Human endonuclease specific for apurinic/apyrimidinic sites in DNA. Partial purification and characterization of multiple forms from placenta.

Six chromatographically distinct forms of endonuclease active on apurinic and apyrimidinic sites in DNA have been purified away from DNA phosphatases, DNA N-glycosidases, and other DNases of human placenta. The forms seem to be monomeric proteins of 27,000 to 31,000 daltons, and although catalytically similar, they can be distinguished from one another on the basis of substrate Km and the effects of small molecules such as ATP. Analysis of enzymatic activity on a spectrum of damaged DNA substrates indicates that the enzyme forms probably act at an appreciable rate only adjacent to the phosphodiester bond of a deoxyribose lacking a base (purine or pyrimidine) in duplex DNA; such sites can be formed by treating the DNA with acid, alkylating agents, DNA N-glycosidases, and, probably, x-rays and OsO4. The incision is made so as to form a deoxyribose 5'-phosphate and a 3'-hydroxynucleotide.     

Human kidney alpha-L-iduronidase: purification and characterization.

α-l-Iduronidase has been purified 25,000-fold from the soluble proteins of human kidney by chromatography on heparin-Sepharose, hydroxylapatite, and Bio-Gel P-100. The α-l-iduronidase activity is associated with 80% of the protein in the most purified preparation. It has a molecular weight of 60,000 ± 6500, determined by sedimentation equilibrium, and can be dissociated by reduction into subunits of molecular weight 31,000 ± 6500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate in the presence of dithiothreitol. It contains glucosamine and binds to concanavalin A. The pH optimum, K_m and V_max for two substrates, phenyl iduronide and [³H]anhydromannitol iduronide, were found to be 4.0, 1.05 mm, 16 μmol/mg protein/min, and 4·5, 9 mm and 270 μmol/mg protein/min, respectively. The enzyme is of the low uptake, noncorrective form with respect to fibroblasts cultured from the skin of patients with Hurler syndrome. It is inhibited by 10⁶ m p-chloromercuribenzoate and 10^?3 m Cu²⁺, but is not significantly affected by other divalent cations, EDTA, or sulfhydryl compounds. Antibodies to α-l-iduronidase have been raised in goats.     

Human alpha-N-acetylglucosaminidase. 1. Purification and properties.

alpha-N-Acetylglucosaminidase was purified from human urine to a state of apparent homogeneity. alpha-N-Acetylglucosaminidase is a glycoprrotein with an extensive charge heterogeneity. The molecular weight determined by gel filtration is 307000. Polycarylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate indicates molecular weight heterogeneity of isocharged forms of the purified enzyme. The enzyme has a pH optimum of 4.5 +/- 0.3 and KM and V values of 0.14-0.74 mM, and 1.04-3.68 mumol mg-1 min-1 for three aryl 2-acetamido-2-deoxy-alpha-D-glucosides and UDP-N-acetylglucosamine. Heparan sulfate, heparin and dermatan sulfate are competitive inhibitors. The enzyme is inhibited by Hg2+ and Cu2+. --SH-protective reagents and thiol reagents have no effect on the enzyme activity. Heating at 65 degrees C and pH values below 5 inactivate the enzyme rapidly.     

The purification and characterization of multiple forms of mouse submaxillary gland renin

Five forms of renin, A₀, A, C, D and E, from mouse submaxillary gland were purified by a two-step procedure including chromatography on the immunoaffinity column and CM-cellulose column. Four renin fractions, A₀, A, C and E were purified to homogeneity by the criteria of polyacrylamide gel electrophoresis, analytical isoelectric focusing and Ouchterlony double immunodiffusion. All these forms of renin have molecular weights of 40 000 as determined by gel filtration on Sephadex G-100 column. No high molecular weight renin could be demonstrated. Individual renin fractions showed similar angiotensin I formation activity, 52–158 ng angiotensin I/ng protein per h. No other protease activity could be detected with hemoglobin or casein as substrate. These purified proteins showed a discrete pattern of migration under polyacrylamide gel electrophoresis. Under denaturing condition in SDS-gel electrophoresis, all but fraction D showed a protein band with a molecular weight of 30 000. Fraction D showed a major component with molecular weight of 33 000. The isoelectric points of these renin forms varied from 5.46 to 5.76. They all reacted with antibody raised against renin A and showed similar pressor response activity with 20 ng quantities of the purified proteins. The closely related characteristics of these five forms of renin were further demonstrated by their similarity in peptide mapping patterns after limited digestion with Staphylococcus aureus V8 protease. The data suggest that these proteins are homologous proteins.     

