Total Proteolytic Activity Research Articles

Effect of Pod Removal on Leaf Photosynthesis and Soluble Protein Composition of Field-Grown Soybeans

Well nodulated, field-grown soybeans (Glycine max [L.] Merr. var Williams) were depodded just prior to seed development and near mid pod-fill. Both treatments caused a considerable increase in leaf dry weight, suggesting continued photosynthate production following pod removal. Moreover, depodding had a marked effect on leaf soluble protein without affecting total proteolytic activity. Early depodding caused a 50% increase in leaf protein, and both early and late depodding caused the retention of protein for several weeks following the decline in control leaves. But despite this retention of protein, leaves of depodded plants showed no difference in the onset of the irreversible decline in photosynthesis. Therefore, although depodding delayed the loss of leaf chlorophyll and protein, it did not delay the onset of functional leaf senescence and in fact, actually appeared to enhance the rate of decline in photosynthesis. There was a good correlation between the irreversible decline in ribulose bisphosphate carboxylase (activity and amount) and that of photosynthesis. In contrast, the correlation did not seem as good between stomatal closure and the onset of the irreversible decline in photosynthesis. The reason total soluble protein remained high following depodding while carboxylase, which normally comprised 40% of the soluble protein, declined was because several polypeptides increased in amounts sufficient to offset the loss of carboxylase. This change in leaf protein composition indicates a change in leaf function; this is discussed in terms of other recent findings.

Pancreatic and Intestinal Response to Dietary Guar Gum in Rats

Rats were fed a fiber-free semi-purified diet or one containing 10% guar gum, a hydrophilic galactomannan, or a laboratory stock diet. The presence of guar gum in the diet decreased food intake and body weight gain perhaps due to distension of the gastrointestinal tract. Relative to the group fed fiber-free diet, liver weight was smaller and pancreas weight larger in the groups consuming guar gum or the stock diet. The latter 2 diets were hypocholesterolemic relative to the fiber-free diet. In both the unfed and fed state the wet weight of the intestine was significantly greater in the rats consuming guar gum. The greatest difference was in the wet weights of the small intestine in the fed animals. In the pancreas, there were no notable differences in digestive enzyme activity between the groups fed guar and fiber-free diet. However, in the intestine, lipase, amylase and total proteolytic activity were significantly greater in the rats fed guar gum. This elevation of enzyme activity in the intestine could be due to a slower rate of enzyme degradation or to enhancement of the enzyme secretion. The ability of guar gum to increase the volume of intestinal contents may be important in its slowing of absorption.

Comparative Inhibition of Trypsins from Several Species by Soybean Trypsin Inhibitors

Inhibition by soybean trypsin inhibitor (SBTI) preparations of trypsins and of total proteolytic activity from several species was compared. Rat, monkey, human, bovine, porcine and mink trypsins were all inhibited 90–100% by SBTI, although there were species differences at low SBTI concentrations. Extent of inhibition of trypsin from the various species did not correlate with SBTI-induced pancreatic enlargement. Total proteolytic activity was inhibited up to 40% in rat, monkey, and human preparations by SBTI, and inhibition curves were similar. SBTIs extracted from unheated soy protein isolate (USPI) or raw soy flour (RSF) were approximately equal in their ability to inhibit human trypsin. SBTI extracted from heated soy protein isolate (SPI) was slightly less effective in inhibiting human trypsin than SBTI from either RSF or USPI.

Progressive reduction in protein synthesis during involution and aging of the mouse thymus

Total proteolytic activity and protein synthesis were measured in cell-free extracts of the thymus during its involution in 1–30-week-old C57B1/6J mice, and in 10-, 12-, 14-, and 24-month-old adult mice of the same strain. The mean specific activity of the proteolytic enzymes exhibited no significant change during involution or throughout the mean life-span of the animals. In contrast, the rate of protein synthesis decreased rapidly during involution, and continued to decrease at a slower rate during aging of the mice.

Lysosomal damage under nitrite intoxication in rainbow trout ( Salmo gairdneri rich.)

