Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells

A Mr 95,000 matrix metalloproteinase (MMP) produced by rat mammary carcinoma cells has been isolated and characterized. The MMP was secreted in a proteolytically inactive form that was free from bound tissue inhibitor of metalloproteinases. The enzyme was highly glycosylated as evident from an apparent drop of Mr from 95,000 to 83,000 after treatment with N-glycanase. Rotary shadowing electron micrographs of purified proenzyme preparations revealed a uniform set of ellipsoidal molecules. Treatment of the proenzyme with 1% SDS resulted in generation of catalytic activity and exposed a cryptic unpaired Cys residue. The latent proenzyme may be activated in at least three additional ways: either spontaneously upon storage, by treatment with organomercurials, or by limited proteolysis by trypsin. Each mode of activation yielded a distinct pattern of cleavage of the enzyme. The activated enzyme cleaved gelatin (denatured type I collagen) and native type IV and V collagen at 30-37 degrees C. Noncollagenous proteins including alpha 1-proteinase inhibitor, casein, and fibrinogen also were cleaved. The rat mammary carcinoma cell line that produces the Mr 95,000 MMP is composed of two distinct (epithelial- and myoepithelial-like) cell types. The enzyme is expressed constitutively by the epithelial cells. This suggests that expression of the Mr 95,000 MMP is regulated differently from that of interstitial collagenase, which is produced by the epithelial cells only in response to specific inductive factor(s) from the myoepithelial-like cells. Monoclonal antibodies raised against the purified latent Mr 95,000 form of the enzyme bind specifically to the Mr 95,000 MMP and have been used to localize the enzyme to the Golgi region and cytoplasmic granules of the epithelial cells.     

Molecular properties of pyrophosphate:fructose-6-phosphate phosphotransferase from potato tuber

Pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) from potato tubers has been purified to homogeneity. The enzyme contains two polypeptides with apparent relative molecular mass (Mr) values of 65,000 and 60,000. These polypeptides give different peptide fragments after limited proteolytic digestion. Antibodies raised against each polypeptide separately are specific for that polypeptide, but both antisera are capable of immunoprecipitating native PFP activity. These antibodies also recognize similar pairs of polypeptides in a range of other plant tissues that contain PFP activity. Based on gel filtration, the Mr value of potato tuber PFP is 265,000. This suggests that the enzyme is a heterotetramer composed of two polypeptides with Mr values of 65,000 and 60,000. In the presence of pyrophosphate, potato PFP dissociates into a 130,000 dimer.     

The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.

A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.     

SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages

We have reported that SV40-transformed human lung fibroblasts secrete a 92-kDa metalloprotease which is not detectable in the parental cell line IMR-90. We now present the complete structure of this enzyme along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes. A similar, perhaps identical, enzyme can be released by polymorphonuclear cells. The preproenzyme is synthesized as a polypeptide of predicted Mr 78,426 containing a 19 amino-acid-long signal peptide and secreted as a single 92,000 glycosylated proenzyme. The purified proenzyme complexes noncovalently with the tissue inhibitor of metalloproteases (TIMP) and can be activated by organomercurials. Activation with phenylmercuric chloride results in removal of 73 amino acids from the NH2 terminus of the proenzyme, yielding an active form capable of digesting native types IV and V collagen. The in vitro substrate specificity of the enzyme using these substrates was indistinguishable from that of the 72-kDa type IV collagenase. The 92-kDa type IV collagenase consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-1, and a unique 54-amino-acid-long proline-rich domain homologous to the alpha 2 chain of type V collagen.     

