Calcium Phosphate Gel Research Articles

Stimulation by Human Placental Conditioned Medium of Hemopoietic Colony Formation by Human Marrow Cells

Medium conditioned by human placental tissue was found to stimulate granulocytic and monocytic colony formation by human marrow cells in semisolid agar cultures. The colony-stimulating activity of unfractionated conditioned medium was equivalent to the activity of standard peripheral blood underlayers. Placentas were a reliable source of active material, and one placenta provided enough material to stimulate 5,000-10,000 cultures of normal or leukemic cells. The colony-stimulating factor in human placental conditioned medium (CSFHPCM) was concentrated and purified 1800-fold using ammonium sulfate precipitation, calcium phosphate gel absorption, DEAE-cellulose batch absorption, gel filtration on Sephadex G-150, and polyarcylamide gel gel electrophoresis. The active factor behaved on gel filtration as a macromolecule with an apparent molecular weight of 30,000 daltons. The active factor in placental conditioned medium was not dependent on the presence of adherent marrow cells with endogenous colony-stimulating activity.

Stimulation by human placental conditioned medium of hemopoietic colony formation by human marrow cells

Medium conditioned by human placental tissue was found to stimulate granulocytic and monocytic colony formation by human marrow cells in semisolid agar cultures. The colony-stimulating activity of unfractionated conditioned medium was equivalent to the activity of standard peripheral blood underlayers. Placentas were a reliable source of active material, and one placenta provided enough material to stimulate 5,000–10,000 cultures of normal or leukemic cells. The colony- stimulating factor in human placental conditioned medium (CSFHPCM) was concentrated and purified 1800-fold using ammonium sulfate precipitation, calcium phosphate gel absorption, DEAE-cellulose batch absorption, gel filtration on Sephadex G-150, and polyarcylamide gel gel electrophoresis. The active factor behaved on gel filtration as a macromolecule with an apparent molecular weight of 30,000 daltons. The active factor in placental conditioned medium was not dependent on the presence of adherent marrow cells with endogenous colony-stimulating activity.

Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain

1. 1.|On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. 2.|The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. 3.|The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. 4.|The phosphatase showed a high affinity for triphosphoinositide. Without added Mg 2+, the K m was 25 μM and V was 33 μmol P i released/min/mg protein. 5. 5.|The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg 2+, the K m values for triphosphoinositide and diphosphoinositide were 5 μM and 25 μM respectively and V was the same for each substrate. 6. 6.|Both Mg 2+ and Ca 2+ activated the enzyme. While Ca 2+ produced maximum activation at 100 μM, a much higher concentration of Mg 2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg 2+ or Ca 2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. 7.|The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 inactivated it almost completely, whereas an increase in pH to 8.0 halved the activity.

Pig liver fatty acid synthetase: Purification and physicochemical properties

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 m m dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of S o 20,w = 13.3 S and D o 20,w = 2.60 × 10 −7cm 2/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10 −7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of M w ( s D ) = 478,000 and sedimentation equilibrium data yielded a value of M w = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.

Isolation and properties of a new, soluble, hemoprotein (H-450) from pig liver

A new soluble hemoprotein, designated as H-450, has been purified from pig liver. The absolute absorption spectrum of H-450 shows maxima at 550 and 428 nm. The dithionite-reduced H-450 has absorption peaks at 572, 540, and 450 nm; the unique Soret band at 450 nm is the basis for our tentative designation of this new hemoprotein as H-450 (hemoprotein 450). The spectrum of dithionite-reduced H-450 at 77 K gives two alpha peaks (571 and 566 nm), three beta peaks (546, 537, and 529 nm), and a Soret band at 449 nm. The prosthetic group of H-450 has been identified as protoheme IX. Gel electrophoresis experiments show that H-450 is composed of two nonidentical subunits, alpha and beta (mol wts = 61 000 and 45 000). H-450 contains 1 mol of heme/alphabeta dimer of 106 000 molecular weight. Preliminary sedimentation equilibrium experiments suggest a minimum molecular weight of 218 000 for the native protein. This corresponds to a tetramer, alpha2beta2 containing two heme groups. H-450 is not reduced by reduced nicotinamide adenine dinucleotide (NADH), NADH phosphate, ascorbate, or ferrocyanide. Neither reduced nor oxidized H-450 binds CO, 1 mM cyahide, or 1 mM azide. Dithionite-reduced H-450 is autoxidizable. The molar extinction coefficient of native H-450 is 261 X 103 at 280 nm and 263 X 103 at 428 nm. The purification procedure involves homogenization, high-speed centrifugation, ammonium sulfate fractionation, diethylaminoethylcellulose chromatography, density gradient centrifugation, a calcium phosphate gel step, and a second density gradient centrifugation. The procedure yeilds approximately 2 mg of purified protein from 750 g of pig liver.

