Tryptic Degradation Research Articles

Phospholipid Exchange between Membranes: PURIFICATION OF BOVINE BRAIN PROTEINS THAT PREFERENTIALLY CATALYZE THE TRANSFER OF PHOSPHATIDYLINOSITOL

Using an assay that measures the transfer of phosphatidyl-[3H]inositol from rat liver microsomes to liposomes consisting of phosphatidylcholine (98 mole %) and phosphatidylinositol (2 mole %), two proteins have been isolated from homogenates of bovine brain cortical tissue which specifically stimulate the above phospholipid exchange. Protein I, purified 508-fold, has a molecular weight of approximately 29,000 and an isoelectric point of 5.2, and protein II, purified 426-fold, has a molecular weight of approximately 30,000 and an isoelectric point of 5.5. Among the phospholipids examined in various transfer systems, both proteins exhibited a marked preference for phosphatidylinositol transfer; phosphatidylcholine, but not phosphatidylethanolamine, could also be transferred between membranes. The capacities to transfer phosphatidylinotisol and phosphatidylcholine were inhibited simultaneously by tryptic degradation of each protein; however, protein II was significantly more sensitive to this treatment than protein I. The effects of membrane concentration (microsomal protein or liposomal phospholipid) on relative rates of phosphatidylinositol transfer have been determined. Estimations of Km and V with respect to each membrane have been made. These properties of the brain phospholipid transfer proteins are discussed in relationship to those of other proteins which function in the redistribution of phospholipid between membranes.

Use of the c-terminal octapeptide of cholecystokinin for gallbladder evacuation in cholecystography.

USE OF THE C-TERMINAL OCTAPEPTIDE OF CHOLECYSTOKININ FOR GALLBLADDER EVACUATION IN CHOLECYSTOGRAPHYPAVO HEDNER and ANDERS LUNDERQUISTAudio Available | Share

Spontaneous and tryptic degradation of virus particles and structural components of adenoviruses.

SUMMARY Electrophoretic analyses of the polypeptide components of adenovirus indicate that both the hexon and the penton base are degraded during storage of either purified virus preparations or isolated antigens. These two protein components are also degraded by trypsin and the products of tryptic digestion of native hexons were examined in detail. They were shown to consist of six polypeptide species which may result from tryptic action at three distinct sites on the hexon polypeptide. Both spontaneously degraded and trypsinized hexon and penton base proteins retained their morphological and antigenic characteristics.

Proteolytic degradation of exocrine and serum immunoglobulins.

The susceptibility of exocrine and serum immunoglobulins and antibodies to proteolytic degradation was assessed. Colostral and duodenal fluid exocrine 11S IgA, monomeric serum IgA, and IgG were digested with trypsin, chymotrypsin, or duodenal fluid. Exocrine IgA was more resistant to digestion than were the serum immunoglobulins. Under conditions of the experiments, most of colostral IgA retained its 11S quaternary structure, including the secretory piece; the portion degraded was reduced almost entirely to peptides. The superior resistance of exocrine IgA was also demonstrated by digestion of serum IgG and nasal exocrine IgA diphtheria antitoxins with trypsin or duodenal fluid. Selective precipitation of trypsin-digested antitoxins with antibodies to heavy chains, light chains, or secretory piece revealed that the differences in susceptibility to digestion were due to differences in lability of the Fc portions of the IgA and IgG antibody molecules. The Fc portions of IgG antibody molecules were degraded or cleaved from the Fab units of the molecules, whereas the Fc-like portions of IgA antibody molecules remained associated with their Fab-like units and the secretory piece. On the other hand, trypsin treatment did not affect the antigen binding ability of the Fab parts of either the exocrine IgA or IgG antibodies. The Fc-like portions of exocrine IgA may be protected from tryptic degradation by the quaternary structure of the 11S molecules, which includes a dimer of 7S IgA subunits and the secretory piece.

Further purification of a polypeptide demonstrating enterogastrone activity.

1. The further purification of a polypeptide having potent enterogastrone activity, without CCK-PZ effects, is described.2. The material was inhibitory when doses of 1.0 mug/kg.hr were administered intravenously. Amino acid analyses demonstrated the absence of proline, a high content of glutamine and a preponderance of lysine over arginine. Tryptic degradation destroys the inhibitory effect of the polypeptide. Further studies must be performed before the physiological status of the polypeptide can be ascertained.

Human calcitonin. IV. Synthesis of calcitonin M

AbstractA preliminary account is giver of the synthesis of calcitonin M (I), isolated from human C‐cell tumour tissue [2] [3]. Identity of the synthetic and the natural hormone was established by thin‐layer chromatography, thin‐layer electrophoresis and conversion to oxidation products, as well as by reference to the pattern of tryptic degradation and by comparing the biological activity of the two hormones. The findings also afforded additional confirmation of the results of structural elucidation [1].In the synthesis of I, use was made of methods described previously [4] for the preparation of porcine α‐thyrocalcitonin, and also of a new method [5] which easily permits the formation of cyclic cystine peptides.

Properties of the immunogenic and sensitizing activities of Bordetella pertussis in mice: I. Enhancement of peptone shock

The capacity of pertussis vaccine to enhance peptone shock in mice paralleled the immunogenic and histamine-sensitizing activities in fractionation and was similarly labile to heat and to tryptic degradation. It is, therefore, suggested that the immunogen itself may be responsible for these sensitizing activities. Elicitation of heightened susceptibility to peptone shock by the administration of a beta-adrenergic blocking agent, propranolol, would suggest that pertussis vaccine may induce this effect, as had earlier been reported and is presently confirmed, with histamine, via autonomic nervous pathways. Several significant differences were found, however, between pertussis-mediated hypersensitivity to histamine and to peptone, including degree of sensitivity engendered, mouse strain specificity, and duration of symptoms.