1. 1. In Salmo gairdneri specimens exposed to 450 μg/l NO 2N (nearly 36 hr LC 50) for various exposure times (12–72 hr) some parameters of liver lysosomal function were examined. 2. 2. In vivo both total proteolytic activity and single protease activities were inhibited, the inhibition increasing with the lengthening of exposure time. In vitro no analogous effect was observed. 3. 3. Lysosomal membranes showed an increased fragility correlated with the physiological conditions of treated animals. 4. 4. Data were considered as the results of a nitrite mediated effect on functional and structural proteins.

Activities and partial purification of extracellular proteases of Bacteroides nodosus from virulent and benign footrot.

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55 degrees C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2CO3 and thioglycollic acid, the total proteolytic activity and its stability at 55 degrees C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8.8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8.8, suggest that this property may be used to distinguish virulent and benign strains.

Extracellular Toxins of Pseudomonas Aeruginosa III. Radioimmunoassay for Detection of Alkaline protease

A sensitive and specific solid phase radioimmunoassay (RIA) for detection of the alkaline protease (AP) of Pseudomonas aeruginosa (PA) was developed and the RIA was used to assay 10 PA strains of various origin and serotype. A great strain variability of AP production was found which differed from 180 to 0.1 microgram per ml of culture supernatant fluid (CSF). The AP concentrations were compared to the total proteolytic activity of the CSF and the results were discussed in relation to cystic fibrosis patients suffering from chronical PA lung infections.

Proteolytic enzyme activity decline in starving fish alevins and larvae

An experiment to identify the point of no return of fish alevins and larvae has been made using the proteolytic activity as an index. Larvae of Coregonus pollan and Salmo gairdneri were starved or fed with zooplankton as a control and proteolytic enzymes measured. The lowest value of the total proteolytic activity coincides with the point of no return expressed in degree-days based on survival trials.

Extracellular Toxins of Pseudomonas aeruginosa IV. Radioimmunoassay for Detection of Elastase

A sensitive and specific solid phase radioimmunoassay (RIA) for detection of the elastase (Ela) of Pseudomonas aeruginosa (PA) was developed and the RIA was used to assay 10 PA strains of various origin and serotype. A great strain variability of Ela production was found which differed from 94.1 to 0.1 microgram per ml of culture supernatant fluid (CSF). The Ela and alkaline protease (AP) concentrations were converted to proteolytic activity and combined. The sum of the calculated enzymatic values of Ela and AP correlated well with the experimentally determined values of total proteolytic activity of the CSF.

Effects of ammonia on liver lysosomal functionality in Salmo gairdneri Rich.

Salmo gairdneri specimens were exposed for 4-48 hours to three different concentrations of un-ionized ammonia (UIA). An increase in lysosomal sensitivity to osmotic shock and in total proteolytic activity occurs in the liver. The amount of this increase depends upon the exposure time or upon environmental and tissue ammonia levels. On the other hand ammonia does not affect lysosome hydrolases activity in vitro.

Partial Purification and Characterization of Endoproteinases from Senescing Barley Leaves

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP(1)) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP(2)) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP(2) contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [(14)C]ribulose-1,5-bisphosphate carboxylase ([(14)C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [(14)C]RuBPCase biphasically. A third endoproteinase (EP(3)), not detected by standard proteolytic assays, was observed when [(14)C]RuBPCase was the substrate.