Purification of a soluble rat liver protein that stimulates microsomal 4-methyl sterol oxidase activity.

A soluble rat liver protein that stimulates microsomal methyl sterol oxidase activity has been isolated and purified to homogeneity by salt fractionation, differential heat inactivation, two calcium phosphate gel association, and Sephadex filtration. The protein, as isolated, may be dissociated with detergent into subunits with a molecular weight of approximately 10,300, as determined electrophoretically. In addition to this stimulatory protein, postmicrosomal supernatant fraction of rat liver contains a low molecular weight methyl sterol oxidase inhibitor, possibly cholesterol ester, which is removed during protein purification. Purified soluble protein enhances the observed rate of oxidative attack of the methyl sterol substrates, 4, 4-dimethyl-5alpha-cholest-7-en-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol. Addition of increasing amounts of either partially purified or homogeneous soluble protein yields hyperbolic stimulation, from which a K'm of about 7 muM has been calculated (based on a monomeric molecular weight of 10,300). Sequential additions of the soluble protein yield equal increments of stimulation. These results are consistent with the suggestion that the soluble protein may be a reactant in the oxidative process. Methyl sterol oxidase is inhitited in vitro by cholesterol, several oxygenated sterols, and cholesterol esters. The extent of inhibition is much greater when the soluble protein is present in the incubation. The inhibition is competitive with respect to methyl sterol substrate; cholesterol succinate, a water-soluble ester, is strongly inhibitory, K'i (ester)/K'm(substrate) approximately 0.2. Since end product inhibition of methyl sterol oxidase may be produced by accumulation of cholesterol or cholesterol metabolites, the soluble protein may participate in regulation of the activity of some microsomal enzymes of cholesterol biosynthesis.

Renal angiotensin I-converting enzyme as a mixture of sialo- and asialo-enzyme, and a rapid purification method.

Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme. The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2 mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].

Cytosol 5′-nucleotidase from chicken liver purification and some properties

1. 1. 5′-Nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) from the cytosol of chicken liver has been purified 1860-fold with an overall yield of 20% by a combination of precipitation at pH 5.3, (NH 4) 2SO 4 fractionation, calcium phosphate gel adsorption, phosphocellulose chromatography and gel filtration with Sephadex G-200. The enzyme has been shown to be highly purified, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This is the first time it has been possible to obtain a purified 5′-nucleotidase from the cytosol of animal tissue. 2. 2. An S 20,w of 9.7 S for 5′-nucleotidase was obtained by the use of sucrose density gradient centrifugation and a Stokes radius of 5.1 nm was estimated by gel filtration techniques. From these values and the assumed partial specific volume of 0.725 cm 3/g, the molecular weight of the enzyme was calculated to be 205 000. One major band, corresponding to a molecular weight of 51 000, was detected after sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicating that the native enzyme was composed of four identical subunits. 3. 3. Some properties of the purified enzyme, including pH optimum, Mg 2+ dependency and substrate specificity, resembled closely those of the partially purified enzyme from chicken liver acetone powder as reported by Itoh, R., Mitsui, A. and Tsushima, K. (1967) Biochim. Biophys. Acta 146, 151–159.

Purification and partial characterization of human angiotensinogen

Renin substrate was initially extracted from human plasma by (NH 4) 2SO 4 followed by chromatography on Sephadex G-150, DEAE cellulose, calcium phosphate gel, isoelectric focusing and preparative polyacrylamide gel electrophoresis. On the basis of one mol of angiotensin per mol of substrate, the purity of the preparation is in excess of 95%. On anlytical polyacrylamide gel electrophoresis in the presence of 1% sodium dodecyl sulfate or 8 M urea, the protein appears homogenous. In addition, the purified protein shows only one preciptin line against anti-normal human serum on either Ouchterlony immunodiffision or immunoelectrophoresis. The biological activity appears similar to “native” renin substrate since the K m is the same as that reported for the renin reaction in whole plasma. The molecular weight was determined as 110 000 by gel filtration and polyacrylamide gel electrophoresis; amino acid analysis of the human substrate differs from that reported for hog, especially in the Asp, Glu and Gly composition.

