Motility of triton-demembranated sea urchin sperm flagella during digestion by trypsin

The survival curves for a population of reactivated spermatozoa exposed to digestion by trypsin indicate that a large number of trypsin-sensitive targets must be digested before the flagellum disintegrates. Changes in flagellar movement during trypsin digestion can be very small, especially when the spermatozoa are reactivated at 0.25 M KCl. They are not the changes which would be expected if elastic resistance of the trypsin-sensitive structures responsible for maintaining the integrity of the axoneme is a significant determinant of flagellar bend amplitude. By carrying out trypsin digestion under a variety of conditions, at least six distinct effects of trypsin digestion on parameters of flagellar movement have been detected. These include a gradual increase in the rate of sliding between tubules, gradual and abrupt changes in beat frequency accompanied by reciprocal decreases in bend angle, changes in the symmetry and planarity of bending, and selective interference with mechanisms for bend initiation and bend propagation

Developments in plant breeding for improved nutritional quality of soya beans II. Anti-nutritional factors

Nutritional value of most plant materials is limited by the presence of numerous naturally occurring compounds which interfere with nutrient digestion and absorption. Although processing is employed widely in removal of these factors, selection of cultivars of soya beans with inherently low levels would have a considerable impact on efficiency of non-ruminant livestock production. The review considers the role of plant breeding in achieving this objective. The most abundant trypsin inhibitors are the Kunitz and the Bowman Birk inhibitors, containing 181 and 71 amino acids respectively. The Kunitz inhibitor is present at a concentration of 1.4g/kg of total seed contents and the Bowman Birk inhibitor 1.6g/kg. A large number of isoforms of the Bowman Birk inhibitor have been described in soya bean cultivars and it has been shown that the general properties of the inhibitor are, in fact, attributable to different isoforms. Nulls for both Bowman-Birk and Kunitz trypsin inhibitors have been identified, allowing new low trypsin inhibitor cultivars to be produced. However, research into breeding for low trypsin inhibitor cultivars currently has limited application as trypsin inhibitors contribute a major proportion of the methionine content of soya beans. Trypsin inhibitors are thought to be involved in the regulation of and protection against unwanted proteolysis in plant tissues and also act as a defense mechanism against attack from diseases, insects and animals. Hence, in breeding programes for low trypsin inhibitor cultivars, alternative protection for growing plants must be considered. Use of soya beans in non-ruminant animal feeds is limited by the flatulence associated with their consumption

Optimisation of tennis string production from bovine intestine : a thesis presented in partial fulfilment of the requirements for the degree of Master in Technology at Massey University, Palmerston North, New Zealand

The collagen and elastin content of the beef thread samples ranged from between 47-70% and 1.2-2.5% respectively. Amino acid analysis showed that the collagen present was probably collagen Type I while the non-collagenous proteins predominantly were globulins with a small amount of albumins. Putative "strong" and "weak" batches of threads could not be differentiated on the basis of collagen content or mechanical properties such as ductility, ultimate tensile strength or Young's modulus. Treatment of "strong" or "weak" threads with three different processes, sodium carbonate, sodium carbonate/EDTA and sodium hydroxide, gave no significant differences in products The sodium carbonate/EDTA process can remove 31.9% of non-collagenous proteins over the three stages of the process. The shrinkage temperature and ductility were lowered while the ultimate tensile strength. Young's modulus and diameter are increased by the processing. Threads given three successive trypsin treatments had 47.4% (2% trypsin) and 36.2% (0.6% trypsin) of non-collagenous proteins had removed. Properties of the treated threads from this treatment gave similar trends to threads from the sodium carbonate/EDTA process except that enzyme treatment resulted smaller thread diameters. Moreover, when the treated threads from the second and third high concentration trypsin treatments were heated, they stretched rather than shrank. This phenomenon was unexpected and apparently has not been previously reported in the literature. On subjecting threads which had had three successive trypsin treatments to the sodium carbonate/EDTA process, the stretch temperature phenomenon was abolished and the normal shrinkage temperature property of collagen was restored. However, the shrinkage temperature of the thread from the integrated trypsin -sodium carbonate/EDTA process was significantly lower than that from the sodium carbonate/EDTA process alone. This integrated process does not affect the tensile strength properties, but the diameter of the treated threads using the higher trypsin rate is significantly smaller than the starting materials. However, threads from the integrated process using lower trypsin seemed to show a trend toward smaller diameters but this observation could not be shown to be statistically significant. It is suggested that two trypsin treatments integrated into a sodium carbonate/EDTA process could be an optimum process provided the smaller diameter trend of wet thread can translate into smaller diameter in dry string

Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (K-a = 8.9 x 10(5) M-1) is about 600-fold lower than that for bovine trypsin (K-a = 5.1 x 10(8) M-1); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (K-a = 7.1 x 10(8) M-1). Amino acid sequence alignment of BLCT, human beta II-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences

Trypsin digest of cancer cells surface stimulates anti-tumor immune response better than cancer cells themselves

Antigens expressed on the surface of cancer cells are accessible targets for both humoral and cell-mediated immune responses, and are therefore potential candidates for vaccine development. Treating surface of live human breast adenocarcinoma cells (MCF-7) with trypsin yields a digest that contains 0.7% of total cell protein. Despite this difference, the trypsin digest stimulates in cytotoxicity assays anti-tumor response which kills 10-40% more cancer cells than those stimulated with cells themselves. From these results, we concluded that trypsin digest obtained from live cancer cells contains the essential antigens to induce an immune-mediated anti-tumor effect, and therefore, is candidate for anti-tumor vaccine development

Chorionic Structures in Maternal Blood

Chorionic villi are the exchange structures of the placenta where human fetuses receive oxygen and nutrients from maternal blood, this article reports an improvement of a published method to recover them from the blood of pregnant, women, quotes their number and illustrates their size and trypsin impregnation. The reasons to explain the presence of chorionic structures and villi in the circulating maternal blood, the possible significance of their existence in maternal blood and the mechanisms to remove them are discussed. Their size and trypsin impregnation are illustrated. This paper discusses the significance of the presence of such allogeneic structures in the circulating blood of pregnant women and presents a brief discussion on the role of trypsin and its inhibitor in pregnancy homeostasis

Immobilisation of enzymes to Perloza cellulose resin : this thesis was presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University /

The studies reported in this thesis describe the use of Perloza™ beaded cellulose resin as a solid support for enzyme immobilisation via covalent binding. The aim of the project was to extend the uses for Perloza™ and to compare the use of well known solid support activation chemistries with a recently developed one for Perloza™. Preparations such as these have potential industrial uses. Three attachment chemistries were studied. The first activation employed 1,1-carbodiimidazole (CDI) then direct attachment of enzyme. The second again used CDI activation followed by attachment of a 6-aminocaproic acid spacer arm and then the enzyme. The final method used was attachment of a diol and subsequent oxidation to an aldehyde. The diol/aldehyde method had the advantage over the CDI methods of being based on aqueous chemistries. The two CDI based methods require extensive use of dry organic solvents. The enzymes investigated in this study were trypsin, chymotrypsin. α-amylase, horseradish peroxidase (HRPO) and alcohol dehydrogenase (ADH). Trypsin was immobilised successtully by all three chemistries. All preparations retained significant activity after immobilisation at room temperature as judged by the chromogenic substrate specific for trypsin N-α-benzoyl-DL-arginine-p-nitroanilide.HC1 (BAPNA). Measurable activity was retained in different studies from between 2 to 7 days at 60°C. The activity of immobilised trypsin with a synthetic peptide substrate was comparable to the activity of free trypsin with the same substrate. Chymotrypsin was also successfully immobilised using all three chemistries. Each preparation showed significant retention of activity after immobilisation as judged by the chromogentic substrate N-glutaryl-L.-phenylalanine-p-nitroanilide (GAPNA). Stabilisation to heating at 60°C was less successful than with trypsin but significant activity was still retained for between 3 and 6 hours. The activity of immobilised preparations with a peptide substrate was comparable to free chymotrypsin. α-Amylase, horseradish peroxidase and alcohol dehydrogenase were studied less extensively than trypsin and chymotrypsin. Nevertheless all three enzymes were successfully immobilised onto Perloza™-CDI-ACA and Perloza™-Diol/Aldehyde. Difficulty was encountered in achieving significant levels of any enzyme immobilisation to Perloza™-CDI for all three enzymes. Subsequent activity assays showed HRPO and α-amylase retained significant activity on all three resin preparations. ADH showed no measurable activity on Perloza™-CDI and very little activity on Perloza™- CDI-ACA and Perloza™-Diol/Aldehyde. Investigations have shown that enzymes can be immobilised on Perloza™ with retention of significant amounts of normal activity at room temperature and improved stability compared with free enzyme at high temperature. Comparisons of the CDI activations with the diol/aldeyde chemistry showed better performance by the latter in trypsin immobilisation and similar performance for chymotrypsin immobilisation. Horseradish peroxidase and ™-amylase were successfully immobilised using CDI/ACA and diol/aldehyde chemistries with the CDI/ACA giving higher initial specific activities than the diol/aldehyde preparation. Alcohol dehydrogenase was also successfully immobilised but gave no measurable activity

Dextran enzyme imine complexes : a preliminary study : this thesis was presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

