Crystalline Trypsin Research Articles

THE ACTION OF PEPSIN AND TRYPSIN ON THE EGG SHELL OF ACHETA DOMESTICUS (L.) (ORTHOPTERA:GYLLIDAE)

The maternal endocuticle, or exochorion, of the swollen egg shell of Acheta domesticus (L.) is digested by crystalline trypsin only after preliminary treatment with pepsin. Pepsin appears to digest the protein moiety of a mucoprotein, thereby exposing the endocuticle to attack by crystalline trypsin. The postulated mucoprotein would cover the egg and presumably lubricate its passage down the genital ducts. Purified 'trypsin', a mixture of proteolytic (and other) enzymes, will digest the endocuticle without previous pepsin treatment. Newly laid eggs are unaffected by these enzymes.

Elastase activity in human pancreatic secretions.

IN 1949, Balo and Banga1 first reported the isolation of the pancreatic enzyme elastase, which was unique in its ability to render soluble elastin (an important structural component of arteries, veins, skin and lung). This enzyme was first believed to be mucolytic in action, but it is now established that it is also a potent proteolytic agent2. Kokas, Foldes and Banga3, in a series of acute experiments, cannulated the main pancreatic duct of dogs and reported demonstrable elastolytic activity in canine pancreatic secretions. Grant and Robbins4 in a similar experiment reported that elastase in the pancreas is inactive. They hypothesize that the inactive ‘proelastase’ must be activated (by small amounts of crystalline trypsin or enterokinase) before elastolytic activity can be demonstrated.

Pancreatic enzyme secretion produced in the rat by trypsin inhibitors.

In previous experiments, rats fed soybean trypsin inhibitor exhibited an exaggerated secretion of principal pancreatic digestive enzymes, as measured by the enzyme activities of the pancreas and intestine shortly after feeding. The present experiments studied the effect of trypsin inhibitors from egg white and lima bean on this pancreatic response in the rat. The influence of atropine and bilateral vagotomy on such secretion was also investigated. Both egg white and lima bean trypsin inhibitors evoked pancreatic enzyme secretion similar to that produced by soybean trypsin inhibitor. Inactivation of the inhibitors by reaction with crystalline trypsin prior to feeding did not impair the response. The physiological activity of these substances appeared to be closely associated with their trypsin inhibitor activity, yet inactivation of endogenous trypsin in the small intestine seemed not to be necessary for the response. Although vagotomy or a parasympathetic blocking agent did not suppress the pancreatic response to soybean trypsin inhibitor, no evidence for the release of a humoral factor by the inhibitor was obtained by cross-circulation observations.

Studies on the digestive proteinase of clothes moth larvae ( Tineola bisselliella)—II : Digestion of wool and other substrates by Tineola proteinase and comparison with trypsin

The digestibility of undenatured wool and other substrates by partially purified clothes moth larval proteinase has been compared with that by trypsin. Under the experimental conditions over 30 per cent of wool was digested whereas crystalline trypsin had no effect. Wool was completely digested in 2 hr when 0·125 M cysteine was added to the Tineola enzyme. The clothes moth enzyme can be considered as having distinct keratinolytic activity, but the amount of wool digestion observed in the absence of reducing agents does not account for all of the digestion which occurs in the insect. The role this enzyme in digestion of wool by clothes moth larvae is discussed.

An Evaluation of Serum "Trypsin" Tests

Abstract When crystalline trypsin was dissolved at various concentrations in pooled serum, the rates of hydrolysis of α-benzoyl-1-arginineamide (BAA) and N-α-benzoyl-dl-arginine-β-naphthylamide (BANA) were not linear functions of the trypsin concentration. Also, the plot of trypsin activity in relation to trypsin concentration differed with different sera. The apparent tryptic activity toward these substrates is felt to be a function of the concentration of serum trypsin inhibitor and, with BANA only, some unidentifed proenzyme which is activated by trypsin. Therefore, it is concluded that it is not feasible to measure serum trypsin concentration with these substrates.

The effect of trypsin on measles virus antigenicity.

SummaryDigestion of whole measles fluids with crystalline trypsin at either 37° or 45° results in a 2- to 3-fold increase in amount of sedimentable complement fixing antigen. This treatment, however, markedly reduces the ability of these fluids to induce formation of neutralizing or complement fixing antibodies when injected in guinea pigs.

