heparin-Sepharose Column Chromatography Research Articles

Purification and characterization of SepII a new restriction endonuclease from Staphylococcus epidermidis

A Type II restriction enzyme SepII has been purified to apparent homogeneity from the gram-positive coccus, Staphylococcus epidermidis. The purification included an ammonium sulfate precipitation followed by Q-sepharose, heparin-sepharose and MonoQ column chromatography on an FPLC system. SDS-PAGE analysis showed a denatured molecular weight of 29 kDa. The effects of temperature, pH, NaCl, Mn 2+, Ca 2+, and Mg 2+ ion concentrations were studied to determine the optimal reaction conditions. The enzyme exhibits near maximal levels of activity between pH 8–10, at 10–20 mM MgCl 2, 100–150 mM NaCl and 1 mM DTT. The results also show that in NEB Buffer 3 the enzyme is active over a broad temperature range from 0 to 70 °C, and in the absence of DNA, enzyme thermostability is observed up to 50 °C for 20 min, while most of the original activity is conserved in 50% glycerol for weeks at room temperature. Single and double digestion in presence of commercial restriction enzymes of known DNA substrates (lambda, pBR322, pET21, pTrcHisB, pPB67) showed that the purified SepII recognized and cleaved the same site as EcoRV. Genomic DNA modification status was also determined.

Isolation, purification and properties of new restriction endonucleases from Bacillus badius and Bacillus lentus

We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Phix174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Phix174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg2+ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 degrees C (pH 8.0) with 100 mM NaCl and 10 mM Mg2+ concentrations.

DNA Aggregating Protein from Carp (Cyprinus capio) Eggs

Hemagglutinating activity for glutaraldehyde-fixed, trypsinized human and rabbit erythrocytes was obtained from carp (Ciprinus capio) egg extract. This activity was inhibited by sulfated glycans such as heparin and fucoidan, and salmon testis DNA (stDNA), but not by mono- and oligosaccharides. The active fraction from hydroxyapatite column, HA400, caused aggregation of stDNA at a concentration of 125 microg/ml. HA400-induced aggregation was inhibited by heparin and fucoidan at the concentrations of 25 and 50 microg/ml, respectively. HA400 also bound to short size single- or double-stranded oligonucleotide in dot blot hybridization analysis using 32P-labeled probe. HA400 was further purified by heparin-Sepharose column chromatography, and consequently, active fractions (HS2, HS3 and HS4) were obtained. Hemagglutinating activities of HS2 and HS4 were 16 and 32 times higher than that of HS3, respectively. In contrast, HS3 showed the highest DNA aggregating activity among these fractions, indicating that the activities of DNA aggregation and hemagglutination were separated by heparin-Sepharose column chromatography. HS3-induced stDNA aggregation was inhibited by 0.5 M NaCl or above. Southwestern blot analysis revealed that the DNA aggregating protein (DAP) possessing oligonucleotide-binding capacity was the 18 kDa protein. The N-terminal amino acid sequence of DAP was LKEGECEVCVGFLGR having partial homology with DNA-binding protein HU-alpha, but showed no homology with nucleohistone proteins. These results indicate that there is a novel, non-histone DAP in carp eggs.

Modulation of Hepatitis C Virus RNA-dependent RNA Polymerase Activity by Structure-based Site-directed Mutagenesis

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNA-dependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 A contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the Lambda 1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive pre-initiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template.

Application of rice (Oryza sativa L.) suspension culture in studying senescence in vitro (I). Single strand preferring nuclease activity

Single Strand-Preferring Nucleases (SSPNs) have been implicated in the triggering and progress of cell death pathways, including senescence in higher plants, though the biological roles of such enzymes are still obscure. In the present study, heterotrophic cell suspension cultures of Oryza sativa L. (rice) cv Taipei 309 were used to investigate changes in Single Strand-Preferring Nuclease activity associated with cell death in vitro . An acid nuclease activity (pH 5.5) was found which was strongly stimulated in the presence of 10 mM Zn 2+ and inhibited by 10 mM EDTA and EGTA. An increase in SSPN activity was concomitant with a loss of cell viability, total protein and the onset of stationary phase of growth in the cell cultures. Using DNA-SDS-PAGE two major SSPNs were detected with the molecular weights of 26 KDa and 53.5 KDa. There are possibilities that these two enzymes represent monomeric and dimeric forms of the same nuclease or they are two different enzymes. The 26 KDa nuclease was partially purified using heparin-Sepharose column chromatography. The results confirm the value of plant cell suspension cultures for the investigation of the molecular process underlying plant cell ageing, senescence processes and programmed cell death (PCD). Possible senescence-associated roles of SSPNs are discussed.

Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10(11) and 9.8 x 10(12)/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.

Production of recombinant human midkine in yeast, Pichia pastoris

Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of the AOX1 gene promoter. The expression of midkine was efficiently induced by methanol in a high cell density fermentation. Approximately 0.3 g/ l culture of midkine accumulated in the cells by 72 h after induction. When the cells were disrupted, midkine was recovered in an insoluble form, and was insoluble even in the presence of 7 M urea. The precipitate was dissolved in the buffer solution (pH 8) containing 8 M guanidine hydrochloride, 10 mM dithiothreitol, 1 mM EDTA and 50 mM Tris-Cl, and then, midkine was renatured by dialysis at high concentration against the buffer solution (pH 8) containing 0.5 M sodium chloride and 20 mM Tris-Cl. The renatured midkine was recovered using a SP-Sepharose column, and purified further by Heparin-Sepharose column chromatography. Approximately 64 mg/ l culture of the purified midkine was obtained. The amino acid sequence of amino-terminus and the amino acid composition of midkine were the same as those of Met-midkine that has a methionine residue at the amino-teminus. Mass spectrometry of purified Met-midkine showed a mass of 13370.7 Da (average), almost the theoretical mass for it. The Met-midkine enhanced the proliferation of Chinese hamster ovary (CHO) cells.

Production of Recombinant Human Midkine in Yeast, Pichia pastoris.

Recombinant human midkine was expressed in the cells of Pichia pastoris under the control of the AOX1 gene promoter. The expression of midkine was efficiently induced by methanol in a high cell density fermentation. Approximately 0.3 g/l culture of midkine accumulated in the cells by 72 h after induction. When the cells were disrupted, midkine was recovered in an insoluble form, and was insoluble even in the presence of 7 M urea. The precipitate was dissolved in the buffer solution (pH 8) containing 8 M guanidine hydrochloride, 10 mM dithiothreitol, 1 mM EDTA and 50 mM Tris-Cl, and then, midkine was renatured by dialysis at high concentration against the buffer solution (pH 8) containing 0.5 M sodium chloride and 20 mM Tris-Cl. The renatured midkine was recovered using a SP-Sepharose column, and purified further by Heparin-Sepharose column chromatography. Approximately 64 mg/l culture of the purified midkine was obtained. The amino acid sequence of amino-terminus and the amino acid composition of midkine were the same as those of Met-midkine that has a methionine residue at the amino-teminus. Mass spectrometry of purified Met-midkine showed a mass of 13370.7 Da (average), almost the theoretical mass for it. The Met-midkine enhanced the proliferation of Chinese hamster ovary (CHO) cells.

Studies on functions of the 63-kDa A- and 74-kDa B'(delta)-regulatory subunits in human erythrocyte protein phosphatase 2A: dissociation and reassociation of the subunits.

A heterodimeric form, CA, of protein-serine/threonine phosphatase (PP) 2A purified from human erythrocytes was dissociated into a 34-kDa catalytic subunit C and 63-kDa inactive subunit A by Sephacryl S-200 gel filtration in the presence of 6 M urea. Reassociation of the C- and A-subunits in the absence of urea suppressed the PP activity of the C subunit toward phosphorylase a, P-H2B histone, and P-H1 histone in the presence or absence of 20 mM MnCl(2) or 50 mM Mg(CH(3)COO)(2), but stimulated the PP activity toward P-H1 histone in the presence of 200 mM NaCl and the Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity toward P-Tyr-Glu copolymers. The 74-kDa inactive B'(delta) subunit was isolated from a heterotrimeric form, CAB'(delta), of PP2A partially purified from human erythrocytes, by heparin-Sepharose column chromatography. The B'(delta) subunit reassociated with CA and suppressed the PP- and PTP-activities of CA. The B'(delta) subunit did not associate with the isolated C subunit directly, and had no effect on the activities of the C subunit, indicating that the A subunit is essential for the association of the B'(delta) subunit with CA and the resulting suppression of the PP- and PTP-activities.

Tissue and subcellular distributions, and characterization of rat brain protein phosphatase 2A containing a 72-kDa delta/B" subunit.

