Proteolysis using enzymes in vitro

A variety of methods were tested for their ability to completely hydrolyse proteins to free amino acids in vitro. The most successful method was found to be that using the enzyme mixture papain, prolidase and aminopeptidase-M, which together completely hydrolysed the test substrates ovalbumin, BSA and insulin (B chain). A range of enzymes were coupled to insert supports, and although such immobilization simplified handling of the enzymes, the coupled enzymes proved less efficient for proteolysis than their soluble counterparts. Amino acid analysis and SDS polyacrylamide gel electrophoresis were used to identify the end products of proteolysis. The use of a pH-stat was found unsuitable for the measurement of proteolysis. In vitro proteolysis was used to study the digestibility of glycoprotein II, purified from Phaseolus vulgaris. Denatured glycoprotein II was fully digested by the enzymic method, although in its native form, the glycoprotein was only partially digested. The digestibility of trypsin inhibitor, purified from Phaseolus vulgaris was also studied. Both in its native form, and after heat treatment, the trypsin inhibitor was poorly hydrolysed. It was also found that native trypsin had some resistance to digestion conferred upon it by the presence of the inhibitor. Active trypsin inhibitor was detected in the faeces of rats whose diet included the inhibitor. The significance of these findings in vitro and in vivo were discussed. The use of proteolysis in vitro to measure the availability of amino acids was found comparable with standard assays for the measurement of available lysine and methionine

Morphological and Chemical Mechanisms of Elongated Mineral Particle Toxicities

Much of our understanding regarding the mechanisms for induction of disease following inhalation of respirable elongated mineral particles (REMP) is based on studies involving the biological effects of asbestos fibers. The factors governing the disease potential of an exposure include duration and frequency of exposures; tissue-specific dose over time; impacts on dose persistence from in vivo REMP dissolution, comminution, and clearance; individual susceptibility; and the mineral type and surface characteristics. The mechanisms associated with asbestos particle toxicity involve two facets for each particle's contribution: (1) the physical features of the inhaled REMP, which include width, length, aspect ratio, and effective surface area available for cell contact; and (2) the surface chemical composition and reactivity of the individual fiber/elongated particle. Studies in cell-free systems and with cultured cells suggest an important way in which REMP from asbestos damage cellular molecules or influence cellular processes. This may involve an unfortunate combination of the ability of REMP to chemically generate potentially damaging reactive oxygen species, through surface iron, and the interaction of the unique surfaces with cell membranes to trigger membrane receptor activation. Together these events appear to lead to a cascade of cellular events, including the production of damaging reactive nitrogen species, which may contribute to the disease process. Thus, there is a need to be more cognizant of the potential impact that the total surface area of REMP contributes to the generation of events resulting in pathological changes in biological systems. The information presented has applicability to inhaled dusts, in general, and specifically to respirable elongated mineral particles