Trypsin Therapy in Pulmonary Diseases, 1955–1960

The role of trypsin as a therapeutic modality for the treatment of inflammation and edema, to liquefy purulent debris and inspissated thick, viscid mucus, has been established.'@5 Clinical observations described6 to document the efficacy of buccally administered trypsin tablets, given in conjunction with intramuscular trypsin, for the rapid control of the signs and symptoms of respiratory infections. Liquefication of sputum and rapid dimunition of cough were observed3 in patients with acute and chronic bronchitis and bronchial asthma following the use of intramuscular trypsin. It has been reported'3 that treatment with intramuscular trypsin, given simultaneously with antibiotics, effected a clinical cure within a period varying from three to six weeks in cases of tuberculous lymphad enitis and bone tuberculosis (sternum and first rib). The author con cluded that trypsin, given simultaneously with antituberculosis drugs and antibiotics, appears to be the treatment of choice in tuberculosis lymphadenitis and secondary sinuses, or bone tuberculosis. After two months of combined treatment with trypsin and antituber culosis drugs and antibiotics, there was a complete resolution of the pul monary lesion in a patient with miliary tuberculosis and tuberculous meningitis. This result prompted the suggestion'4 that trypsin in sesame oil be considered as an adjunct to antituberculosis drugs and antibiotics in the management of miliary and meningeal tuberculosis. Our interest in the proteolytic enzymes was initiated in 1955. With passage of time, new pharmaceutical forms of crystalline trypsin were developed in an endeavor to promote patient cooperation. Our studies started with crystalline trypsin in sesame oil (Parenzyme). In 1957, we used buccal tablets of crystalline trypsin (Parenzyme B); in 1958-59, trypsin in aqueous solution (Parenzyme A) and finally in 1959 an enteric coated tablet of trypsin for oral administration (Orenzyme). We will present our experiences with the various available forms of trypsin along with our results in four groups of patients.

Effect of Trypsin on a Susceptible Strain of Influenza A Virus

Summary The study dealt with a trypsin-sensitive line of influenza A virus. Incubation of this virus with 0.25% crude (Difco 1:250) or crystalline trypsin for 1 hr at 37°C completely destroyed the hemagglutinins and reduced the egg infectivity by more than 99%. A second line of virus derived from the same isolate as the trypsin-sensitive line and closely related to it antigenically, and several other strains of influenza A, showed no significant decrease in either hemagglutinins or infectivity when treated in a similar manner. Crude trypsin inactivated the sensitive virus at a more rapid rate than did crystalline trypsin. The results presented indicate that the crude product contains some heat labile constituent(s) which alone is incapable of inactivating the virus but which does make it more readily available to trypsin action. All preparations of the sensitive virus appeared to contain a small percentage of trypsinresistant particles, but subcultures of these particles in embryonated eggs always yielded virus populations composed primarily of trypsinsensitive virus, and in that respect they were indistinguishable from the original virus suspension. By employing special procedures, a stable line of virus that possessed greatly increased resistance to trypsin was obtained from the sensitive strain. Repeated passage of the sensitive virus at either high or low dilution caused no significant variation in its susceptibility to trypsin, which suggests that this property might serve as an easily recognized and stable marker for genetic studies.

Chromatographic studies on trypsin, trypsinogen and the activation process

Crystalline preparations of trypsin and trypsinogen were chromatographed on carboxymethylcellulose at pH 3.2 using gradient elution of increasing salt concentration. Crystalline trypsin contained an inert component which could be quantitatively precipitated with 0.05 M CaCl 2 at pH 8. This permitted the recovery of trypsin which was chromatographically homogeneous. A chromatographic study of the autocatalytic activation of trypsinogen in the presence and absence of calcium ions suggested that this inert protein may be the result of activation in the absence of calcium ions. It was also demonstrated that this Chromatographic technique could be applied to the large-scale preparation of trypsinogen which is relatively low in trypsin.

Photoreactivation of transforming DNA by an enzyme from bakers' yeast.

