Isaac Schenkein

Proteolytic enzymes from the mouse submaxillary gland. Specificity restricted to arginine residues.

Abstract The mouse submaxillary proteases (A + D), the isolation and properties of which were previously described by us, hydrolyze only arginyl bonds in proteins. Formol titrations and peptide mapping on digests of polyarginine, polylysine, lysozyme, histone, and insulin suggested this specificity. Amino acid compositions of peptides from lysozyme and insulin showed that most but not all arginyl bonds were hydrolyzed but that no lysyl bonds were split. The proteases should be useful in the sequencing of proteins.

Increased Nerve-Growth-Stimulating Activity in Disseminated Neurofibromatosis

NERVE-GROWTH factors have been isolated from a variety of substances such as sarcoma, snake venoms and the spinal-axial region of the chick embryo. The best source has been the submandibular saliva...

Allergy to human seminal plasma.

THE infrequency of the syndrome of allergy to human ejaculate1 prompts us to report another such case. In addition, we present immunologic data including histamine release, and the finding that the...

A Model for the Regulation of Antibody Synthesis by Serum Antibody

Publisher Summary This chapter discusses a model for the regulation of antibody synthesis by serum antibody. The immune response (ImR) after removal of specific antibody was studied. In these experiments, antibody was removed specifically in two ways: (1) by successive exposure of aliquots of plasma of immunized animals to solid phase immunoadsorbents followed by return of the adsorbed plasma to them; (2) by exchange transfusion of animals immunized to two antigens with blood containing antibody of one specificity only, thereby depleting the other antibody specifically. Following the removal, there was a marked rise in serum levels of specific antibody, which frequently reached peak titers exceeding those prior to removal. The assay used in these studies was phage neutralization which is affected by the binding affinity of antibody. These experiments were repeated using an antibody assay that would allow estimation in molar terms. The results obtained support a model in which the level of serum antibody regulates the ImR by means of equilibrium with antibody in an immunogen-containing compartment.

Esteroproteolytic enzymes from the submaxillary gland: Kinetics and other physicochemical properties☆

Abstract Two esteroproteolytic enzymes (A and D) have been isolated from the mouse submaxillary gland and shown to be pure by ultracentrifugation, immunoelectrophoresis, acrylamide-gel electrophoresis, and amino acid analyses. The enzymes have molecular weights of approximately 30,000 and are structurally and antigenically related. Narrow pH optima between 7.5 and 8.0 are exhibited by both enzymes. The “pK1s” are between 6.0 and 6.5 and the “pK2s” are near 9.0. A marked preference for arginine-containing esters is shown by both enzymes. The maximum specific activity of enzyme A on p-tosylarginine methyl ester (TAME) at pH 8 was 2500–3000 μ m min−1 mg−1 and for enzyme D, 400–600 μ m min−1 mg−1. With TAME as substrate, the Km for enzyme A was 8 × 10−4 m at 25 °C and 6 × 10−4 m at 37 °C. For D, Km was 3 × 10−4 at 25 °C and 2 × 10−4 m at 37 °C. An apparent activation of enzyme D by tosylarginine (TA), a product of TAME hydrolysis, and all α-amino acids examined was due to removal of an inhibitor by chelation. This effect could be duplicated by 8-hydroxyquinoline and diethyldithiocarbamate but not by EDTA. Enzyme A was not affected by these substances to any remarkable extent. Several divalent ions proved to be potent inhibitors of enzyme D. Both enzymes are inactivated by the active site reagents diisopropyl phosphofluoridate and tosyllysine chloromethylketone but much less rapidly than is trypsin. Nitrophenyl-4-guanidionobenzoate reacts with a burst of nitrophenol liberation but with a rapid continuing hydrolysis. One active site per molecule is indicated. Enzyme D is inactivated by urea, reversibly at 10 m and with maximal permanent losses at 6 m . Autolysis of the unfolded form by the native enzyme when they coexist at intermediate urea concentrations appears to occur. Identity of enzyme D and the epithelial growth factor binding protein is demonstrated.