Autodigestion in Crude Extracts of Soybean Leaves and Isolated Chloroplasts as a Measure of Proteolytic Activity

Two methods of measuring protein breakdown resulting from self-digestion during incubation in extracts of soybean leaves were examined. The release of free alpha-amino-nitrogen was measured with ninhydrin, and the disappearance of the large subunit of ribulose bisphosphate carboxylase (RuBPcase) was followed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rates of protein breakdown were measured as a function of temperature, pH, and leaf developmental stage and in the presence of various proteinase inhibitors. These treatments had differential effects on apparent proteolysis, depending on the method used. Determination of the ratio of alpha-amino-nitrogen plus peptide bond-nitrogen to alpha-amino-nitrogen indicated that the ninhydrin method detected the activity of exopeptidases preferentially. The disappearance of the large subunit of RuBPCase as shown on gels was due primarily to the activity of endopeptidases. The sensitivity of the two types of proteolytic degradation to proteinase inhibitors differed.Determination of temporal changes in proteolytic activity during leaf development showed that total proteolytic activity, measured by either method, increased during leaf expansion and maturation and decreased during senescence. Incubation of intact isolated chloroplasts at 37 C resulted in the breakdown of the large subunit of RuBPcase, although the chloroplasts contained no measurable proteinase activity as determined by the release of alpha-amino-nitrogen during the incubation. No acid proteinase (pH 4.5) activity was detected in the chloroplasts when hemoglobin was used as a substrate. These results indicate that the proteinases which break down RuBPCase in isolated chloroplasts may not be detectable by conventional assay procedures.

Changes in Small Intestinal Digestive Enzyme Activity and Bile Acids with Dietary Cellulose in Rats

Rats were fed semipurified diets which contained either 20% cellulose, Solka Floc, (HF) or no fiber (C) for 10 days. In the intestinal contents, the HF group had lower activity per milligram contents of trypsin, chymotrypsin, amylase, lipase and total proteolytic activity. Total activity of enzymes in the intestinal contents was also lower, except for chymotrypsin and leucine amino peptidase. Bile acid levels per milligram were lower in the HF group but the total amount was greater. The total weight of intestinal contents was greater in the HF group and total amount of protein present was elevated. The results indicate that a source of dietary fiber, cellulose, can affect the availability of enzymes and bile acids in the small intestine.

Evolution des activités protéolytiques digestives chez les Jeunes Mulgilidae

Abstract Proteolytic activity in the stomach, pyloric caeca and intestine decreases with increasing size in the three fish species Mugil auratus, M. capito and M. saliens . Differences between these species are found mainly in the gastric proteolytic activity which appears to be related to diet. This activity is pronounced in M. saliens whose diet is mainly carnivorous. Comparison of regression lines relating gastric proteolytic activity to size reveals differences which distinguish M. auratus from both of the other species. Total proteolytic activity exhibits high variability depending on the types of diet.

Inhibition of Human and Rat Pancreatic Proteinases by Crude and Purified Soybean Proteinase Inhibitors

Effects of proteinase inhibitors on total proteolytic activity and trypsin and chymotrypsin activity in human pancreatic juice were determined separately. Purified inhibitors as well as crude extracts of raw soybeans completely inhibited trypsin and chymotrypsin activity while 40 to 50% of the total proteolytic activity remained. Inhibition experiments with 1,10-o-phenanthroline showed that this residual proteolytic activity was due mainly to carboxypeptidase A and B. Comparative studies with rat pancreatic enzymes demonstrated certain similarities between the corresponding enzymes from rat and man. However, differences were revealed which indicate that the rat enzymes must be used with great caution when applied as models for the human proteinases when studying effects of soybean inhibitors.

Fructose 1,6-bisphosphatase and total proteolytic activity in the liver of Salmo gairdneri: Effects of pH and ammonia

1. In the livers of Salmo gairdneri specimens exposed to sublethal concentrations of un-ionized ammonia (UIA), changes were seen to occur in the properties of fructose 1,6-bisphosphatase along with an increase in total proteolytic activity and free aminoacid concentration. 2. The increase in environmental pH leads to similar though less marked effects. 3. In line with the studies carried out by other workers, the changes observed in this gluconeogenic enzyme are believed to be brought about by lysosomal proteases, which may be assigned the value of indicators of environmental stress.