Ultraviolet-inactivated Hemophilus influenzae transforming DNA recovers its activity when mixed with cell-free extracts of bakers' yeast and exposed to visible light. The active agent in the extract is not used up in the reaction, and purification has not separated it into more than one non-dialyzable component. It differs from the agent in Escherichia coli extract, which produces very similar photoreactivation, but which can be resolved into non-dialyzable and dialyzable components, the latter being used up during illumination. The yeast agent can be salted out of solution and recovered quantitatively; it is inactivated by crystalline trypsin and chymotrypsin and by brief heating at 60 degrees C.-all facts suggesting that it is an enzyme for which ultraviolet lesions in the DNA serve as substrate. The kinetics of recovery are also consistent with such an assumption. This enzyme is unusual both because it is involved in a light-dependent reaction and because it has a non-destructive action on DNA outside an intact cell.

Purification and some properties of a protease from pea seeds, Pisum sativum L, s.sp. arvense A. and G.

The purification of a protease from pea seeds (about 350-fold) is described and some properties of the purified enzyme for which the name arvensin is proposed are discussed. The enzyme acts optimally on casein at pH 8.0; it is inhibited by some salts of heavy metals, but do not by iodoacetamide. It somewhat resembles trypsin. The specific activity of the most pure preparation is about a quarter of that of crystalline trypsin.

Peptide maps obtained with tryptic digests of the proteins of some strains of tobacco mosaic virus

Proteins were separated from five strains of tobacco mosaic virus (TMV) by treatment with acetic acid. The isolated proteins were digested with crystalline trypsin at pH 7.8 and the resultant peptides were separated into fractions, soluble and insoluble, respectively, at pH 4.5. The soluble peptides, comprising from 60 to 76% of the material, depending on the strain, were separated by a combination of paper electrophoresis and paper chromatography. Peptide spots were located by spraying with various indicator reagents. “Maps” thus obtained of the peptides from five strains of TMV showed many features in common and several striking differences.

Electrophoretic heterogeneity of trypsin.

IN a previous communication1 it was reported that crystalline trypsin, when submitted to paper electrophoresis at pH 2.6, shows the presence of three different and proteolitically active fractions, even in the absence of calcium ions.

ENZYMATIC DIGESTION, AN AID IN PRIMARY ISOLATION OF BACTERIA FROM DISEASED TISSUES

Animportant function ofanydiagnostic laboratory istheisolation ofpathogenic microorganisms fromtissue specimens andtheir identification. A reviewofourexperience overthe pastfewyearsindicated thatourstandard methods forprimary isolation ofpathogens sometimes failed todemonstrate thepresence ofsuch organisms, particularly wherecasehistories strongly indicated bacterial infection. Itwaspostulated thatourroutine methods maynotbeadequate torelease bacteria from tissues whensuchorganisms maybesurrounded by normalorpathological cellular ortissue barriers. Ithasbeenknownformanyyearsthatthe intercellular matrix oftissues canbedigested withcontrolled amounts ofproteolytic andhydrolytic enzymes(Monn6, 1946; Day,1947; Holtfreter, 1948; Essner etal., 1954). Living microorganisms contained within thesetissues will resist digestion, whereas deadcells arerapidly digested (Northrop etal., 1948). Ourobjective wastodetermine whether diseased tissue specimens,whentreated withcertain proteolytic enzymes, wouldyield pathogenic microorganisms whichotherwise mightnotbe recovered by routine methods. MATERIALS AND METHODS Tissue specimens. Specimens werereceived from clinically diseased animals, ininsulated containers, refrigerated during shipment either with dryice orice. Enzymes. Crystalline trypsin andexperimental enzymeX-108(Cyanamid) wereusedinthis study. Bothwerereceived asdriedpowders in vials. X-108isa powerful proteinase isolated from fermentation beerofCondiobolus brefeldianus. In 1Presentaddress: HenryPhippsInstitute, University ofPennsylvania, Philadelphia, Penn

Mode of action of trypsin on Bacillus megaterium.

SummaryOur previous results, demonstrating with crude trypsin the digestibility of B. megaterium and the non-digestibility of B. cereus, could be confirmed with crystalline trypsin only by pretreating the bacteria with fat solvents or by adding lipase to it. The surface of B. cereus is different from that of B. megaterium; a lipid material can be removed from the surface of the later one which renders the bacterial protoplasts accessible to the action of proteolytic enzymes.