Membrane glycoproteins involved in hepatocyte adhesion to collagen type I

Liver membrane glycoproteins with affinity for immobilized collagen type I were subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroelution of the separated proteins. Electroeluted glycoproteins with ability to neutralize the inhibitory effect of anti-CollCAM antibodies on hepatocyte adhesion to collagen were collected from several consecutive runs and used to raise a high titer antiserum, denoted anti-CollCAM II. IgG from this antiserum inhibited the attachment of hepatocytes to dishes coated with collagen type I, but not to fibronectin- or collagen type IV-coated dishes. When the antibodies were immobilized to Sepharose CL-4B they bound three sets of glycoproteins with apparent Mr's of 105,000, 115,000, and 130,000 as analyzed by SDS-PAGE under nonreducing (NR) conditions. Upon reduction (R) the glycoproteins migrated with apparent Mr's of 115,000, 130,000, and 160,000, respectively. The Mr 105,000-115,000 (NR) glycoproteins effectively neutralized the inhibitory effect exerted by both anti-CollCAM and anti-CollCAM II antibodies, on hepatocyte spreading and attachment to collagen type I substrates. Peptide mapping suggested the Mr 160,000 (R) species to be different from the Mr 115,000 (R).     

Action of the major protease from Entamoeba histolytica on proteins of the extracellular matrix

The action of the major protease from the parasitic protozoon Entamoeba histolytica, a cysteine protease of Mr 27,000-29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated alpha 2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.     

Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.     

Analysis of cholecystokinin-binding proteins using endo-beta-N-acetylglucosaminidase F

We have previously shown that the cholecystokinin (CCK)-binding proteins in rat pancreatic plasma membranes consist of a major Mr 85,000 and minor Mr 55,000 and Mr 130,000 species as revealed by affinity labeling with 125I-CCK-33 using the cross-linker, disuccinimidyl suberate. The glycoprotein nature of these species was investigated using endoglycosidase F (endo F) and neuraminidase treatment and wheat germ agglutinin-agarose chromatography. Treatment of affinity-labeled membranes with endo F resulted in increased electrophoretic mobilities of all three binding proteins, indicating removal of N-linked oligosaccharide side chains. Endo F treatment of each protein in gel slices indicated the following cleavage relationships: Mr 85,000----65,000; Mr 55,000----45,000; Mr 130,000---- 110,000. Using limiting enzyme conditions to digest each protein contained in excised SDS gel slices, three and four products, respectively, were identified for the Mr 85,000 and 55,000 proteins. Similar treatment of the Mr 130,000 protein revealed only the Mr 110,000 product. These results indicated that the Mr 85,000 protein has at least three, the Mr 55,000 protein has at least four, and the Mr 130,000 protein has at least one, N-linked oligosaccharide side chain(s) on their polypeptide backbone. Neuraminidase treatment of affinity-labeled membranes caused slight increases in the electrophoretic mobilities of all three proteins, indicating the presence of sialic acid residues. Solubilization of affinity-labeled membranes in Nonidet P-40 followed by affinity chromatography on wheat germ agglutinin-agarose revealed that all three CCK-binding proteins specifically interact with this lectin and can be eluted with N-acetyl- D-glucosamine. Analysis of the proteins present in the eluted fractions by silver staining indicated a significant enrichment for proteins having molecular weights corresponding to the major CCK-binding proteins in comparison to the pattern of native membranes. Taken together, these studies provide definitive evidence that the CCK- binding proteins in rat pancreas are (sialo)glycoproteins.     

Antibody-linked polymerase assay on protein blots: a novel method for identifying polymerases following SDS-polyacrylamide gel electrophoresis

We describe a method for correlating polymerase activity with a particular polypeptide band in an SDS-polyacrylamide gel which does not require renaturation of the SDS-denatured enzyme. The method involves the following steps: (i) transfer of proteins from an SDS-polyacrylamide gel onto nitrocellulose; (ii) incubation with excess antiserum raised against a partially purified polymerase preparation to link one Fab site of an antibody molecule to the denatured enzyme on the nitrocellulose; (iii) binding of native polymerase to the other Fab site of the antibody molecule in the immune complex to generate a specific polymerase 'sandwich'; (iv) assaying of the nitrocellulose filter for antibody-linked native polymerase activity using an appropriate template and a radioactive substrate followed by treatment with trichloroacetic acid to precipitate in situ the radioactive product. The essential feature of this method is that the use of both non-specific anti-polymerase serum and a partially purified enzyme preparation is sufficient to allow identification of a specific protein following SDS-polyacrylamide gel electrophoresis. This antibody-linked polymerase assay has been developed to identify a 130,000-dalton RNA-dependent RNA polymerase from cowpea leaves. Possible applications of this type of assay as a tool for identifying a wide variety of proteins are discussed.     

Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum.

Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells.     

Characterization of cell surface glycoproteins recognized by the granulocyte-specific monoclonal antibody, AHN-1

Major surface-iodinated proteins of Mr 105,000 and 145,000 of normal human neutrophils are immunoprecipitated by a number of monoclonal antibodies (AHN-1 to AHN-6), which react specifically with granulocytes among peripheral blood cells and selectively inhibit phagocytosis. These proteins, and an Mr 60,000 component, were purified by monoclonal antibody affinity chromatography, molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Each of the three purified proteins was immunoprecipitated by all six antibodies. Nevertheless, tryptic peptide maps of the three proteins indicated that each was a distinct component. AHN-1 to AHN-6 also bound to glycolipid fractions of human neutrophils, and the binding of each antibody to human neutrophils was blocked by the carbohydrate sequences, lacto-N-fucopentaose III. The data indicate that a predominant antigenic determinant of human neutrophils is lacto-N-fucopentaose III, or related carbohydrates, present on three distinct proteins as well as glycolipids. At least one of these molecules appears to be involved in the process of phagocytosis.     

Maturational changes in terminal glycosylation of small intestinal microvillar proteins in the rat

Studies were performed to identify rat intestinal microvillar proteins which undergo changes in terminal glycosylation during postnatal development. Pulse-labeling with [3H]fucose or N-[3H]acetylgalactosamine showed significantly higher incorporation into purified microvillar membranes of weanling than suckling rats. In contrast, the incorporation of [3H]sialic acid after pulse-labeling with N-[3H]acetylmanosamine was higher in suckling rats. SDS-polyacrylamide gel electrophoresis revealed these developmental differences in radioactive sugar incorporation to involve mainly proteins above Mr 90,000. 125I-labeled peanut lectin autoradiography revealed an Mr greater than 330,000 binding protein in suckling rats. Neuraminidase treatment of the membranes revealed the presence of sialyl-substituted sites in this protein in suckling, weaning and weanling animals, but the unmasking of sites decreased with advancing maturation. 125I-labeled Ulex europeus I autoradiography showed marked increases in binding of this lectin to Mr 66,000, 92,000, 130,000, 150,000 and greater than 330,000 proteins from weaning to weanling periods. Similar age-related increases in soybean lectin binding to Mr 130,000-150,000, and greater than 330,000 proteins were demonstrated by affinity chromatography. The Mr values of the major lectin-binding proteins were close to those reported for several hydrolases (trehalase, alkaline phosphatase, sucrase-isomaltase and glucoamylase). Comparison of the Coomassie blue-stained electrophoretograms from each age-group against the corresponding autoradiograms of lection-binding proteins led us to conclude that, while the content of these proteins in the membrane achieve their mature levels at or before weaning, their terminal glycosylation (desialylation, fucosylation, N-acetylgalactosamination) is not fully established until later development.     

Identification of a type V collagenolytic enzyme

A neutral metal protease has been identified which cleaves native type V collagen under conditions where pepsinized type IV collagen or the interstitial collagens are not significantly degraded. The enzyme is secreted into the media of cultured M50-76 reticulum cell sarcoma (malignant macrophages) and leiomyosarcoma tumor cells. Biosynthetically labeled type V collagen prepared from organ cultures of human amnion membrane is used for a routine assay of type V collagenolytic activity. The partially purified enzyme a) exists in a latent form requiring trypsin activation for maximum activity; b) has a molecular weight estimated by molecular sieve chromatography of approximately 80,000 daltons; c) is inhibited by EDTA but not phenylmethylsulfonyl fluoride; and d) produces specific cleavage products of both A and B collagen chains.     

Purification, properties, and evidence for two subtypes of human placenta hexokinase type I

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with Mr 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration (Mr 118,000), preparative isoelectric focusing (pI 5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.     

Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.     