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

Purification and characterization of low density lipoprotein containing apolipoprotein B-48 from the plasma of an apoE-deficient patient

The plasma low density lipoprotein (LDL) of an apolipoprotein (apo) E-deficient patient was reported to contain apoB-48 (D. Kurosaka et al., Atherosclerosis 1988;88:15), which is a structural protein of chylomicrons and is not present in the fasting plasma LDL of normal subjects. We separated the LDLs containing apoB-48 (apoB-48 LDL) and apoB-100 (apoB-100 LDL) from the plasma of this patient using heparin-sepharose column chromatography. The apoB-48 LDL contained apoA-I, apoA-IV, and apoCs besides apoB-48. There were no differences in size and lipid composition of the apoB-48 and the apoB-100 LDLs, both of which contained more triglyceride than LDL from normal subjects. After incubation with apoE, the apoB-48 LDL contained twice the amount of apoE than the apoB-100 LDL and showed a marked decrease in apoA-I and apoA-IV content. These results suggest that lipoproteins containing apoB-48 in normal subjects can be quickly taken up through an apoE-mediated receptor mechanism after receiving apoE in exchange for apoA-I and A-IV.

Phosphatidylinositol 4,5-Bisphosphate Binding to the Pleckstrin Homology Domain of Phospholipase C-δ1 Enhances Enzyme Activity

The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some co-complexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-delta1 (PLCdelta1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in a dose-dependent manner with an EC50 = 1 microM (0.3 mol%), up to 9-fold higher when 5 microM (1.5 mol%) of PIP2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 microM of PI with a molar ratio of PI:PS:PC = 1:5:5). Stimulation was specific for PIP2, since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP2-mediated stimulation was, however, inhibited by inositol 1,4, 5-triphosphate (IP3) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLCdelta1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP2-mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP2. Analysis of PIP2-stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (Ks) of PLCdelta1 for the PI/PS/PC vesicles was reduced from 558 microM to 53 microM, and the interfacial Michaelis constant (Km) was reduced from 0.21 to 0.06 by PIP2. The maximum rate of PI hydrolysis (Vmax) was not affected by PIP2. These results demonstrate that a major function of the PH domain of PLCdelta1 is to modulate enzyme activity. Further, our results identify PIP2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP2.

Midkine, a heparin-binding growth/differentiation factor, exhibits nerve cell adhesion and guidance activity for neurite outgrowth in vitro.

By means of a baculovirus expression system, a large amount of mouse midkine (MK) was produced. The protein was purified to homogeneity by heparin-Sepharose column chromatography. The purified protein was of a mature type; the signal peptide was cleaved at the expected site. To examine the neurite-guiding activity of MK, rat embryonic brain cells (embryonic days 17-18) were cultured on plates coated with purified MK in a grid pattern. The cells attached to and extended their neurites along the substrate pattern. This interaction was strongly inhibited by heparin, but not by other glycosaminoglycans. Treatment of the cells with heparitinase was effective for inhibiting their adhesion to the substrate. These data suggest that the heparin-like domain on cell surface heparan sulfate proteoglycan is the primary site for MK binding upon interaction with nerve cells.

Angiotensin II-modified LDL is taken up by macrophages via the scavenger receptor, leading to cellular cholesterol accumulation.

The incidence of myocardial infarction is significantly higher in hypertensive patients with increased plasma concentration of angiotensin (Ang) II. Ang II was shown to bind to LDL in vitro, and in the present study we showed its binding to LDL in vivo. Ang II (10(-7) mol/L) was incubated with LDL for 3 hours at 37 degrees C, followed by reseparation of the modified lipoprotein (Ang II-LDL) and its incubation with J-774 A.1 macrophages. Binding of Ang II to LDL significantly increased the lipoprotein protein degradation (by 25%) and its cell association (by 75%) compared with nontreated LDL. Unlike Ang II-LDL, both Ang I-LDL and Ang III-LDL were taken up by macrophages similar to native LDL. The lipid composition and size of Ang II-LDL were similar to those of native LDL, and it was not aggregated. Ang II-LDL was not oxidized, as the contents of malondialdehyde and peroxides were not different from those found in native LDL. On heparin-Sepharose column chromatography, Ang II-LDL was eluted in the void volume, like acetylated LDL (Ac-LDL) and unlike native LDL, which binds to heparin. The cellular degradation of Ang II-125I-labeled LDL by J-774 A.1 macrophages of Ang II-125I-labeled LDL by J-774 A.1 macrophages was studied in the presence of a 50-fold excess of nonlabeled native LDL, Ang II-LDL, Ac-LDL, or oxidized LDL (Ox-LDL). Whereas native LDL had no effect on the degradation of Ang II-125I-LDL by the macrophages, Ac-LDL, Ox-LDL, and Ang II-LDL reduced the cellular uptake of the lipoprotein by 77%, 82%, and 87%, respectively. Similarly, fucoidin but not free Ang II reduced macrophage degradation of the labeled Ang II-LDL. We conclude that Ang II can modify LDL to a form that is not oxidized or aggregated but is still taken up at an enhanced rate by macrophages via the scavenger receptor.