Gastric secretory function in coeliac disease

Volume, total titrable acidity, total proteolytic activity and pepsin activity have been determined in 14 coeliac patients and in 8 controls of comparable ages and body weights. Basal secretion (B.O.), total outputs (T. O.) and peak outputs (P.O.) after pentagastrin injection have been determined. Peak outputs (values 60 min/kg) of these parameters are as follows: volume 5.0+/-1.7 ml in coeliacs, 4.3+/-1.2 ml in controls; total titrable acidity 406.1+/-155.0 mEq in patients, 296.1+/-182.4 in conttrols; total proteolytic activity 962.1+/-501.1 micronEq in coeliacs, 569.6+/-272.2 in controls; pepsin activity 789.1+/-521.8 micronEq in patients, 447.6+/-150.4 in controls.

Breakdown of Ribulose Bisphosphate Carboxylase and Change in Proteolytic Activity during Dark-induced Senescence of Wheat Seedlings

When 8-day-old wheat seedlings (Triticum aestivum L. var. Chris) are placed in the dark the fully expanded primary leaves undergo the normal changes associated with senescence, for example, loss of chlorophyll, soluble protein, and photosynthetic capacity (Wittenbach 1977 Plant Physiol. 59: 1039-1042). Senescence in this leaf is completely reversible when plants are transferred to the light during the first 2 days, but thereafter it becomes an irreversible process. During the reversible stage of senescence the loss of ribulose bisphosphate carboxylase (RuBPCase) quantitated immunochemically, accounted for 80% of the total loss of soluble protein. There was no significant change in RuBPCase activity per milligram of antibody-recognized carboxylase during this stage despite an apparent decline in specific activity on a milligram of soluble protein basis. With the onset of the irreversible stage of senescence there was a rapid decline in activity per milligram of carboxylase, suggesting a loss of active sites. There was no increase in total proteolytic activity during the reversible stage of senescence despite the loss of carboxylase, indicating that this initial loss was not due to an increase in total activity. An 80% increase in proteolytic activity was correlated with the onset of the irreversible stage and the rapid decline in RuBPCase activity per milligram of carboxylase. Delaying senescence with zeatin reduced the rate of loss of carboxylase and delayed both the onset of the irreversible stage and the increase in proteolytic activity to the same degree, suggesting that these events are closely related. The main proteinases present in wheat and responsible for the increase in activity are the thiol proteinases. These proteinases have a high affinity for RuBPCase, exhibiting an apparent K(m) at 38 C of 1.8 x 10(-7)m. The K(m) for casein was 1.1 x 10(-6)m. If casein is representative of noncarboxylase protein, then the higher affinity for carboxylase may provide an explanation for its apparent preferential loss during the reversible stage of senescence.

Survey of Major Proteases in Germinating Peanut Seeds by Affinity Chromatography

Summary Peanut seeds were germinated for 6 days before the cotyledons were extracted and the protease activity was examined. To determine papain activity an extract was passed through an activated thiol-Sepharose gel. The eluate contained 0.1% of total protease activity applied to the column. Another extract was passed through a soyabean trypsin inhibitor-Sepharose gel to determine trypsin activity. Only 0.03% of the total proteolytic activity was eluted. The eluate from a concanavaline A-Sepharose gel did not show any proteolytic activity. Those results show that nearly all of the proteolytic activity in germinating peanut seeds is probably associated with peptidases.

A study of the application of proteolytic enzymes to fish food

A study was carried out to determine the effect of adding bovine trypsin to the diet of common carp fry. When added in the same and three times the quantity normally found in natural foods it increased the proteolytic activity by the same proportion. After 58 days of the experimental diet there was no deleterious effect on the survival of fish. Food utilization, stated in terms of feeding coefficient, apparent net protein utilization (NPU), protein efficiency ratio (PER) and NPU was from 1.63 to 1.79, 19.3 to 21.3, 1.32 to 1.46 and 46.0 to 50.5 respectively. The percentage composition of bond amino acid in all fish groups was found to be the same after the experiment. However, a considerable decrease in the total proteolytic activity in the hepatopancreas was observed for these fish which were given bovine trypsin in the diet. These results, with other recently published material suggest the possibility of a new approach to the study of the application of proteolytic enzymes to fish food.