Calcium-activated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms

Three forms of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) were extensively purified from rat liver homogenate. Subcellular fractionation analysis indicated that the majority (approximately 85%) of the activity was associated with particulate fractions of the liver. Among these, the microsomal and nuclear fractions accounted for approximately 63% and approximately 10% of total activity. The remaining 15% of protein kinase C was recovered in the soluble fraction following differential centrifugation. It was also found that most of the membrane-associated protein kinase C was latent, with 4-6-fold stimulation with detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate, octyl beta-glucoside, or Triton X-100. The activity of both the bound form and the soluble enzyme was enhanced by the addition of Ca2+ and phosphatidylserine, when histone H1 was used as substrate. The bound protein kinase C activity was dissociated by homogenization of liver in buffer containing ethylene glycol bis(beta-aminoethyl ether)-N,-N,N',N'-tetraacetic acid, ethylenediaminetetraacetic acid, and various proteolytic inhibitors, and the solubilized extract was used to purify multiple forms of the enzyme. The purification procedure sequentially utilized (NH4)2SO4 fractionation, ion-exchange chromatography on DEAE-cellulose, gel permeation chromatography on Fractogel TSK HW-55 (F), ion-exchange chromatography on hydroxylapatite, gel permeation chromatography on Ultrogel AcA34, and affinity chromatography on polyacrylamide-immobilized phosphatidylserine. On hydroxylapatite columns, protein kinase C activity was resolved into three isoenzymic forms designated C-I, C-II, and C-III. The molecular weights of the three isoenzymic forms were in the range of 208,000-225,000 as shown by chromatography on calibrated Ultrogel AcA34 columns and sucrose density gradient centrifugation. Furthermore, all three isoenzymes demonstrated a single peak with a sedimentation coefficient (s20.w) in the range of 9.0-9.2. However, with polyacrylamide gel electrophoresis, all the forms showed a single protein component with average molecular weight of 64K, suggesting that the native isoenzymes may be composed by subunits. Finally, all three isoenzymes exhibited nearly identical enzymatic properties.     

Cytosolic aspartate aminotransferases from different chicken tissues: purification and characterization of their multiple forms

Cytosolic aspartate aminotransferases from chicken heart, liver, spleen, skeletal muscle and breast muscle differed in number of their molecular forms, detected by polyacrylamide gel electrophoresis and specific staining. The number of molecular forms varied from tissue to tissue but the electrophoretic mobilities of a given form in all tissues were analogous. Within a single tissue most of the enzyme activity was present as the lowest-running band (alpha form) and the rest was distributed in minor bands termed (B,tau, alpha and epsilon forms). We report a method for the purification of cytosolic aspartate aminotransferases from various chicken tissues. The procedure can be carried out in one week and allows the obtention of isolated molecular forms of the enzyme, independently of the tissue under study. Separation of multiple forms was also achieved by chromatofocusing. The isoelectric points determined by this method for a given form in all five tissues were analogous and differed from those of the molecular forms of the enzyme from other origins. An Mr of 100,000 was obtained for all molecular forms of the five chicken tissues studied.     

S-Adenosylhomocysteine hydrolase from human placenta. Affinity purification and characterization.

S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4',5'-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4',5'-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.     

Human tumor necrosis factor. Production, purification, and characterization

Human tumor necrosis factor (TNF) was purified to homogeneity from serum-free tissue culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 4 beta-phorbol 12-myristate 13-acetate. The purification scheme consisted of controlled-pore glass and DEAE-cellulose chromatography, Mono Q-fast-protein liquid chromatography, and reverse-phase high performance liquid chromatography. The purified protein was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The specific activity of purified tumor necrosis factor is approximately 10(8) units/mg. The protein has a molecular weight of approximately 17,000, an isoelectric point of 5.3, and contains two cysteines involved in a disulfide bridge. Approximately 50% homology between TNF and another cytolytic lymphokine, lymphotoxin, exists when the NH2-terminal 34 residues of TNF and internal sequence generated by tryptic, Staphylococcus aureus V8 protease, and chymotryptic digests of TNF are aligned with the complete amino acid sequence of lymphotoxin.     

Purification and partial characterization of alpha-N-acetylglucosaminidase from human liver.

