Milton Levy

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.

New methods for the preparation of biospecific adsorbents and immobilized enzymes utilizing trichloro-s-triazine☆

Abstract Methods for preparing biospecific adsorbents and immobilized enzymes utilizing Sepharose CL as a support and trichloro-s-triazine as the linking agent are described. The difficulties encountered during conventional aqueous and mixed aqueous-phase reactions of trichloro-s-triazine with insoluble polyols, particularly reagent hydrolysis, are avoided by performing the activation reactions in anhydrous organic phase and replacing the second chlorine on the triazine ring by an aromatic amine. Ligands can be coupled to the activated support in either aqueous or organic phase. The methods have been applied to the attachment of a number of different enzymes, proteins, and small-molecule ligands to Sepharose. The superiority of the triazine linkage to the cyanogen bromide linkage is demonstrated.

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.

Purification and properties of pig liver esterase

Abstract A homogenous preparation of pig liver aliesterase (EC 3.1.1.1) was obtained by the use of preparative starch-gel electrophoresis. The esterolytic activity of this preparation is markedly accelerated at high substrate concentration. Two active sites on the molecule may account for this unusual kinetic behavior. The sites may be identical as in a monomer-dimer equilibrium or they may be nonidentical and different in function. The esterase apparently is neither stereospecific nor stereoselective, and it did not exhibit an asymmetric active site. The enzyme hydrolyzes esters that are nonionic at a much greater rate than charged analogs. Half-esters of dicarboxylic acids are neither substrates nor inhibitors. Apparently an anionic charge on an ester inhibits the association between the esterase and ester.

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.

Estrogen-dependent trypsin-like activity in the rat uterus. Localization of activity in the 12,000g pellet and nucleus.

Abstract Direct evidence was obtained for the presence of hormone-stimulated trypsin-like protease activity in the rat uterus. Ovariectomized rats were either untreated (U), treated with estradiol (E), or estradiol plus progesterone (EP). The uteri were excised and subcellular fractions were prepared. Each fraction was assayed for protease activity using protamine as substrate, the cleavage products being quantitated fluorometrically following reaction with 4-phenylspiro[furan-2(3H),1′-phthalan]-3,3′dione (Fluram). Fractions from U rats yielded negative results, whereas the 12,000 g pellets and nuclei from the uteri of E and EP rats exhibited appreciable activities. No significant increase in protease activity was observed in thymus and diaphragm following hormone treatment, indicating organ specificity. The enzyme (or enzymes) from the 12,000 g pellet was solubilized and some characteristics were determined. The apparent K m is about 1.0 × 10 −6 m , the temperature optimum is about 44 °C and maximum velocity is achieved in the alkaline range (pH ~ 8.5). The protease is a plasminogen activator and is inhibited by diisopropyl fluorophosphate, Antipain, and Leupeptin. These properties resemble those of trypsin.

Contribution of E-Amino Groups to Ninhydrin Color Production in Proteins

The ninhydrin color given by model compounds and by characterized proteins shows a consistent contribution from the ε-amino group of lysine of about 67 percent of that of an α-amino group, except in free lysine or in N-terminal lysine, where the ε-amino group makes a small contribution (7 to 10 percent) to the total color. This information can be applied to structure determination of Nε peptides of lysine.

Spectrophotometric and stoichiometric titrations of ribonuclease in eight molar urea.

Abstract Stoichiometric and spectrophotometric titration curves of ribonuclease in 8 M urea indicate that the six tyrosyl hydroxyls are equally capable of dissociating hydrogen ions and follow a monotonic titration curve with p K (int) of 10.67 and w = 0.018. That three of these groups in water show much lower potencies as hydrogen-ion donors than the other three is presumed to result from intramolecular hydrogen bonding. The low value of w , the electrostatic interaction factor in urea, suggests “unfolding” of the molecule of ribonuclease, and the rather low p K (int) of the six groups suggests that hydrogen bonding of the hydroxyls to urea occurs in the urea solutions. Since ribonuclease is active in 8 M urea solution, the structure involved in hydrogen bonding of the tyrosyl hydroxyls is not essential to enzyme action.

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.