Phosphocellulose Column Chromatography Research Articles

Isolation and characterization of BliAI, an isoschizomer of ClaI from Bacillus licheniformis

The restriction endonuclease BliAI, an isoschizomer of ClaI, which recognizes the sequence 5'- AT¯CGAT - 3', was purified from a natural isolate identified as Bacillus licheniformis. The restriction endonuclease was isolated from cell extracts using single-step purification by phosphocellulose column chromatography. The restriction endonuclease is active at 37oC and over a wide range of pH and salt concentration. The molecular weight of the purified restriction enzyme is consistent with a value of 39 kDa.

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.

Characterization of the quantitatively major collagen in the mantle of oyster Crassostrea gigas

A quantitatively major collagen was isolated from the pepsin-solubilized collagen preparation of the mantle by differential salt precipitation and phosphocellulose column chromatography, and its constituent α components (α1 and α2) were purified by phosphocellulose column chromatography. The subunits were demonstrated to be genetically distinct from each other by peptide mapping and amino acid analysis. The amino acid composition calculated from those of the α1 and α2 components in 2∶1 ratio coincided well with that of the major collagen from the mantle. These results suggest that the major collagen in the mantle of the oyster may have a heterotrimer structure (α1)2 α2.

Bsu2413I and Bfi2411I, two new thermophilic type II restriction endonucleases from Bacillus subtilis and Bacillus firmus: isolation and partial purification. Thermophilic endonucleases from two Bacillus species.

Two new thermophilic type II restriction endonucleases, which we designated as Bsu2413I and Bfi2411I, have been isolated from gram-positive thermophilic bacteria Bacillus subtilis strain 2413 and Bacillus firmus strain 2411 respectively and partially purified. The restriction endonucleases were extracted from cell extracts and purified using single step purification through phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights of 33 and 67 kDa for the Bsu2413I and 39 and 67 kDa for the Bfi2411I. The partially purified Bsu2413I enzyme restricted pBR322 DNA into two fragments of 3250 and 1100 bp whereas Bfi2411I enzyme restricted pBR322 DNA into two fragments of 3500 and 800 bp. The activity of both endonucleases was assayed at 55 degrees C and they required Mg+2 as cofactor like other type II restriction endonucleases.

Bme585 I [5′-CCCGC(4/6)-3′], a new isoschizomer of restriction endonuclease Fau I, isolated from a strain of Bacillus mesentericus

Bme585 I is a new member of the restriction endonuclease type IIS family. It was partially purified from the heterothrophic, mesophilic bacterial strain Bacillus mesentericus 585 by ammonium sulphate precipitation and phosphocellulose column chromatography. Bme585 I is a monomeric protein with a molecular mass of 62 kD. The enzyme is active over a broad pH range from 7.0 to 8.8, has a temperature optimum of 37°C and tolerance of NaCl in reaction buffer from 0 to 400 mM. Bme585 I recognizes the asymmetric sequence 5′-CCCGC(4/6)-3′ and is therefore an isoschizomer of restriction endonuclease Fau I.

Purification and characterization of novel salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15.

Salt-active acharan sulfate lyase (no EC number) has been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with GAG degrading enzymes. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, diethylaminoethyl (DEAE)-cellulose, CM-Sephadex C-50, HA ultrogel and phosphocellulose column chromatography with the final specific activity of 81.33 micro mol x min-1 x mg-1. The purified salt-active acharan sulfate lyase was activated to 5.3-fold by salts (KCl and NaCl). The molecular weight of salt-active acharan sulfate lyase was 94 kDa by SDS/PAGE and gel filtration. The salt-active acharan sulfate lyase showed optimal activity at pH 7.2 and 40 degrees C. Salt-active acharan sulfate lyase activity was potently inhibited by Cu2+, Ni2+ and Zn2+. This enzyme was inhibited by some agents, butanediol and p-chloromercuric sulfonic acid, which modify arginine and cysteine residues. The purified Bacteroidal salt-active acharan sulfate lyase acted to the greatest extent on acharan sulfate, to a lesser extent on heparan sulfate and heparin. The biochemical properties of the purified salt-active acharan sulfate lyase are different from those of the previously purified heparin lyases. However, these findings suggest that the purified salt-active acharan sulfate lyase may belong to heparin lyase II.

Dissociation of DNA polymerase alpha-primase complex during meiosis in Coprinus cinereus.

