Synthetic Trypsin Inhibitor Research Articles

The requirement of a trypsin-like acrosomal enzyme for fertilization in the domestic fowl.

Summary. The effect of a synthetic, irreversible trypsin inhibitor (TLCK: tosyl-l-lysine chloromethyl ketone) upon the fertilizing capacity of spermatozoa from four individual cocks was evaluated. Recently ejaculated spermatozoa were incubated with TLCK for 20 min at 37° C before insemination. The inhibitor was used at two concentrations that did not affect sperm motility. The mean fertility for the unincubated control, non-TLCK-incubated control and 50 μg TLCK-incubated semen groups was 91·8, 85·4 and 51·9%, respectively. Using 75 μg TLCK, the mean fertility for the unincubated control, incubated control and treated semen groups was 93·1, 74·1 and 24·4%, respectively. These results demonstrate the effectiveness of a synthetic trypsin inhibitor in successfully reducing fertilization in vivo and thus suggest the need for an active acrosomal trypsin-like enzyme for fertilization of the hen's ovum.

The kinetics of inhibition of rabbit sperm acrosomal proteinase by 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK).

The interaction of acrosomal proteinase, a trypsin-like enzyme extracted from rabbit epididymal spermatozoa, BAEE (N-benzoyl-L-arginine ethyl ester), a synthetic substrate, and TLCK (L-1-chloro-3-tosylamido-7-amino-2-heptanone), a synthetic trypsin inhibitor, was analyzed by kinetic methods. TLCK is a competitive inhibitor, forming a reversible complex at the active site of the proteinase. The Michaelis (Km) and dissociation (Ki) constants are similar to those for trypsin. With time, the proteinase-TLCK complex becomes irreversible. TLCK thus labels the enzyme active site, a mechanism previously established for its inhibition of trypsin.

Blockage of fertilization in Rana pipiens by trypsin inhibitors.

Fertilization of Rana pipiens ova was blocked when spermatozoa were incubated with either naturally-occurring or synthetic trypsin inhibitors immediately prior to insemination. One synthetic inhibitor, 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride (TLCK), had an ED50, of approximately 0.275 mg/ml. Washing TLCK-treated spermatozoa prior to insemination did not eliminate the block to fertilization. At effective doses, TLCK-treated spermatozoa had normal motility. These findings suggest that, in R. pipiens, sperm enzymes similar to trypsin are necessary for fertilization.

2274. Latest development in the soya-bean saga: Kakade, M. L., Simons, Nancy & Liener, I. E. (1970). Nutritional effects induced in rats by feeding natural and synthetic trypsin inhibitors. J. Nutr.100, 1003

2274. Latest development in the soya-bean saga: Kakade, M. L., Simons, Nancy & Liener, I. E. (1970). Nutritional effects induced in rats by feeding natural and synthetic trypsin inhibitors. J. Nutr.100, 1003

Amino-acid composition of crayfish trypsin.

THE homology in primary structure of mammalian pancreatic serine proteases is well established1,2. Progress has now been made in the elucidation of the evolutionary history of this group of proteases. Neurath and his co-workers3 studied the amino-acid composition of trypsinogen isolated from the pancreas of the spiny Pacific dogfish and presented a preliminary examination of the primary structure of this enzyme. From these data a high degree of homology as compared with bovine trypsinogen can be inferred. It is now of interest what will be found beyond the gap that separates vertebrate and invertebrate animals. Invertebrate proteases are difficult to investigate because of the small amounts of material obtainable from these animals. In the crayfish Astacus leptodactylus we found an enzyme resembling mammalian trypsin in many respects4,5. It exhibits a distinct tryptic cleavage specificity when tried on the oxidized B-chain of insulin and is inhibited by all natural and synthetic trypsin inhibitors including TLCK. It is a serine protease and possesses the sequence –Asp–Ser–Gly– around the active centre serine residue6. In addition, extended immunological studies have been carried out on the crayfish trypsins of three species which revealed that they do occur as iso-enzymes varying in number and electrophoretic mobility7.

Nutritional Effects Induced in Rats by Feeding Natural and Synthetic Trypsin Inhibitors

In order to evaluate and compare the nutritional effects induced in rats by feeding navy bean trypsin inhibitor (NBTI), a natural inhibitor, with p-aminobenzamidine (p-ABA), rats were fed the following diets: 1) basal diet consisting of heated navy bean meal (control), 2) basal plus navy bean trypsin inhibitor (NBTI), and 3) basal plus p-aminobenzamidine (p-ABA), a synthetic trypsin inhibitor. Diets 2 and 3 caused growth inhibition, reduced protein efficiency ratio (PER), and pancreatic enlargement as compared to control. The supplementation of diets 2 and 3 with cystine increased growth and PER to a limited extent, whereas they were markedly stimulated by antibiotics. Supplementation with cystine or antibiotics or both had no effect either on the size or enzyme secretion of pancreas. In general, enzyme activities in pancreas and intestine were higher on diet 3 followed by diets 2 and 1. The fecal excretion of representative amino acids under study was similar in rats fed diets 1, 2, and 3. This was also true when antibiotics were added to the above diets. It appears that the nutritional effects induced in rats by feeding a natural or synthetic trypsin inhibitor are similar. The possible action of antibiotics and the failure of cystine supplement in improving the PER of diets containing the inhibitors are discussed.

The break-down of 131-I-gamma-globulin in the digestive tract of the new-born pig.

1. The intestinal absorption of [(131)I]porcine and bovine serum gamma-globulin after oral administration has been investigated in conscious pigs less than 20 hr old. Absorption was measured by the concentration of (131)I in venous blood during the 6 hr after feeding and also by the distribution of (131)I between homogenates of the alimentary tract and the rest of the animal at the end of the experiment.2. The concentration of (131)I in the blood was always low after feeding [(131)I]gamma-globulin, although a large proportion of the isotope fed was found to have left the alimentary tract. This indicated that much of the [(131)I]-gamma-globulin had been hydrolysed into fragments of low mol.wt. which were not retained in the plasma. There were no significant differences between results obtained with homologous and heterologous gamma-globulin.3. Examination by gel-filtration confirmed that, after feeding [(131)I]-serum gamma-globulin, much of the (131)I in the plasma was associated with material of mol.wt. less than 12,400 and demonstrated that the break-down of bovine gamma-globulin was comparable with that of homologous gamma-globulin.4. Comparison of the absorption of [(131)I]serum gamma-globulin from colostrum with that from a chloride solution with a similar Na(+) and K(+) concentration showed that, although the blood concentration remained low, colostrum reduced the hydrolysis of the labelled protein.5. This effect of colostrum could be simulated by the addition to the chloride solution of either the synthetic trypsin inhibitor Trasylol or a higher concentration of unlabelled protein.6. Gel-filtration of samples of the contents of the stomach, duodenum and terminal ileum after feeding [(131)I]serum gamma-globulin showed that proteolysis occurred at all these sites.