Phaseolus vulgaris cytoplasmic leucyl-tRNA synthetase. Purification and comparison of its catalytic, structural, and immunological properties with those of the chloroplastic enzyme

The cytoplasmic leucyl-tRNA synthetase was purified from bean (Phaseolus vulgaris) leaves. After ammonium sulfate fractionation and chromatography on Sephadex G-50, DEAE-cellulose, hydroxylapatite, and phosphocellulose, complete purification was achieved by blue Sepharose CL-6B chromatography using specific elution with pure yeast tRNALeu1. The enzyme was purified 1050-fold and had a specific activity of 940 nmol of leucyl-tRNA formed/min/mg of protein. Polyacrylamide gel electrophoresis of the native enzyme showed one band, but the denatured enzyme showed two bands. These two protein bands are structurally related. The smallest protein appears to be a cleavage product from the largest one, suggesting the presence of a sensitive cleavage site in the cytoplasmic leucyl-tRNA synthetase. The cytoplasmic enzyme is a monomer (Mr = 130,000), larger than its chloroplastic counterpart (Mr = 120,000). The two enzymes differ in their substrate (tRNA) specificity, tryptic peptide map, and amino acid composition. Antibodies were raised against the cytoplasmic enzyme and against the chloroplastic enzyme and no cross-immunological reaction was detected, showing that the two enzymes do not share any antigenic determinant. Taken together, these results suggest that P. vulgaris cytoplasmic and chloroplastic leucyl-tRNA synthetases are coded for by different genes.     

Purification and characterization of nucleoside diphosphatase from rat-liver microsomes. Evidence for metalloenzyme and glycoprotein

Nucleoside diphosphatase was purified from rat liver microsomes more than 3000-fold with a 16% yield using a procedure including concanavalin-A--Sepharose and phenyl-Sepharose column chromatography. The purified enzyme had a specific activity of 2500 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation, and a Stokes' radius of 4.8 nm was estimated by the gel filtration technique. Its molecular weight is 130,000, but only one single band of Mr 65,000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrates. The enzyme had a pH optimum of 7.5, an isoelectric point of 4.85 and a Km of 2.5 mM for UDP. On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65,000-Mr subunit. Metal-free nucleoside diphosphatase has been prepared. The activity of the metal-free enzyme was restored by the addition of several divalent cations, zinc being the most effective.     

zeta-Crystallin is a major protein in the lens of Camelus dromedarius

Camel (Camelus dromedarius) lenses contain a protein with an apparent subunit Mr 38,000 that constitutes approximately 8-13% of the total protein. The protein has been purified and has a native Mr 140,000 as determined by gel filtration. This is consistent with its being a tetramer. The protein reacts with antibodies raised against both guinea pig zeta-crystallin and peptides corresponding to amino acids 1-10 and 295-308, but not to antibodies raised against amino acids 320-328 of zeta-crystallin. Based on these criteria it is concluded that this protein, which is a major constituent of camel lens, is zeta-crystallin. This may be the first example of a protein (enzyme) being independently utilized as a crystallin in the lens of species from two mammalian orders.     

Transforming growth factor beta 1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s).

Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins.     

Purification and determination of the NH2-terminal amino acid sequence of uracil-DNA glycosylase from human placenta

Uracil-DNA glycosylase has been purified approximately 130,000-fold from extracts of human placenta. Although all of the uracil-DNA glycosylase activity coeluted through six chromatographic steps, at least four distinct peaks of activity were resolved in the final purification on a Mono S column. Each of the peaks containing uracil-DNA glycosylase activity contained two peptides of Mr = 29,000 and Mr = 26,500, respectively, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experimental evidence indicated that the Mr = 29,000 peptide was the uracil-DNA glycosylase enzyme. The amino-terminal sequence of each peptide was determined after blotting of the peptides from the gel onto Polybrene GF/C paper. The sequences were not related to each other, and neither was any significant homology to other proteins found. Uracil-DNA glycosylase had a molecular turnover number of approximately 600/min and apparent Km value of 2 microM. The enzyme is a basic protein and was stimulated about 10-fold by 60-70 mM NaCl whereas higher concentrations were inhibitory.