Studies on the mechanism of the degradation of human γ-globulin by trypsin

The presence of 0.05 M N-ethylmaleimide resulted in a marked inhibition of the production of 3.5 S fragments from normal human 7 S γ-globulin by trypsin. The presence of 0.1 M iodoacetamide also resulted in an inhibition of the fragmentation process. However, the component distribution of the resulting digests differed in that the principal component of the N-ethylmaleimide inhibited digests sedimented at the same rate as the unfragmented γ-globulin, while that of the iodoacetamide inhibited digests sedimented at a somewhat reduced rate. Reduction of the N-ethylmaleimide or iodoacetamide inhibited digests by 0.1 M 2-mercaptoethanol resulted in the conversion of faster sedimenting materials to 3.5 S fragments. Inhibition of the production of 3.5 S fragments by tryptic degradation in the absence of added sulfhydryl blocking reagent was also observed following prior treatment of the γ-globulin by N-ethylmaleimide. The findings of this study provide evidence that the free sulfhydryl groups of human γ-globulin are involved in the splitting of disulfide bonds during the fragmentation of the γ-globulin by trypsin. Rabbit 7 S γ-globulin differed from human γ-globulin in that treatment by trypsin did not produce 3.5 S fragments except in the presence of a reducing reagent.

INHIBITION BY TRYPSIN INHIBITORS OF DISSOCIATION OF EMBRYONIC TISSUE BY TRYPSIN.

TRYPSIN is used extensively for dissociation of tissues and in the preparation of suspensions of living cells1. The cell-dissociating effect of this enzyme has been attributed to tryptic degradation of cell binding materials1; however, theoretically, other possibilities exist and the question whether the characteristic enzymatic properties of trypsin are actually involved in cell separation seems not to have been adequately tested, though such information would be of some general interest. Furthermore, since trypsin attacks selectively peptide linkages adjacent to lysyl and arginyl residues, clarification of the relationship between its cell-dissociating activity and its lytic properties would be useful in considerations of the mechanism of cell binding. Trypsin inhibitors offer a direct approach to this matter. It was reported that trypsin inactivated by diisopropylflurophosphate (DFP) did not dissociate adult rat liver cells2. In the work recorded here, the effects of soybean and ovomucoid trypsin inhibitors on the dissociation by trypsin of embryonic tissue were examined. The interaction between these inhibitors and trypsin is considered to be specific and stoichiometric3–5 at concentrations equimolar to and higher than that of the enzyme, the inhibitors rapidly and completely abolish its tryptic activity. According to available results3,5, the molecular weights of trypsin and trypsin inhibitors used in this investigation were of the following order of magnitude: trypsin—24,000; soybean trypsin inhibitor—24,000; ovomucoid trypsin inhibitor—28,000.

Immunological investigation of human γ-globulin degraded by chymotrypsin

Chymotryptic hydrolysate of human γ-globulin was separated by means of filtration on Sephadex gels. Gels with a low degree of cross-linking (G-75, G-100) were suitable for separation of components with precipitation properties. The obtained fractions were studied by immunoelectrophoresis and compared with fragments of γ-globulin isolated after tryptic degradation.

Isolation of immunologically active fragments of normal human γ-globulin after tryptic degradation

After degradation of human γ-globulin with trypsin several products with retained precipitation properties can be demonstrated by immunoelectrophoresis. A group separation of these products can be obtained by gel filtration with Sephadex G-75. Further purification of two fragments is possible by means of column electrophoresis or ion-exchange chromatography. One of the fragments shows retained antibody activity.

Hemoglobin. X. Analysis of the alpha- and beta-chains of normal adult hemoglobin by its tryptic degradation products

Hemoglobin. X. Analysis of the alpha- and beta-chains of normal adult hemoglobin by its tryptic degradation products

An immunological comparison of immunoglobulins from human blood serum, urine and milk using diffusion-in-gel methods

γ-Globulins from normal human serum and urine and immunoglobulins from normal human milk have been compared by means of diffusion-in-gel methods. The normal immunoglobulins have also been compared with fragments of serum 7 S γ-globulin obtained by tryptic degradation and with two Bence-Jones proteins. Normal serum and urine were found to contain two or three low molecular weight γ-globulins (γ l-globulins) which were antigenically deficient compared to 7 S γ-globulin. Two serum γ l-globulins gave reactions of interference with two corresponding urine γ l-globulins. One of the γ l-globulins gave a reaction of interference with a milk immunoglobulin. The other γ l-globulin gave a reaction of partial interference with another milk immunoglobulin and was antigenically deficient compared to this protein. The γ l-globulins gave reactions of interference with the S-fragment of 7 S γ-globulin obtained after tryptic degradation. In comparative immunoelectrophoretic studies, reactions of interference were obtained between one Bence-Jones protein and two urine γ l-globulin precipitates and between another Bence-Jones protein and a third urine γ l-globulin precipitate. Some differences observed between the behaviour of the serum and urine γ l-globulins as well as possible relationships between γ l-globulins and Bence-Jones proteins are discussed.

The preparation, purification, and amino acid sequence of a polypeptide renin substrate.

A purified preparation of a polypeptide renin substrate prepared by tryptic degradation of the protein renin substrate has been analyzed by the fluorodinitrobenzene method and after degradation with renin, carboxypeptidase, and phenylisothiocyanate, has been found to possess the amino acid sequence; asp-arg-val-tyr-ileu-his-pro-phe-his-leu-leu-val-tyr-ser. The first 10 of these amino acids constitutes hypertensin I which is released by cleavage of the leucyl-leucine bond by renin. The remaining 4 amino acids, leu, val, tyr, ser, apparently link hypertensin I to the protein renin substrate.