A deficiency in alpha-N-acetylglucosaminidase is known as mucopolysaccharidosis IIIB or Sanfilippo B syndrome. We purified this enzyme almost 39,000-fold from liver to homogeneity with 3% recovery. Use of concanavalin A (Con A)-Sepharose and heparin-Sepharose resulted in 13.4-fold and 11.6-fold purifications of the enzymatic activity, respectively. The molecular mass was estimated to be 300 kDa by gel filtration and 80 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The isoelectric point was 5.1, optimal pH was 4.5, and the Km for p-nitrophenyl alpha-N-acetylglucosamine was 0.13-0.20 mM. The purified enzyme was stable at 50 degrees C for 1 h and within the pH range of 6.5-8.5. Anti-serum against the purified enzyme raised in BALB/c mice inhibited the activities of alpha-N-acetylglucosaminidase.     

A novel dihydrodiol dehydrogenase in bovine liver cytosol: purification and characterization of multiple forms of dihydrodiol dehydrogenase.

Three enzymes (DD1, DD2, and DD3) having dihydrodiol dehydrogenase activity were purified to homogeneity from bovine cytosol. DD1 and DD2 were identified as 3 alpha-hydroxysteroid dehydrogenase and high-Km aldehyde reductase, respectively, as judged from their molecular weights, substrate specificities and inhibitor sensitivities. DD3 was a unique enzyme which could specifically catalyze the dehydrogenation of trans-benzenedihydrodiol and trans-naphthalenedihydrodiol without any activity toward the other tested alcohols, aldehydes, ketones, and quinones. The Km value of DD3 (0.18 mM) for benzenedihydrodiol was lower than those of other dihydrodiol dehydrogenases so far reported. DD3 immunologically crossreacted with DD1, but showed no crossreactivity with DD2. Additionally, DD3 was inhibited in a competitive manner, with a low Ki value of 1 microM, by androsterone, which was a good substrate for DD1. It was assumed that DD3 is a novel enzyme which is specific to dihydrodiols, exhibiting similarity to DD1 in immunological and structural properties.     

Human placenta: de visu demonstration of interstitial Cajal-like cells

Traditional interstitial cells of Cajal (ICC) are present in the digestive tube and are supposed to act as pacemakers and neuromodulators. However, interstitial Cajal-like cells (ICLCs) were found outside the gastrointestinal tract, in various organs (e.g. ureter, bladder, fallopian tube, uterus, pancreas, mammary gland, myocardium etc.) and looking for such ICLC is a priority in our laboratories.We report here unequivocal visual evidence that ICLCs are present in the mesenchymal tissue of the villi from human term placenta.The following methods were used: a. vital staining with methylene blue (cryosections); b. silver impregnation (paraffin sections); c. Epon-embedded sections (approximately 1 microm) of glutaraldehyde/osmium fixed tissue, stained with toluidine blue; d. primary cell cultures (or second-passage cells) to reveal the characteristic, very long, moniliform cell processes and mitochondrial localization at dilations (molecular fluorescence probe: Mito Tracker Green); e. immunofluorescence for c-kit/CD117 marker or other characteristic proteins; f. transmission electron microscopy to establish the identity of ICLC.     

Various forms of mouse lactoferrins: purification and characterization

This work was conducted to study the microheterogeneity of mouse lactoferrin (LF). Two forms, LF₁ and LF₂, could be purified from uterine luminal fluid by ion-exchange HPLC on a Protein PAK SP 5PW column. Another form, LF₃, was purified from the epididymis homogenate by affinity chromatography on a column of Protein A-Sepharose coupled with the purified LF₂ antibody that was prepared to give no crossreaction with serum albumin. Both LF₁ and LF₂ showed a M_r 74 000 band while LF₃ gave a M_r 70 000 band on reducing SDS–PAGE. All of them were reduced to a M_r 68 000 band after they had been digested with N-glycosidase F. The data from automated Edman degradation confirmed the completely identical 19 amino acid sequences in the N-terminal regions of these three LFs, except the lack of N-terminal Lys–Ala of LF₂/LF₃ in LF₁. LF in tissue homogenates was immunodetected by Western blot procedure using the purified LF₂ antibody. Different amounts of LF with a molecular mass of the 70 000 or 74 000 were distributed in the non-sexual organs such as kidney, spleen, lung, heart and liver and the sexual glands including epididymis, vagina, uterus, ovary and prostate. No LF was detected in stomach, intestine, testis and seminal vesicle.     

Isolation, purification, and characterization of pregnancy-specific protein B from elk and moose placenta.

