of Chlamydomonas reinhardtii was compared with release by trypsin of ribosomes from endoplasmic reticulum of rat liver. Rough thylakoids could be stripped of ribosomes with trypsin. Ribosomes were released as a mixture of polysomes, monosomes, and subunits. Chymotrypsin was much less effective than trypsin. Released ribosomes behaved like intact particles on zone velocity centrifugation, but showed extensive modification of their polypeptides. Stripped thylakoids sedimented as membranes, but also showed extensive modification of their polypeptides. Some ribosomes could be released in polysomes from rough endoplasmic reticulum by trypsin, provided the incubation mixture contained rat liver extract as a source of RNAase inhibitor. In its absence, ribosomes were released predominantly as monosomes, as reported by others. The rat liver extract inhibited release of polysomes from rough thylakoids by trypsin. Thus, the difference in form in which ribosomes are released from thylakoids, and endoplasmic reticulum by trypsin may be due to the high RNAase content of the latter.

Animals↗
PubMed1971

Isolation and immunochemical studies of dog trypsin.

Dog trypsin (EC 3.4.4.4) was isolated from dog pancreatic juice on SP-Sephadex C-50. The preparation was homogeneous on disc electrophoresis at pH 4.3. On agarose gel electrophoresis at pH 8.6, dog pancreas trypsinogen had the mobility of an alpha 2-globulin and trypsin the mobility of a beta-globulin. On gel filtration on Sephadex G-75 at pH 4.0, dog trypsin was eluted in the same fractions as bovine trypsin. It was inhibited by soybean trypsin inhibitor. Rabbit anti-dog trypsin inhibited the caseinolytic activity of bovine trypsin by about 60%.

Animals↗
PubMed1991

Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene.

Streptomyces griseus trypsin (E.C. 3.4.21.4) is one of the major extracellular proteinase, which is secreted by S. griseus. The gene encoding S. griseus trypsin was isolated from a S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing the gene for S. griseus trypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividans. Deduced amino acid sequence from the nucleotide sequence suggests that S. griseus trypsin is produced as a precursor, consisting of three portions; an amino-terminal pre sequence (32 amino acid residues), a pro sequence (4 residues), and the mature trypsin. The S. griseus trypsin consists of 223 amino acids with a computed molecular weight of 23,112. The existence of proline at the pro and mature junction suggests that the processing of S. griseus trypsin is non-autocatalytic.

Amino Acid Sequence↗
PubMed1979

Plasma trypsin in chronic pancreatitis and pancreatic adenocarcinoma.

We have used a simple and precise radioimmunoassay to measure trypsin in human plasma. Fasting plasma trypsin concentrations were extremely low in patients with chronic pancreatitis with steatorrhoea (5 +/- 2 ng/ml) when compared to healthy controls (86 +/- 7 ng/ml, p less than 0.001). In patients with chronic pancreatitis but no steatorrhoea basal plasma trypsin levels were similar to those of the normal controls (99 +/- 25 ng/ml). A small but significant postprandial rise in plasma trypsin concentrations was observed in normal subjects (mean increment 15 +/- 4%, p less than 0.005, paired t test) but was absent in patients with chronic pancreatitis with steatorrhoea. In contrast to exocrine deficient chronic pancreatitis, other malabsorptive conditions associated with steatorrhoea (active coeliac disease and acute tropical sprue) demonstrated mean fasting trypsin concentrations similar to controls. Patients with adenocarcinoma of the pancreas had basal trypsin concentrations similar to healthy subjects as did patients with adenocarcinoma of the stomach, colon, rectum, brochus, and breast. In some cases measurement of plasma trypsin may be of help in the differential diagnosis of steatorrhoea.

Adenocarcinoma↗
PubMed1988

Enhanced urinary trypsin inhibitory activity in gentamicin-induced nephrotoxicity in rats.

We delineated in rats, the relationship between trypsin inhibitory activity in the urine and the nephrotoxic effects of gentamicin, eg, proteinuria and deterioration of glomerular filtration rate (GFR), measured by creatinine clearance. Gentamicin, 70 mg/kg per day, was injected intraperitoneally for 6-10 successive days. Serum and urine gentamicin levels were determined by a microbiological test. Trypsin inhibitory activity was assayed by the casein digestion method. The results showed a steady increase in urinary trypsin inhibitory activity starting from the fourth injection day. The increased levels of urinary trypsin inhibitory activity were associated with increased levels of urinary gentamicin excretion (r = 0.36, p less than 0.02, n = 50 after the fourth injection day), and were significantly higher than in control groups (p less than 0.001). The urinary trypsin inhibitory activity was inversely correlated with the GFR (r = -0.45, p less than 0.01, after the second injection day). The serum trypsin inhibitory activity remained unchanged throughout the study period in all groups. These data suggest that increased urinary trypsin inhibitory activity may be involved in the pathogenesis of gentamicin-induced nephrotoxicity.

Animals↗
PubMed1986

Trypsin suppression of pancreatic enzyme secretion. Differential effect on cholecystokinin release and the enteropancreatic reflex.