The use of glucose oxidase as a generator of H2O2 in the enzymatic radioiodination of components of cell surfaces

Glucose oxidase generates H2O2 via the conversion of glucose to gluconolactone in the presence of molecular oxygen. Coupling of this reaction to the lactoperoxidase-catalyzed substitution of iodine for hydrogen on the phenolic side chains of proteins present on the surfaces of murine myeloma cells leads to a very marked increase in incorporation of radioactivity as well as a significant improvement of cell survival.

Proteases from mouse submaxillary gland

Abstract Two proteases (“A” and “D”) from mouse submaxillary gland have been isolated in high purity. At pH 8.5 “D” is more negative than “A” and is also smaller. Amino acid composition differs only in 6 constituents. “D” is activated 100–300% when acting on tosyl arginine methyl esther by tosyl arginine and all α-amino acids. Both enzymes have a strong preference for arginyl bonds.

Synthesis and intracellular transport of immunoglobulin in secretory and nonsecretory cells.

The availability of clones of cells in the form of transplantable tumors or established lines in tissue culture (myelomas and lymphomas) has afforded the opportunity to study the cellular mechanisms which underly the synthesis intracellular transport, and secretion of immunoglobulin (Ig) . Using techniques of subcellular fractionation and electron microscopy, it has become possible to elucidate many features of the secretory pathway. A model is proposed which is consistent with most of the available evidence and which seeks to interpret the sites of Ig assembly, carbohydrate addition, and segregation of Ig from other molecules not destined for secretion from the cell. The model suggests that the secretory pathway for Ig in plasmacytic cells is similar to that utilized by other secretory cells in general. Recent experiments also suggest that some lymphocytic cells which synthesize but do not secrete Ig utilize many features of the secretory pathway of plasmacytic cells. Small amounts of Ig are synthesized and appear to be transported to the plasma membrane but are not released from the cell. These results and other considerations suggest that these cells may represent neoplastic clones of antigen recognition cells with Ig receptors on the plasma membrane. If our interpretation is correct, the genesis of the antigen-specific Ig receptor of lymphocytes involves a stage of intracellular transport which is similar to that for secreted Ig. Our present understanding of events which characterize the intracellular life of the Ig molecule in these two types of lymphoid cells are discussed below.

Sexual Dimorphism of Mouse Submaxillary Glands and Its Relationship to Nerve Growth Stimulating Protein.

Summary A protein obtained from the mouse submaxillary gland, which enhances the growth of sympathetic and spinal ganglia and their processes, is 4-30 times more concentrated in the male than in the female sub-maxillary gland, an organ which shows a sexual dimorphism in histological appearance and in several physiological differences. The masculinization of the gland which occurs during pregnancy and lactation, as a result of increased androgen secretion during that time, has been shown to be correlated to a logarithmic increase in concentration of nerve growth protein, to the point where it may equal that of the male. It is suggested that this increase may serve the function of aiding the development of parts of the nervous system of the suckling mouse.

Nerve Growth Factor of Very High Yield and Specific Activity

Nerve growth factor has been isolated from submaxillary glands of mnature male mice at specific activities about a million times, and in yields of biological activity ten million times, greater than best previous results. The major improvement in the isolation is related to the separation of a highly active tosylarginine methyl esterase present in cruder preparations. The new nerve growth factor may be an entity different from the older one, although no gross differences in the qualitative aspects of their actions are apparent on superficial examination of chick ganglia influenced by them. The neurites which develop from a ganglion in the presence of nerve growth factor are of nearly equal length. The amount of nerve growth factor determines the number of neurites but not the extent of individual development. The amount of the new nerve growth factor which evokes the appearance of a hundred neurites from a single ganglion appears to be about ten molecules. Since each neurite seems to arise from a different neuron each molecule of nerve growth factor must affect several cells. This result can be rationalized by a catalytic mechanism or by indirect action of nerve growth factor through a hypothetical cell which produces a neurite evocator on contact with the molecule of nerve growth factor.