Trypsin fibers

Leuba, S.H., Bustamante, C., Zlatanova, J., and van Holde, K. (1998) Contributions of linker histones and histone H3 to chromatin structure scanning force microscopy studies on trypsinized fibers. Biophys. J. 74(6), 2823-2829. 

Animal cell cultures that are initiated from cells removed directly from the animal are called primary cultures (Figure 2). Primary cultures include both explant cultures (i.e., cultures initiated from small pieces of intact tissue), as well as cultures initiated from preparations of individual or dispersed cells (obtained from intact tissue by mechanical or proteolytic dismption). Nerve fiber explant cultures in blood plasma were among the earliest types of tissue cultures (Harrison, 1907). Cells grow out from such tissue explants and form a single layer of cells completely filling the tissue culture vessel surface. Such cell cultures are called confluent monolayers. Confluent monolayers can then be treated with trypsin, so as to remove the individual cells from the culture vessel surface. The resulting cell suspension is then transferred into other culture containers, so that more viable monolayer... 

When myosin is digested with trypsin, two myosin fragments (meromyosins) are generated. L ht mero-myosin (LMM) consists of aggregated, insoluble a-he-hcal fibers from the tail of myosin (Figure 49 ). LMM... 

There is evidence that protease inhibitors selectively regulate the activity of specific digestive enzymes at the level of gene expression (Rosewicz et al., 1989). Specifically, soybean trypsin inhibitor increases secretion of proteases, including a form of trypsin that is resistant to inhibition but does not cause an increase in amylase secretion. Although the relationships between protease inhibitors and exocrine pancreatic secretion have received the most attention, pancreatic secretion is increased when potato fiber is added to the diet (Jacob et al., 2000), although the mechanism and signaling pathway have not been elucidated. 

In order to produce soy protein, soybeans are first dehulled, flaked, and defatted to make white flakes . Soy protein concentrates are obtained by removing a portion of the carbohydrates from defatted and dehulled soybeans. Alcohol extraction is the method most commonly used to manufacture soy protein concentrates even though it results in the loss of isoflavones. Soy protein concentrates retain most of the fiber in the original soybean and must contain at least 65% protein on a moisture-free basis to meet quality standards. The most concentrated source of soy protein is soy protein isolates (or isolated soy protein, ISP), which is required to be at least 90% protein on a moisture-free basis. It is heat-treated during processing to insure inactivation of trypsin inhibitors. Most isolated soy protein is manufactured by water extraction from defatted and dehulled soybeans and it retains the natural isoflavones. 

Several examples of the binding of enzymes to poly(vinyl alcohol) are in the literature. These could possibly be used to treat enzyme deficiency diseases. In a recent example, trypsin was immobilized on poly(vinyl alcohol) fibers using maleic dialdehyde or bromal. While the reaction was more complete with bromal, the reaction with maleic dialdehyde gave a better support which showed decreasing activity with increasing enzyme content. The activity of the bromal activated system was independant of the enzyme content (52 ). Trypsin and papain were attached to poly(vinyl alcohol) by the reaction sequence shown in Equation 13. In this case, the crosslinked poly(vinyl alcohol) is treated by the 1,3-dioxalone derivative and then converted to either the isothiocyanate or the diazonium salt for coupling with the enzyme. The bound enzymes showed significant, altho reduced, activity in each case (53). 

Fig. 5. SFM images of chicken erythrocyte chromatin fibers. (A) Untrypsinized, linker histone-containing control fibers, and (B) linker histone-stripped fibers. The stripping of linker histones destroys both the three-dimensional interactions of adjacent nucleosomes and the zig-zag arrangement of consecutive nucleosomes. Trypsinization of the N-terminal histone tails of the linker histones and core histone H3 result in the loss of the three-dimensional association of the consecutive nucleosomes, but does not destroy the zig-zag configuration. Imaging of fibers deposited onto mica was performed in air under conditions of ambient humidity and temperature (from Ref. [32]). Full width of each image corresponds to 500 nm.

Regardless of terminology, pectin qualifies as fiber under these definitions. Kertesz (52) showed that human saliva, gastric juices, trypsin, and pepsin were ineffective in hydrolyzing pectin. Werch and Ivy (53) demonstrated that pectin passes relatively unchanged through the stomach and ileum. 

Soy protein preparations contain a variety of biologically active compounds including saponins, fibers, trypsin inhibitors, and isoflavones (Potter, 2000). Hamsters and rats fed ethanol-extracted soy protein isolates had no ability to lower plasma cholesterol compared to intact soy protein isolates (Lucas et al., 2001 Ni et al., 1999). Extraction with ethanol is a treatment that would remove saponins, isoflavones, and other phytochemicals from the protein. Although one study showed that ethanol washing did not... 