Previously, the activity of DNA polymerase alpha was found in the meiotic prophase I including non-S phase stages, in the basidiomycetes, Coprinus cinereus. To study DNA polymerase alpha during meiosis, we cloned cDNAs for the C. cinereus DNA polymerase alpha catalytic subunit (p140) and C. cinereus primase small subunit (p48). Northern analysis indicated that both p140 and p48 are expressed not only at S phase but also during the leptotene/zygotene stages of meiotic prophase I. In situ immuno-staining of cells at meiotic prophase I revealed a sub population of p48 that does not colocalize with p140 in nuclei. We also purified the pol alpha-primase complex from meiotic cells by column chromatography and characterized its biochemical properties. We found a subpopulation of primase that was separated from the pol alpha-primase complex by phosphocellulose column chromatography. Glycerol gradient density sedimentation results indicated that the amount of intact pol alpha-primase complex in crude extract is reduced, and that a smaller complex appears upon meiotic development. These results suggest that the form of the DNA polymerase alpha-primase complex is altered during meiotic development.

Molecular species of collagen in pectoral fin cartilage of skate ( Raja kenojei)

Soluble collagen was prepared from the pectoral fin cartilage of skate ( Raja kenojei) by limited pepsin digestion. It was fractionated into three fractions by differential salt precipitation. All collagen fractions were further purified by phosphocellulose column chromatography and were characterized with respect to solubility, mobility on SDS–PAGE, peptide map, and amino acid composition. The resultant data indicate the distribution of three molecular species of collagen, corresponding to Type I and II as major collagens and Type XI as a minor collagen, respectively.

Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium.

Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and Sephacryl S-300 column chromatography with a final specific activity of 45.7 micromol.min-1.mg-1. Chondroitin AC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and phosphocellulose column chromatography with a final specific activity of 57.03 micromol.min-1.mg-1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel filtration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel filtration. Chondroitin ABC and AC lyases showed optimal activity at pH 7.0 and 40 degrees C, and 5.7-6.0 and 45-50 degrees C, respectively. Both chondroitin lyases were potently inhibited by Cu2+, Zn2+, and p-chloromercuriphenyl sulfonic acid. The purified Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4-sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6-sulfate). The purified chondroitin AC lyase acted to the greatest extent on chondroitin sulfate A, and to a lesser extent on chondroitin C and hyaluronic acid. They did not act on heparin and heparan sulfate. These findings suggest that the biochemical properties of these purified chondroitin lyases are different from those of the previously purified chondroitin lyases.

Existence of two molecular species of collagen in the muscle layer of the ascidian ( Halocynthia roretzi)

The molecular species of collagen in the muscle layer of the ascidian Halocynthia roretzi was examined by biochemical techniques. Two types of collagen which showed distinct patterns from each other on SDS-PAGE were isolated from the pepsin-solubilized collagen by differential salt precipitation and phosphocellulose column chromatography. They were demonstrated to be genetically distinct from each other by peptide mapping and by amino acid analysis. These results indicate that at least two molecular species of collagen are present in the muscle layer of the ascidian.

Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I, from Bacillus subtilis. Bsu121I, a type II restriction endonuclease from Bacillus subtilis.

A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified. The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease. The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp. The endonuclease activity required Mg(+2) as cofactor like other type II endonucleases.

Molecular species of collagen from wing muscle of skate ( Raja kenojei )

Soluble collagen was prepared from skate ( Raja kenojei ) wing muscle by limited pepsin digestion. It was fractionated into two fractions, major and minor, by differential ammonium sulfate precipitation. Two collagen types were purified from the major and minor collagen fractions by phosphocellulose column chromatography and were identified as Type I and V collagens, respectively, by SDS-PAGE, peptide mapping, and amino acid analysis.

Structure of DNA polymerase delta from Saccharomyces cerevisiae.