Pregnancy-specific protein B (PSPB) was isolated, purified, and partially characterized from elk and moose placenta. The procedure, which was monitored by bovine PSPB (bPSPB) RIA, included homogenization and extraction in aqueous solution, acidic and ammonium sulfate precipitation, and ion exchange, gel filtration, and affinity chromatographies. The estimated molecular sizes of moose PSPB (mPSPB) were 58 kDa and 31 kDa, and of elk PSPB (ePSPB) were 57 kDa, 45 kDa, and 31 kDa by SDS-PAGE. The isoelectric points of mPSPB were 4.8, 6.6, and 6.7, and of ePSPB were 4.8, 4.9, 6.1, and 6.2 as determined by isoelectric focusing and two-dimensional gel electrophoresis. The carbohydrate contents of mPSPB and ePSPB were approximately 3.15% and 4.98%, respectively. Although ePSPB and mPSPB were recognized by anti-bPSPB in an Ouchterlony double immunodiffusion test, they were found to share identical epitopes and partial identities compared to bPSPB. After treatment at different temperatures (20-60 degrees C) for 1 h, the immunoreactivities of ePSPB and mPSPB in serum were very stable. Only ePSPB in serum treated at 60 degrees C lost some immunoreactivity. After alteration of serum pH (pH 3-11) for 2 h, the immunoreactivities of ePSPB and mPSPB became lower at pH 3 and 4, and remained stable from pH 5 to 11. These data show that moose and elk PSPB have properties similar to those of bovine and ovine PSPB.     

Human placental chorionic renin: production, purification and characterization

Native human renin, produced from the culture of human chorionic trophoblasts, has been purified to homogeneity on a milligram scale using a five-step purification scheme. The chorion cells secrete 50-200 milliGoldblatt Units of trypsin-activatable prorenin per ml into the medium. The pro-enzyme is partially purified by ammonium sulfate fractionation and chromatographies on QAE-Sephadex and cibracon blue-agarose. Following conversion of prorenin to the active enzyme by porcine trypsin, the renin is purified to homogeneity by affinity chromatography and gel filtration. Chorionic prorenin has a molecular weight of 43,000; the active enzyme 40,000. Both proteins exist as a single polypeptide chain as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions. The average specific activity of six different preparations was found to be 1072 Goldblatt Units/mg. The amino acid composition and N-terminal sequence of the active enzyme has been determined and is identical to the human kidney enzyme. Microheterogeneity of chorionic renin was demonstrated by isoelectrofocusing analysis. The physical characterization of chorionic renin is compared with that reported for the human kidney enzyme.     

NAD biosynthesis in human placenta: purification and characterization of homogeneous NMN adenylyltransferase.

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from human placenta. The purification procedure consists of several chromatographic steps, including dye-ligand, adsorption, and hydrophobic interaction chromatography. The final enzyme preparation is homogeneous as judged by a single silver stainable band on both nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 132,000, as determined by gel filtration on a Superose 12 HR 10/30 fast protein liquid chromatography column. The protein possesses a quaternary structure and is composed of four apparently identical M(r) 33,000 subunits. Isoelectrofocusing experiments give multiple pI values ranging from pH 4.7 to 6.6. Optimum pH study shows a plateau extending from pH 6.0 to pH 9.0. Km values for NMN, ATP, NAD+, and PPi are 38, 23, 67, and 125 microM, respectively. Kinetic analysis reveals a behavior consistent with an ordered sequential Bi-Bi mechanism. Among several metabolites tested only ADP-ribose and beta-NMNH were found to significantly inhibit the enzyme activity.     

Human eosinophil peroxidase: purification and characterization

Human eosinophil peroxidase (EPO) was isolated from granules from granulocytes of a patient with hypereosinophilia. The granules were extracted by means of 0.2 M NaAc, pH 4.0. The purification steps included gel filtration chromatography on Sephadex G-75 superfine and ion-exchange chromatography on CM-Sephadex G-50. The purified protein showed one band on agarose-electrophoresis, a high peroxidase activity, and a 415-nm/280 nm ratio of 1.15. After reduction, EPO showed two bands on SDS-PAGE of m.w. 52,000 and 15,000, respectively. On gel filtration, the unreduced protein had a m.w. of approximately 77,000. Amino acid analyses showed a high content of arginine and aspartic acid. Monospecific antibodies to EPO were prepared in rabbits, and a specific radioimmunoassay was developed. There was an almost linear correlation between the content of EPO measured by the radioimmunoassay and the number of eosinophils in a mixed cell extract from reference material, indicating the eosinophil origin of EPO. The content of EPO was estimated to be 15.0 micrograms/10(6) eosinophils.