Partial purification and characterization of a binding factor specific for initiator tRNA and ribosomal 40S subunits and of an aminoacyl-tRNA hydrolase specific for 40S-bound Met-tRNA f from rat liver

The crude soluble fraction of rat liver cytoplasm promotes the binding of acetylphenylalanyl-tRNA but not of Met-tRNA f to 40S subunits derived from 80S ribosomes. A protein has been extensively purified from the soluble fraction that catalyzes the template-dependent, GTP-independent binding of Met-tRNA f, acetylphenylalanyl-tRNA and phenylalanyl-tRNA but not Met-tRNA m. Purification involves fractionation with ammonium sulfate and chromatography on calcium phosphate gel, DEAE-Sephadex, carboxymethyl cellulose and Sephadex G-200. The optimum Mg 2+ concentration for the binding reaction with Met-tRNA f is between 6 and 8 m m and the optimum temperature is between 10 and 15 °C. The complex formed as a result of the interaction between 40S subunits, acetylphenylalanyl-tRNA and poly(U) is functional; acetylpolyphenylalanine is synthesized when the isolated 40S-poly(U)·acetylphenylalanyl-tRNA complex is incubated with 60S subunits, phenylalanyl-tRNA, elongation factors and GTP. The crude cytoplasmic fraction, which does not stimulate the binding of Met-tRNA f, inhibits the purified factor-promoted binding of this substrate; the factor-independent, high magnesium ion-stimulated binding of Met-tRNA f to 40S subunits is also inhibited. The inhibitory activity can be resolved from the binding factor and is extensively purified by chromatography on calcium phosphate gel and carboxymethyl Sephadex and by electrofocusing. In the presence of 40S subunits, crude and purified preparations of the inhibitory activity hydrolyze Met-tRNA f but not Met-tRNA m or acetylphenylalanyl-tRNA. Free Met-tRNA f is not hydrolyzed. Incubation of hydrolase-containing preparations with the preformed 40S-·Met-tRNA f complex results in the rapid and extensive breakdown of the complex with release of acid-insoluble methionine; the formation of an 80S·substrate complex, by the addition of 60S subunits, protects particle-bound Met-tRNA f.

The implications of multiple forms of phenylalanine hydroxylase in phenylketonuria and related diseases of phenylalanine metabolism

Phenylalanine hydroxylase prepared from rat and human liver occurs in three unique isozymal forms. The enzyme was separated into three fractions on neutral calcium phosphate gel columns. Authenticity of the enzyme activities was confirmed by substrate specificity, Michaelis constants for substrate and cofactor, and pH optima. Differentiation of three forms as suggested by column chromatography was confirmed by rechromatography, gel filtration, density gradient centrifugation, and dissimilar responses to temperature, para-chlorophenylalanine, and pretreatment with lecithin. We have demonstrated that the isozymes mature at different times during intrauterine and extrauterine development and that the enzyme is not mature at birth in the rat. We offer various proposals of the significance of isozymes of this enzyme, particularly with regard to the various states of hyperphenylalaninemia and particularly phenylketonuria.

Purification, properties, and isozyme pattern of galactose-1-phosphate uridyl transferase from calf liver

Galactose-1-phosphate uridyl transferase was purified approximately 2000-fold from calf liver with a yield of 15%. The purification procedure involved ammonium sulfate fractionation, calcium phosphate-gel adsorption, and chromatography on DEAE-cellulose, hydroxylapatite, and Sephadex columns. The purified product demonstrated five protein bands on polyacrylamide-gel electrophoresis. Each band had transferase activity as five peaks of activity were observed on preparative polyacrylamide-gel electrophoresis. Galactose-1-phosphate uridyl transferase showed no requirement for divalent metals for activity. In contrast, it was inhibited by Mg 2+ and other divalent metals. The purified enzyme but not the crude preparation was stimulated by sulfhydryl compounds. The enzyme was completely inhibited by low concentrations of p-hydroxymercuribenzoate.