A model system involving the formation of protein-dextran complexes has been investigated with a view to improving existing methods of drug administration. Activation of the dextran was achieved by periodate oxidation to give levels of 7%, 21% and 56% activated glucose moieties. The protein-dextran complexes were investigated with the prospect of obtaining sustained release of proteins from the dextran in an unmodified form. Covalent conjugation of proteins to carbohydrate polymers is known to confer stability on the protein. The proteins in this study were bound to the dextran through imine bonds. The proteins investigated were lysozyme, trypsin, amylase, alcohol dehydrogenase and catalase. The selection covered a range of molecular weights and varying enzymatic activities. As might be predicted, the speed of complex formation was shown to be greater at the 21% level of activation compared to the 7% activation of dextran in all cases studied. Lysozyme, the smallest protein, readily formed complexes at all three levels of activation. At the 56% level the resulting complex had an extremely high MW, greater than 1MDa. The extensive binding between the dextran and lysozyme molecules resulted in a complex that was inactive and showed no signs of releasing any lysozyme, active or inactive. At the lower levels of activation, complex was formed with relative ease. Upon conjugation lysozyme exhibited only minimal activity. Release of a lysozyme-like species with normal lytic activity was observed. Precautions were taken to minimise possible autolysis in the trypsin study. Once complexed it was postulated that autolysis would be prevented or minimised. Similarly the 56% level of activation appeared to be too high to obtain a viable complex for facile trypsin release. Sustained release of a trypsin-like protein was observed with complexes at the 7% and 21% levels. SEC and SDS-PAGE, in conjunction with a positive BAPNA assay gave support to the released species being trypsin-like. While complexed to the dextran trypsin showed no signs of activity. Released trypsin-like species and unreacted trypsin showed similar tryptic maps from a synthetic peptide, the peptide was designed to show distinctive fragments. α-Amylase, twice the MW of trypsin and over three times the MW of lysozyme, formed complexes with ease at both 7% and 21% levels of activation. Conjugation to dextran did not effect the activity of α-amylase. Over time the release of an α-amylase-like species from the complex was observed. Alcohol dehydrogenase and catalase are both high MW proteins. Complex formation was observed for each protein. Subsequent experiments showed that upon release the proteins appeared to dissociate, most probably into their subunits. It is also possible that the dimers and monomers bound to the dextran. The main advantage of conjugation in this case appeared to be to confer stability on the proteins. The ADH-complex exhibited enzymatic activity. At 7% and 21% activation levels the lower MW proteins formed complexes with dextran that exhibited release of a protein species. The higher MW proteins were possibly stabilised when conjugated to dextran, but dissociated upon release. Investigations have shown that the level of activation chosen affects the extent of binding and therefore the functions of the resultant complex. Thus activation levels can be manipulated depending on the desired result. While lower dextran activation levels appeared to be more suited for smaller MW proteins, there were indications that the larger MW proteins could form beneficial complexes at higher activation levels. Results indicated that conjugation to periodate activated dextran could be extended to further proteins with the possibility of therapeutic or commercial applications

Unfolding simulations reveal the mechanism of extreme unfolding cooperativity in the kinetically stable alpha-lytic protease.

Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease alpha-lytic protease (alphaLP) has done just that, as its unfolding is both very slow (t(1/2) approximately 1 year) and so cooperative that partial unfolding is negligible, providing a functional advantage over its thermodynamically stable homologs, such as trypsin. Previous studies have identified regions of the domain interface as critical to alphaLP unfolding, though a complete description of the unfolding pathway is missing. In order to identify the alphaLP unfolding pathway and the mechanism for its extreme cooperativity, we performed high temperature molecular dynamics unfolding simulations of both alphaLP and trypsin. The simulated alphaLP unfolding pathway produces a robust transition state ensemble consistent with prior biochemical experiments and clearly shows that unfolding proceeds through a preferential disruption of the domain interface. Through a novel method of calculating unfolding cooperativity, we show that alphaLP unfolds extremely cooperatively while trypsin unfolds gradually. Finally, by examining the behavior of both domain interfaces, we propose a model for the differential unfolding cooperativity of alphaLP and trypsin involving three key regions that differ between the kinetically stable and thermodynamically stable classes of serine proteases

Use of microwaves to improve nutritional value of soybeans for future space inhabitants

Whole soybeans from four different varieties at different moisture contents were microwaved for varying times to determine the conditions for maximum destruction of trypsin inhibitor and lipoxygenase activities, and optimal growth of chicks. Microwaving 150 gm samples of soybeans (at 14 to 28% moisture) for 1.5 min was found optimal for reduction of trypsin inhibitor and lipoxygenase activities. Microwaving 1 kgm samples of soybeans for 9 minutes destroyed 82% of the trypsin inhibitor activity and gave optimal chick growth. It should be pointed out that the microwaving time would vary according to the weight of the sample and the power of the microwave oven. The microwave oven used in the above experiments was rated at 650 watts 2450 MHz