The structure of the yeast cell wall. II. Degradative studies with enzymes

In contrast to the use by earlier investigators of alkali in degrading the walls of yeast cells, various enzymes have now been employed for this purpose, the reaction products being characterized with the aid of chromatography. Isolated cell walls were dissolved completely by enzyme preparations fromHelix pomatiaand to various extents up to about 50% by enzymes present in malt, by crystalline trypsin or by crystalline papain. In the case of the malt and snail enzymes, the rapidity of the reactions was directly proportional to the phosphorus content of the cell wall. Both glucose and N-acetylglucosamine were formed using either the malt or the snail enzymes, but no dialyzable products were detected following the action of trypsin or papain. In all cases the non-dialyzable products included both mannan and glucan fractions. Using papain, an initial phase of rapid reaction led to almost complete loss of the negative charge associated with phosphate ions. Effectively the whole of the phosphate thereby dissolved was subsequently recovered in the form of a complex with mannan and protein, which therefore probably forms part of the wall surface. On the basis of such observations, the major constituents of the wall are envisaged as consisting of an insoluble glucan matrix (ca. 50%) attached by means of a protein ‘cement’ (ca. 7%) to mannan (ca. 20%) and also to soluble glucan (ca. 10%). From the magnitude of the charge density at both the inner and outer surfaces of the wall, it is tentatively concluded that a relatively large fraction of each is occupied by mannan and a comparatively small fraction by protein.

The Use of Trypsin in the Therapy of Tuberculous Lymphadenitis and Tuberculous Fistulae

This report deals with nine cases of tuberculous lymphadenitis treated with trypsin in sesame oil and uninterrupted antituberculosis drugs. Follow-up periods varied from eight to 16 months, including histopathological evaluation of the results of treatment. Antituberculous drugs in tuberculous lymphadenitis have been in general disappointing. The natural course of tuberculous lymphadenitis with its alternating periods of quiescence and relapse make it difficult to evaluate those few cases that have met with apparently successful treatment. The disappointment is more deeply felt as other forms of primary tuberculous and fresh exudative lesions have responded so well to the drug now in use. We felt that any additional factor that will enhance the phagocytic power of the mononuclear cells1. 3, � and open up vascular channels to the site of the causative factor of the disease for the drugs in use may be helpful in overcoming the infection.2’ 6 With this in mind, pure crystalline trypsin in sesame oil,t each ml. containing 5 mg., was administered intramuscularly simultaneously with various antituberculous drugs. Methods: We selected only those cases where unequivocal evidence of tuberculous etiology was present and which under prolonged drug treatment did not improve or become progressively worse. In every case we had biopsy or culture for Koch’s bacillus before the start of treatment, where feasible during treatment, and in all cases at the end of treatment. Each case had x-ray films of the neck, chest, spine and appropriate bones. Routine blood studies, urine examinations, liver function tests, and ECG were performed at the start, during, and on termination of treatment. The cases were examined daily on the ward. Trypsin was administered intramuscularly in combination with one or more of the following: Rimifon, para-aminosalicylic acid and streptomycin.

The effect of intramuscularly administered trypsin on blood injected into the vitreous of rabbits.

Clearing of vitreous hemorrhages is variable and unpredictable in spite of measures used to accelerate absorption (von Sallmann,1Lepri2). Crystalline trypsin in sesame oil was first studied by Hopen and Campagna (1954)3,4in patients with various ocular conditions. Good results were noted, following its intramuscular administration, in 16 cases of extraocular trauma and in 5 patients with postoperative hyphema. In two of five patients with vitreous hemorrhages improvement was attributed to trypsin. One patient showed complete clearing in eight weeks, whereas the hemorrhage of the second was partially clear in four weeks. The mode of action of trypsin is not certain, but various investigators have stressed the anti-inflammatory and thrombolytic effects.5,6Following the report of Hopen and Campagna, intramuscularly administered trypsin has been tried in this hospital in a small series of patients with diabetic vitreous hemorrhages, and the results observed prompted a controlled experiment on

The use of trypsin (intramuscular) in the treatment of edema due to intestinal anastomosis

Crystalline trypsin in sesame oil given intramuscularly to patients in whom intestinal anastomoses were performed produced an earlier resumption of effective intestinal peristalsis. It also seemed to be effective in releasing retention due to a malfunctioning gastroenterostomy stoma and in reducing the edema around lesions causing intestinal obstruction.

A report on the use of crystalline trypsin in treatment of alveolar osteitis

A report on the use of crystalline trypsin in treatment of alveolar osteitis