DNA polymerase delta (Pol delta) from Saccharomyces cerevisiae consists of three subunits, Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa), present at a 1:1:1 stoichiometry in purified preparations. Previously, based on gel filtration studies of Pol delta, we suggested that the enzyme may be a dimer of catalytic cores, with dimerization mediated by the Pol32 subunit (Burgers, P. M., and Gerik, K. J. (1998) J. Biol. Chem. 273, 19756-19762). We now report on extensive gel filtration, glycerol gradient sedimentation, and analytical equilibrium centrifugation studies of Pol delta and of several subassemblies of Pol delta. The hydrodynamic parameters of these assemblies indicate that (i) Pol32 is a rod-shaped protein with a frictional ratio f/f(0) = 2.22; (ii) any complex containing Pol32 also has an extremely asymmetric shape; (iii) the results of these studies are independent of concentration (varied between 0.1-20 microm); (iv) all complexes are monomeric under the conditions studied (up to 20 microm). Moreover, a two-hybrid analysis of the Pol32 subunit did not detect a Pol32-Pol32 interaction in vivo. Therefore, we conclude that the assembly structure of Pol delta is that of a monomer.

Identification and Characterization of Molecular Species of Collagen in Fish Skin

ABSTRACT: Pepsin‐solubilized collagen (PSC) prepared from the skin of 3 fish species—common horse mackerel, yellow sea bream, and tiger puffer—were separated into 2 fractions, major and minor, by ammonium sulfate precipitation. These collagen fractions were further purified by phosphocellulose column chromatography. From the results of SDS‐PAGE, peptide mapping, and amino‐acid analysis, the purified major and minor collagens were identified to be type I and V collagens, respectively. These results suggest that type V collagen might be widely present in fish skin as a minor collagen.

The inhibitory effect of extract of camellia sinensis and extract of Camellia Ptilophylla Chang on dna polymerase of ehrlich ascites carcinoma cells

Objective: To detect the effect of extract of Camellia Sinensis (ECS) and extract of Camellia Ptilophylla Chang (ECPC) on DNA polymerase (Pol) of Ehrlich ascites tumor cells. Methods: Referring to the method of K.Ono, Pol was extracted from Ehrlich ascites tumor cells in mice. Pol α, β, and γ were separated by phosphocellulose column chromatography and were identified. The effect of ECPC and ECS on Pol was studied. Results: ECPC and ECS were shown to inhibit the activity of Pol α, β, and γ. IC50 values of ECS on Pol α, β, and γ were 10.2 µ g/ml, 9.9 µ g/ml and 28.9 µ g/ml respectively. IC50 values of ECPC on Pol α, Pol β and Pol γ were 5.6 µ g/ml, 15 µ g/ml and 14.7 µ g/ml respectively. The modes of inhibition of ECPC on Pol α, Pol β and Pol γ were noncompetitive with respect to template DNA. The Ki values of ECPC on Pol α, β, and γ were 2.68±0.12 µ g/ml, 2.24±0.12 µ g/ml, 2.56±0.18 µ g/ml. Conclusion: ECPC and ECS were shown to have inhibitory effect on DNA polymerase of tumor cells. The mode of inhibition of ECPC on Pol α, Pol β and Pol γ were noncompetitive with respect to template DNA.

Effects of acetaldehyde on microtubules and hepatic kinesin: a study of the pathogenesis of alcoholic liver disease

In ballooned hepatocytes associated with alcoholic liver disease, secretory proteins are abundant, possibly due to an impairment of the microtubule-dependent vesicular transport system. We studied the effects of ethanol and acetaldehyde on microtubules and hepatic kinesin function. Tubulin and microtubule-associated protein 2 was purified by phosphocellulose and hydroxyapatite column chromatography from microtubule proteins of bovine brain. Hepatic kinesin was purified from rabbit liver. The assembly of microtubules was quantified by spectro-photometer. The kinesin motility assay is done with or without various concentrations of ethanol or acetaldehyde using video-enhanced differential interference contrast microscopy. Ethanol had no effect on the assembly or disassembly of the microtubules or on hepatic kinesin function, even at high concentrations of up to 100 mM. In contrast, acetaldehyde reduced hepatic kinesin function at concentrations greater than or equal to 100 μM and induced microtubular disassembly at concentrations greater than or equal to 200 μM. In alcoholic liver injury, chronic reduction of hepatic kinesin function may cause retention of secretory proteins and hepatocyte swelling. Moreover, reduction of the microtubule concentrations due to acetaldehyde may further impair the vesicular transport system and reduce the cells ability to maintain morphology, in turn accelerating hepatocyte ballooning.

A protein inhibitor of hepatic drug metabolizing enzymes from Ehrlich ascites cells.

