Affinophoresis of trypsins with an anionic affinophore

Affinophoresis (Shimura, K. and Kasai, K. (1982) J. Biochem. 92, 1615–1622) is a newly devised electrophoretic separation technique for biomolecules, using an affinophore. The affinophore is a macromolecular polyelectrolyte bearing affinity ligands. It migrates rapidly in an electric field, and molecules which have affinity for the ligand are carried with it and separated from other molecules. An anionic affinophore for trypsin was synthesized. p-Aminobenzamidine, a competitive inhibitor of trypsin, was coupled to one-fifth of the car?yl groups of polyacrylyl-β-alanyl-β-alanine by the use of water-soluble carbodiimide and the residual car?yls were converted to sulfonate groups by coupling with aminomethanesulfonic acid. Affinophoresis was carried out in 1% agarose gel plates, and the protein bands were detected with Coomassie brilliant blue R250. Enhanced migrations of bovine and Streptomyces griseus trypsins towards the anode were observed with the anionic affinophore. The migrations of inactive forms prepared by active site modifications were scarcely affected. However, the affinophore was not effective for Streptomyces erythreus trypsin, an anionic trypsin, probably because of ionic repulsion between the anionic molecules. S. griseus trypsin was separated from Pronase by affinophoresis.     

Acyl-CoA: sn-Glycerol-3-Phosphate O-Acyltransferase in Rat Brain Mitochondria and Microsomes

Abstract: The subcellular distribution of acyl-CoA: sn -glycerol-3-phosphate O-acyltransferase between brain mitochondria and microsomes was investigated. The activities associated with purified rat brain mitochondrial and microsomal preparations could be distinguished by differences in their acyl-CoA specificity, products of acylation, and sensitivity to N -ethylmaleimide, trypsin, acetone, and polymyxin B. It was concluded that both brain mitochondria and microsomes possess the acyltransferase.     

A bar model for the pump and channel function of the reconstituted Na+,K+-ATPase

Purified Na+,K+-ATPase is treated with trypsin. The altered enzyme is then reconstituted into liposomes and the change in active and passive Na+,K+-fluxes is recorded. Trypsin treatment transforms the slow passive Na+,K+-fluxes into leaks. The leak formation is correlated with the degree of proteolysis and the associated decrease in Na+,K+-ATPase activity. The active Na+,K+-transport capacity decreases in parallel with the passive transport. It is thus proposed that the Na+,K+-ATPase molecule primarily contains unspecific transmembrane tunnels that are rendered ion-selective by transverse bars of specific length (bar model).     

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.     

The enhancement of the human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells by trypsin treatment is ascribed to the binding of trypsin to the Fc fragment

Abstract Human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells was remarkably enhanced by trypsin treatment of the cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of proteins of the cells treated wtrypsin was the same as that of the cells treated with pronase, although pronase treatment had been shown to reduce the activity in our previous study (FEMS Microbiol. Lett. 123, 305–310, 1994). This contradiction was clarified by the finding that trypsin bound the Fc fragment more strongly than the cells, and a small amount of trypsin remained in the cells treated with trypsin and washed well. On the basis of these results, it was concluded that the enhancement of cell activity by trypsin treatment was ascribed to binding of the Fc fragment to trypsin remaining in the trypsin-treated cells.     

Surface properties of sendai virus envelope

Surface properties of Sendai virus envelope membrane have been measured, using both biological and biophysical techniques. Both normal and trypsin-treated virus were studied. SDS gel electrophoresis showed cleavage of the F protein exclusively by trypsin. The major activity change was observed in the hemolysing activity which is an expression of F protein. Hemolysis was reduced to less than 10% of its value for intact virus. ³¹P nuclear magnetic resonance studies of the envelope surface of the native virus showed a highly restricted phospholipid headgroup environment. Interestingly, this restriction was relieved by treatment with trypsin. Thus these data suggest a role of the F protein of Sendai virus in tightly organizing the surface of the viral envelope membrane.     

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 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees 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, Plodiainterpunctella, 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.     

Thermal stabilization of trypsin with glycol chitosan

Glycol chitosan was evaluated as thermoprotectant additive for trypsin in aqueous solutions. Maximal stabilization was achieved by using a polymer/protein ratio of 2 (w/w). The catalytic properties of trypsin were not affected by the presence of the polysaccharide. The enzyme thermostability was increased from 49 °C to 93 °C in the presence of the additive. Trypsin was also 37-fold more stable against incubation at 55 °C and its activation free energy of thermal inactivation was increased by 9.9 kJ/mol when adding glycol chitosan.     

Autoactivation of Mouse Trypsinogens Is Regulated by Chymotrypsin C via Cleavage of the Autolysis Loop

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150–Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149–Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.