Malic enzyme from silkworm eggs-purification and characteristics

1. 1. Malic enzyme (malate dehydrogenase (decarboxylating) E.C. 1.1.1.40) was purified from cytosol of diapausing silkworm eggs by ammonium sulphate fractionation, ion exchange chromatography on DEAE- and phosphocellulose, and calcium phosphate gel negative absorption. 2. 2. The enzyme has an optimum pH at 8.5 and requires Mn or Mg ions for its maximum activity. 3. 3. Pyruvate is the main end product of the reaction. 4. 4. The Km for l-malate and NADP was determined to be 253 μM and 3.3 μM respectively. 5. 5. The enzyme has a mol. wt of 61,000 as determined by gel filtration.

An electrophoretic analysis of the acid-insoluble chromosomal proteins from different organs of maize seedlings

Nonhistone chromosomal proteins (NHCP) appear to play an important role in controlling transcription and therefore development; however, these proteins have been studied relatively little in plants. Here the acid-insoluble NHCP from the epicotyls, mesocotyls and roots of 4–5 day-old corn seedlings are compared and fractioned. Nuclei and thence chromatin were prepared as described (23). After removal of the histones by extraction with H2SO4, the remaining NHCP were dissolved with sodium dodecyl sulfate (SDS) containing mercaptoethanol and electrophoresed on SDS polyacrylamide gels. On the gels, these acid-insoluble NHCP gave at least 27 bands corresponding to molecular weights from 15,000 to 180,000. Although the mass ratios for acid-insoluble NHCP/DNA differed for different tissues, the electrophoretic patterns were indistinguishable. Subsets of the total NHCP were obtained by fractionations employing sequential extractions at different NaCl concentrations, phenol extractions (2 different procedures) and absorption onto calcium phosphate gel; nevertheless, the electrophoretic patterns of corresponding fractions from different organs looked very similar.

Deoxycytidine Kinase: Properties of the Enzyme From Human Leukemic Granulocytes

Deoxycytidine kinase, which phosphorylates deoxycytidine (CdR) and its analog, cytosine arabinoside (ara-C), has been purified 71-fold from human leukemic cells. Biochemical properties of the partially purified enzyme included a molecular weight of 68,000, Kms of 7.8 muM for CdR and 25.6 muM for ara-C, and optimal activity with ATP and GTP as phosphate donors. Ara-C phosphorylation was strongly inhibited by CdR (Ki = 0.17 muM) and dCTP (Ki = 7.3 muM) and was weakly inhibited by ara-CTP (Ki = 0.13 mM). Purification by calcium phosphate gel elution and DEAE chromatography effectively separated this enzyme from cytidine deaminase, which deaminates both CdR and ara-C, and from uridine-cytidine kinase, the enzyme which phosphorylates 5-azacytidine. CdR kinase activity was found to decrease and cytidine deaminase to increase with maturation of normal and leukemic granulocytes. Myeloblasts purified by Ficoll sedimentation revealed an average kinase activity of 15.4 U/mg protein in acute myelocytic leukemia and 12.3 U/mg protein in blastic crisis of chronic myelocytic leukemia (CML). The average ratio of CdR kinase to deaminase activity in crude cell extracts varied from 0.197 in AML and 0.089 in blastic crisis to 0.0004 in normal granulocytes, reflecting the changes which take place with cellular maturation. The absolute levels of kinase and deaminase and the ratio of these two enzymes varied considerably among patients with AML, indicating that quantitative differences may be found in the metabolism of CdR and its analogs in leukemic cells.