Hepatic drug metabolizing enzymes were significantly decreased in Ehrlich ascites tumour-bearing mice. A protein inhibitor of hepatic drug metabolizing enzymes was isolated from Ehrlich ascites cells and purified. This involved ammonium sulphate fractionation (60-80%), DEAE, phosphocellulose, Sephadex G-100 and hydroxyapatite column chromatography. Purification attained was 800-fold. The inhibitory protein was effective in decreasing all the components of hepatic mixed-function-oxidase system and drug metabolizing enzymes both in vivo and in vitro. This novel inhibitor may have potential applications in chemical carcinogenesis.

Changes in fidelity levels of DNA polymerases α-1, α-2, and β during ageing in rats

DNA polymerases (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from the regenerating livers of rats of various ages. The extracts were separated into three DNA polymerase fractions (α-1, α-2, and β) by phosphocellulose column chromatography, and their fidelity levels were then monitored with the synthetic template-primer, poly (dA-dT), poly dA-dT10, or poly dC-poly dG. The fidelity levels of the three DNA polymerases from regenerating liver of rats younger than 20 months were high, while those of DNA polymerases from rats older than 20 months were significantly lower with similar profiles on all three template-primers. On the other hand, the fidelity levels of enzymes from 23- and 26-month-old rats were similar. These results indicate that the levels of error-prone DNA polymerases increase rapidly in the regenerating liver of rats from ages 20 to 23 months. This may due to the amplification of DNA polymerase gene mutations by an error-prone enzyme itself. However, the cells in which mutations in the functional gene occur may undergo cell death because the fidelity levels of the DNA polymerases in the older animals did not increase.

Age-associated changes in the template-reading fidelity of DNA polymerase α from regenerating rat liver

DNA polymerases (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from regenerating livers from young and aged rats. DNA polymerase α was separated and partially purified by DEAE-cellulose column chromatography, polyethyleneglycol precipitation, and phosphocellulose column chromatography, and fidelity levels were then monitored with the synthetic template-primer poly (dG-dC). The fidelity level of the DNA polymerase from regenerating liver of a 4-month-old rat was very high, while that of the DNA polymerase from a 24-month-old rat was significantly decreased. To confirm this result, DNA was synthesized on poly (dG-dC) in a reaction mixture containing [ 32P]dTTP, and the synthetic polynucleotide was purified and digested with HhaI restriction endonuclease. After hydrolysis, the oligonucleotides were developed by two dimensional thin layer chromatography on PEI cellulose plates. Spots containing [ 32P]dTMP were observed when DNA polymerase from a 24 month-old rat was used, but none was found in polynucleotides synthesized using DNA polymerase from a 4 month-old rat. Nearest neighbor analysis suggested that dG-dT and dC-dT pairs were constructed by mis-incorporation due to DNA polymerase α.

Age-related changes in proliferating cell nuclear antigen levels

To clarify the effect of aging on rat liver regeneration, we compared proliferating cell nuclear antigen (PCNA) levels in control and regenerating livers from young and aged rats 48 h after partial hepatectomy. The nucleoplasm and cytoplasm from regenerating livers of 2-month and 24 month-old rats were fractionated by phosphocellulose column chromatography, aliquots of fractions were transferred to nitrocellulose filters and the amounts of PCNA in each fraction were measured by an immunostaining method. Two forms of PCNA, L type (eluted at low concentrations of KCl) and H type (eluted at high KCl concentrations) were observed in the nucleoplasm from both control and regenerating young rat liver. On the other hand, the cytoplasm contained P type (eluted in the pass-through fraction), L type and H type PCNA. In control liver from aged rats, three types of PCNA in the cytoplasm and two types in the nucleoplasm were present at decreased levels. In regenerating liver from young rats, the increases in L type in the cytoplasm and H type in the nucleoplasm were remarkable. However, none of the three PCNA types increased significantly during liver regeneration in aged rats. Treatment with DNase resulted in the disappearance of the H type with a concomitant increase in the P and L types. These results suggest that the H type is a complex form consisting of the P and L types of PCNA and DNA. These results suggest that the increase in the L type in the cytoplasm reflects newly synthesized PCNA production for cellular proliferation and that the increase in the H type in the nucleoplasm is a reflection of binding to DNA and the fundamental role of PCNA itself in liver regeneration in young rats. On the other hand, there was little increase in any of the three types in regenerating liver from 24-month-old rats. Thus, PCNA content may be closely related to the decrease in the rate of cellular proliferation in aged animals.