Quantitative and qualitative changes of extracellular-superoxide dismutase in patients with various diseases

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is the major SOD isozyme in extracellular fluids. It has previously been shown that EC-SOD levels in sera from healthy persons are clearly divided into two discontinuous groups: a lower group (named Group I, below 120 ng/ml) and a higher group (Group II, above 400 ng/ml). The family studies have shown that the high EC-SOD level in healthy persons is genetically transmitted. We report here on the EC-SOD levels in the sera of patients with various diseases. The EC-SOD levels were distinctly higher in patients with renal diseases and moderately higher in liver diseases and diabetes than those in normal healthy persons. In cerebrovascular diseases, heart diseases and acute digestive diseases, significant differences of EC-SOD were not observed. In patients with renal diseases, the increase of EC-SOD was accompanied by the lack of renal function. Serum EC-SOD in Group I healthy persons is known to be heterogeneous with regard to heparin affinity and can be separated into three fractions: A without affinity, B with weak affinity and C with relatively strong heparin affinity, whereas the EC-SOD in Group II is mainly one fraction of C-type. Also in the case of hemodialysis patients, serum EC-SOD in Group I or Group I' (~120–400 ng/ml) was divided into three fractions. EC-SOD in Group II showed two different profiles on heparin-Sepharose column chromatographies: one consisted mainly of EC-SOD C and the other consisted of EC-SOD A and C. It is probable that the high serum EC-SOD level in hemodialysis patients was due to two possible factors: the genetic transmitted factor and unknown pathophysiological factor(s).

Large and Cholesteryl Ester-Rich High-Density Lipoproteins in Cholesteryl Ester Transfer Protein (CETP) Deficiency Can Not Protect Macrophages from Cholesterol Accumulation Induced by Acetylated Low-Density Lipoproteins1

High-density lipoprotein (HDL) has been speculated to have an anti-atherogenic function. Many in vitro studies have demonstrated that HDL has the ability to remove cholesteryl ester (CE) from lipid-laden macrophages. However, the effect of alteration in chemical composition and particle diameter on the in vivo function of HDL is unknown. In the study described here, we have isolated the HDL from patients homozygous for cholesteryl ester transfer protein (CETP) deficiency and examined its function in vitro, in order to clarify the anti-atherogenic property of HDL in CETP-deficient subjects. Apolipoprotein (apo) E-free HDL2 from the patients, separated by heparin-Sepharose column chromatography, was rich in CE, poor in triglycerides (TG), and enlarged in size on 4-30% nondenaturing polyacrylamide gradient gel electrophoresis. In contrast, HDL3 from the patients was normal in size and in its chemical composition. First, we examined the effect of HDL on CE accumulation in macrophages. After mouse peritoneal macrophages had been incubated with both acetylated low-density lipoproteins (Ac-LDL) and HDL, cellular CE content was determined by an enzymatic, fluorometric method. Ac-LDL alone induced a 9-fold accumulation of CE. The addition of apo E-free HDL2 and HDL3 from controls and patients' HDL3 prevented CE accumulation in macrophages, while patients' HDL2 had no preventive effect. We next investigated the in vitro ability of HDL to remove cellular CE from lipid-laden macrophages after incubation with Ac-LDL. After loading of macrophages with cholesterol by Ac-LDL, HDL was added to the culture medium and the cellular CE content was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and Characterization of Platelet-Activating Factor Acetylhydrolase from Peritoneal Fluid Obtained from Guinea Pigs after Endotoxin Shock1

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetyl-hydrolase is an enzyme that hydrolyzes the acetyl ester of PAF. We purified this enzyme, which accumulated in the peritoneal cavity during endotoxin shock, by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl, heparin-Sepharose, Con A-Sepharose, chelating-Sepharose, and MonoQ column chromatographies. We identified a monomeric polypeptide with a molecular weight of approximately 63 kDa on SDS-PAGE. This molecular weight differs from those of previously described acetylhydrolases. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. In addition, the enzyme activity was not inhibited by either pBPB or quinacrine. Accordingly, this enzyme is distinct from phospholipase A2. In addition, this enzyme also hydrolyzed some oxidatively fragmented phospholipids with PAF-like biological activities such as 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-succinoyl-sn-glycero-3-phosphocholine.