Recrystallized trypsin (Armour) had no effect on the purified fibers, but commercial grade trypsin effected the dissolution of the fibers when incubated overnight at 37°C. This was apparently due to the presence in the commercial trypsin preparations of pancreatic elastase (Bald and Banga, 1949). When treated for a short time with a partly purified preparation of elastase, obtained from pancreatin, the elastic fibers were eroded to a degree that the smooth cylindrical structure was lost and under the light microscope the fiber appeared to be composed of a pair of intertwined fibrils each approximately 2 n ln diameter. The process of dissolution then accelerated, and in favorable specimens each fibril was seen to resolve into a pair of similarly intertwined members, each with a diameter of approximately 1 ft. These 4 strands were very friable and under Brownian motion rapidly broke up into short beadlike segments and soon disappeared. 

With elastin preparations as described above, the classic proteolytic enzymes of the pancreas—trypsin and the chymotrypsins—have little activity and do not produce dissolution of the fibers. In the early literature there are many reports of the dissolution of elastin libers by pan-creatin or by impure preparations of trypsin, but it was not until the work of Balo and Banga (1949) that the existence of a separate elastolytic enzyme in the pancreas was recognized. Soon after this Banga (1952) obtained a purified crystalline enzyme which was thought to be substantially free from nonelastolytic components of the pancreatic system of enzymes. 

Although it is now generally agreed that pure preparations of trypsin and chymotrypsin do not digest elastin there have been frequent reports in the early literature that pepsin is capable of dissolving elastic fibers. This was repeated recently by Fisher et al. (1960) who observed moderate elastolytic activity by three different crystalline pepsin preparations at pH 1.2. The action of pepsin was estimated to be approximately one-eighth... 

Partial hydrolysis of elastin by reagents other than organic acids also gives rise to a mixture of soluble proteins similar to a- and /3-elastin. Thus Wood (quoted by Hall et al, 1952) first demonstrated that partial hydrolysis with dilute sodium hydroxide yields a protein which forms a reversible coacervate on raising the temperature of its solutions. Later Wood (1958) showed that on prolonged heating in aqueous solution a-elastin is converted into an insoluble gellike form. Reconstituted fibers of heat-treated a-elastin resembled fibrous elastin in their elastic behavior and X-ray-dif-fraction pattern but imlike purified elastin they were dissolved by 1 % acetic acid at 100°C and by crystalline trypsin. 

Total volume of pancreatic juice, amount or concentration of bicarbonate, and activities of pancreatic enzymes are measured in duodenal contents. The enzyme most commonly measured is trypsin, but amylase, lipase, chy-motrypsin, and elastase may also be evaluated. The Lundh test consists of administering a standardized meal consisting of 5% protein, 6% fat, 15% carbohydrate, and 74% nonnutrient fiber. Advantages of the Lundh meal are that it provides a physiological stimulus to the pancreas and is simple to administer. However, administration of the meal prevents determination of the total enzyme and bicarbonate or secretory volume. Moreover, it provides inadequate or no stimu-... 

It seems reasonable to suppose that the elevated cathepsin activity in dystrophic muscle, by enhancing muscle protein breakdown in vivo, is the cause of the increased rate of protein turnover the increased synthesis could then be seen as an adaptive response to the accelerated breakdown. There is yet, however, no real evidence that this is so the factors which control the breakdown of protein in muscle fibers are probably complex and little understood (PIO). A further possibility, that the proteins of atrophying muscles are in some way more susceptible to breakdown by proteolytic enzymes, seems unlikely Kohn (K9) reported that myosin from denervated rat muscle was digested normally by trypsin, and this was found to be true also of myosin from dystrophic mice and chickens (Kl), whereas Pollack and Bird (P21) stated that the autolytic activity of denervated muscle was not increased relative to the breakdown of hemoglobin by the muscle. 

Wang and Johnson (2001) reported on test measurement methods that were major indicators of soybean oil quality. These tests included peroxide value, anisidine value, FFA content, phospholipid content, total tocopherol content, oxidative stability index, color, and moisture content. For soybean meal, they reported on urease activity, protein dispersibility index (PDI), rumen bypass or rumen undegradable protein, trypsin inhibitor activity, moisture content, residual oil content, protein content, fiber content, color, amino acid profiles, and protein solubility under alkaline (KOH) conditions. 

Factors such as fiber diameter, compactness, and the difficult solubility of collagen would seem of importance in determining such properties as resistance to hydrolytic attack by enzymes. This inertness is probably not entirely derived from the chemistry of collagen but depends also on physical factors which prevent an enzyme from gaining access to its substrate. Pepsin, acting under acid conditions on swollen fibrils, is able to cause degradation (195), and increased temperature or physical maceration facilitates even the attack by trypsin in neutral or slightly alkaline environments (194, 200). 

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