We have recently demonstrated that intraduodenal perfusion of trypsin inhibits phenylalanine-stimulated pancreatic enzyme secretion by suppression of release of cholecystokinin (CCK). It is not known whether trypsin in the duodenum inhibits pancreatic secretion stimulated by a cholinergic mechanism. To investigate this question gastrointestinal intubation and perfusion were performed in 12 healthy subjects. Volume and osmoreceptors in the duodenum, which are known to elicit pancreatic secretion through cholinergic pathways, were stimulated by infusing increasing volumes (1.0, 2.5, and 5.0 ml/min) of normal saline or increasing osmolality (300, 400, 500 mosmol) of NaCl solution. Increasing the rates of intraduodenal perfusion of normal saline or increasing the osmolality of the duodenal perfusates caused a dose-related increase in pancreatic trypsin and chymotrypsin outputs without affecting basal plasma CCK levels (0.9 +/- 0.1 pM). The volume- or osmolality-stimulated pancreatic secretions were abolished by atropine, but not by intraduodenal perfusion of trypsin. In contrast, intraduodenal perfusion of phenylalanine (10 mM) produced a significant increase in plasma CCK levels (6.7 +/- 0.8 pM) and a three- to fourfold increase in pancreatic enzyme outputs. Perfusion of the duodenum with bovine trypsin (1 g/L) reduced the plasma CCK levels to basal values and significantly attenuated the phenylalanine-stimulated enzyme secretion to 63% +/- 4% of control. Simultaneous administration of atropine and intraduodenal perfusion of trypsin completely abolished the pancreatic enzyme response to phenylalanine stimulation. These studies indicate that the intestinal phase of human pancreatic enzyme secretion is under both hormonal and neural control. Intraduodenal trypsin inhibits only pancreatic secretion mediated by CCK release, and not that mediated by cholinergic mechanisms. These observations suggest that feedback regulation of pancreatic enzyme secretion is stimulus specific.

Adult↗
PubMed1992

Increased activity and stability of poly(ethylene glycol)-modified trypsin.

The reaction of trypsin with activated monomethoxypoly(ethylene glycol) with various molecular masses led to the development of a series of poly(ethylene glycol)-modified trypsins (PEG-trypsins). On determining the catalytic properties of PEG-trypsin using N-benzoyl-L-arginine p-nitroanilide as a substrate, a three- to fourfold increase in the maximal velocity of hydrolysis was found to occur, whatever the size of the PEG moiety used. PEG-trypsin with higher molecular mass moieties showed lower Michaelis constant values. The activation of trypsin was neither reversed by nucleophiles such as hydroxylamine, nor prevented when modification was carried out in the presence of benzamidine or in the presence of the polypeptidic soybean trypsin inhibitor. Chemical modification of about 80% of the free amino groups with PEG chains significantly improved the resistance to heat and detergents. This might result from the formation of a highly hydrogen-bonded structure around the enzyme.

Enzyme Activation↗
PubMed1996

Trypsin promotes C6 glioma cell proliferation in serum- and growth factor-free medium.

C6 glioma cells could be successively subcultured and maintained in serum- and growth factor-free medium (SF/GFF medium). C6 cell proliferation in SF/GFF medium was positively correlated with the initial cell density at plating. This correlation disappeared when the medium had been renewed early after cell adhesion (3 h after plating), suggesting that C6 cell growth depends on some diffusible factor in the medium before renewal, and that this factor is not secreted from C6 cells in the assay culture but is transferred from the cell suspension. The supernatant of trypsinized C6 cell suspension (SCS), trypsin-EDTA solution for routine cell harvesting use, and modified trypsin of protein sequencing grade all promoted C6 cell proliferation at, appropriate dilutions or concentrations under SF/GFF conditions. The growth promoting effects of SCS and trypsin-EDTA solution were completely inhibited by soybean trypsin inhibitor. These results demonstrate that the serine protease trypsin has a proliferative effect on C6 cells continuously subcultured in SF/GFF medium. In addition, it is suggested that trypsin used for cell dispersion is transferred from cell suspension into the culture, where it promotes C6 cell growth after passage in our SF/GFF subculture system.

Animals↗
PubMed1984

Trypsin from Greenland cod, Gadus ogac. Isolation and comparative properties.

Trypsin(ogen) was isolated from the pyloric ceca of Greenland cod. Greenland cod trypsin catalyzed hydrolysis of N alpha-benzoyl-DL-arginine p-nitroanilide, tosyl arginine methyl ester and protein and was inhibited by the serine protease inhibitor PMSF and other well-known trypsin inhibitors. Greenland cod trypsin was more stable at alkaline pH than at acid pH; and was inactivated by relatively low thermal treatment. Like other trypsins, the enzyme was rich in potential acidic amino acid residues but poor in basic amino acid residues and had a molecular weight of 23,500; but it had less potential disulfide pairs, less alpha-helix and a lower H phi ave than other trypsins previously characterized. Reactions catalyzed by Greenland cod trypsin were not very responsive to temperature change, such that specific activity was relatively high at low reaction temperature.

Amino Acids↗