Deoxycytidine kinase: properties of the enzyme from human leukemic granulocytes

Deoxycytidine kinase, which phosphorylates deoxycytidine (CdR) and its analog, cytosine arabinoside (ara-C), has been purified 71-fold from human leukemic cells. Biochemical properties of the partially purified enzyme included a molecular weight of 68,000, Kms of 7.8 muM for CdR and 25.6 muM for ara-C, and optimal activity with ATP and GTP as phosphate donors. Ara-C phosphorylation was strongly inhibited by CdR (Ki = 0.17 muM) and dCTP (Ki = 7.3 muM) and was weakly inhibited by ara- CTP (Ki = 0.13 mM). Purification by calcium phosphate gel elution and DEAE chromatography effectively separated this enzyme from cytidine deaminase, which deaminates both CdR and ara-C, and from uridine- cytidine kinase, the enzyme which phosphorylates 5-azacytidine. CdR kinase activity was found to decrease and cytidine deaminase to increase with maturation of normal and leukemic granulocytes. Myeloblasts purified by Ficoll sedimentation revealed an average kinase activity of 15.4 U/mg protein in acute myelocytic leukemia and 12.3 U/mg protein in blastic crisis of chronic myelocytic leukemia (CML). The average ratio of CdR kinase to deaminase activity in crude cell extracts varied from 0.197 in AML and 0.089 in blastic crisis to 0.0004 in normal granulocytes, reflecting the changes which take place with cellular maturation. The absolute levels of kinase and deaminase and the ratio of these two enzymes varied considerably among patients with AML, indicating that quantitative differences may be found in the metabolism of CdR and its analogs in leukemic cells.

Partial purification of rabbit hind brain tryptophan hydroxylase by affinity chromatography

Chromatography of crude homogenates of rabbit hind brains on ε-amino caproyl-D-tryptophan methyl ester-agarose gels provide enzyme fractions with specific activity 7–10 times higher than the starting material. The activity was found to be associated with two distinct components. While nearly forty-fold increase in specific activity can be achieved by purification of the homogenate on calcium phosphate gels prior to affinity chromatography, only a single active component was noted in such prepurified extracts.

Australia Antigen ‘ad’ and ‘ay’ Subtypes. Purification and Partial Characterization

The two commonly-found Australia antigen subtypes have been purified by simple inexpensive, reproducible, physico-chemical methods involving ammonium sulfate precipitation, peptic digestion, calcium phosphate gel fractionation and molecular sieving on Sepharose 4B. The overall recovery of the two subtypes varied between 50 and 60% of the total antigen content on the starting serum. The purified antigens were found homogeneous by discontinuous gel electrophoresis, analytical ultracentrifugation, and isopycnic banding ultracentrifugation. No contaminating serum proteins could be detected by immunoelectrophoresis. The two purified antigen subtypes have similar Stokes radii and buoyant densities. The sedimentation coefficients for the 'ad' and 'ay' subtypes were found to be 33.0 and 40.1 times 10- minus 13 sec, respectively.

Pulmonary angiotensin-converting enzyme. Structural and catalytic properties.

Angiotensin-converting enzyme has been solubilized from a particulate fraction of rabbit lung and purified to apparent homogeneity in 11% yield by a procedure including fractionation with DEAE-cellulose and calcium phosphate gel, elution from Sephadex G-200, and lectin affinity chromatography. The molecular weight estimated by equilibrium sedimentation was approximately 129,000, either in the absence or presence of 6 M guanidine hydrochloride. A slightly higher value of 140,000 determined for the reduced, denatured protein by gel electrophoresis in the presence of sodium dodecyl sulfate and a much higher figure derived from gel filtration are probably due to the glycoprotein nature of the enzyme. Its oligosaccharide content accounted for 26% of the weight calculated from its amino acid and carbohydrate composition. The estimated content of sugar residues per mole was: galactose, 57; N-acetylglucosamine, 53; mannose, 43; N-acetylneuraminic acid, 19; and fucose, 4. Threonine and alanine were identified, respectively, as NH2-terminal and COOH-terminal residues by the dansylation procedure and by digestion with carboxypeptidase A. The enzyme was found to contain approximately 1 g atom of zinc per mol. Km values for hydrolysis of hippurylhistidylleucine and angiotensin I were 2.3 and 0.07 mM, and the corresponding turnover numbers were 15,430 and 792 mol/min/mol at 37 degrees. Bradykinin was also a substrate, and release of its COOH-terminal dipeptide, Phe-Arg, was catalyzed at a comparable rate to that of His-Leu from the COOH terminus of angiotensin I. Enzyme activity required the presence of chloride ions and was inhibited by EDTA and by low concentrations of Bothrops bradykinin-potentiating peptides. In addition, hydrolysis of hippurylhistidylleucine was inhibited competitively by other defined peptides, including di- and tripeptides, which were not substrates.