INHIBITORY EFFECTS OF BESTRABUCIL, A CONJUGATE OF CHLORAMBUCIL AND ESTRADIOL, ON THE PRODUCTION OF ANDROGEN‐INDUCED GROWTH FACTOR(S) BY SHIONOGI CARCINOMA 115 CELLS

It is known that diffusible trophic factors play an important role in both the normal and cancerous growth regulatory processes of hormone-responsive cells such as are found in the prostate and mammary glands. Consequently, it is important to identify whether the production of such growth factors is affected by administration of therapeutic agents. We examined the effect of bestrabucil, a benzoate of an estradiol-chlorambucil conjugate, on the production of growth factor(s) by Shionogi carcinoma 115 (SC-115) cells, an androgen-responsive cultured cancer cell line. We then investigated whether the inhibitory effect found was specific to bestrabucil, or if it was also produced by a mixture of the 2 compounds, estradiol and chlorambucil. Bioassay employing BALB/3T3 cells demonstrated the presence of two kinds of growth factor in the conditioned medium obtained by culturing SC-115 cells in medium containing 10(-8) M testosterone; these factors could be separated by heparin-sepharose column chromatography using 0.5 M NaCl and 1.1 M NaCl. When the SC-115 cells were cultured in medium containing bestrabucil, at a concentration of 10(-5) M, no growth factor activity was detected in the fraction eluted from the heparin-sepharose column by 1.1 M NaCl. At bestrabucil concentrations of 10(-5)-10(-7) M, concentration-dependent inhibition of growth factor production by SC-115 cells could be demonstrated by 3H-thymidine uptake assay. However, this inhibitory effect could not be demonstrated using only a mixture of estradiol and chlorambucil.(ABSTRACT TRUNCATED AT 250 WORDS)

NADPH-cytochrome c reductase from human neutrophil membranes: purification, characterization and localization

Neutrophil-membrane-associated NADPH-cytochrome c reductase and cytochrome b558 were separately eluted and highly purified by a combination of ion-exchange Sepharose, N-amino-octylagarose, 2',5'-ADP-Sepharose and heparin-Sepharose column chromatographies. The purified cytochrome c reductase with an apparent molecular mass of 68 kDa contained FMN and FAD (FMN/FAD approx. 1). Cytochrome b558 prepared in the presence of phospholipids and FAD showed marked O2-.-producing activity (Vmax., 8.53 mumol of O2-./min per mg of cytochrome; Km for NADPH 58.8 microM) in a cell-free assay system consisting of cytosol, arachidonate and GTP[S]. However, when it was obtained without FAD added to the purification process, it had negligible FAD and little or no O2-.-forming activity in the reconstituted system. The NADPH oxidase activity was not markedly stimulated on incubation of the purified reductase with either flavinated or flavin-depleted cytochrome b558 in the cell-free system, suggesting that the reductase is not likely to be involved in neutrophil O2-. generation. The purified reductase cross-reacted with polyclonal antibodies against both hepatic NADPH-cytochrome P-450 reductase and a synthetic peptide, ILVGPGTGIAPFRSF, which indicates residues 529-543 located in the glycine-rich NADPH-binding domain of the P-450 reductase, but cytochrome b558 did not produce any immunoreactive bands to these antibodies. These antibodies also produced a positive reaction with a 76 kDa protein from dimethyl sulphoxide-induced HL-60-cell microsomes. After solubilization of the microsomal membranes, the 76 kDa protein was readily converted into a partially proteolysed form (68 kDa) even in the presence of antiproteases. In addition, the microsomal fraction shows a CO difference spectrum with a peak at about 454 nm and a trough at 476 nm in the presence of dithionite, indicating the presence of a cytochrome P-450-like haemoprotein.

Evidence for two DNA repair enzymes for 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in human cells.

Two DNA repair enzymes for 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine; OH8Gua, oxo8Gua) have been identified in human HeLa cell nuclear extract. One is OH8Gua-glycosylase and the other is OH8Gua-endonuclease that lacks OH8Gua-glycosylase activity. They were separated by heparin-Sepharose column chromatography and characterized by endonuclease nicking assay or by measuring the OH8Gua released from substrate DNA using high pressure liquid chromatography-electrochemical detection. Both OH8Gua repair enzymes act only on the OH8Gua-containing strand of the duplex substrate DNA containing OH8Gua/C, OH8Gua/T, or OH8Gua/G. DNA containing OH8Gua/A base pair was very poor substrate for either enzymes. OH8Gua-endonuclease simultaneously cleaves phosphodiester bonds on both sides of the OH8Gua residue, leaving 5'-hydroxy and 3'-hydroxy groups.