CM-cellulose Column Research Articles

Purification, characterization and developmental changes in the titer of a new larval serum protein of the silkworm, Bombyx mori

A new protein was found as a major component of hemolymph proteins up to day 1 of the last larval instar of Bombyx mori, and was named Bombyx mori larval serum protein (BmLSP). The BmLSP was purified to homogeneity by ammonium sulfate precipitation, CM-cellulose column chromatography and gel permeation chromatography. The molecular weight of BmLSP was estimated to be 30,000 by SDS-PAGE and 25,000 by gel permeation chromatography. The amino acid composition of BmLSP was similar to that of 30 kDa proteins which are the major serum proteins in the older last (fifth) instar larvae. The 20 NH 2-terminal amino acids were sequenced and found to be quite different from those of the 30 kDa proteins. Developmental changes in BmLSP titer were followed throughout post-embryonic life by Western blotting using a specific antiserum against BmLSP. Within 1 day after larval hatching, BmLSP appeared in the hemolymph and remained at an almost constant level until day 1 of the last instar. On day 2 of the last instar, the BmLSP level suddenly fell and then gradually decreased toward larval-pupal metamorphosis. Thus, BmLSP is a true larval serum protein and is different from proteins stored for metamorphosis.

Properties of Cholesterol Oxidase Produced by Rhodococcus equi MIL 1037

Cholesterol oxidase produced by Rhodococcus equi MIL 1037 was purified from the supernatant solution of the culture broth by ammonium sulfate fractionation, DEAE- and CM-cellulose column chromatography, hydroxyapatite column chromatography and Sephadex G-75 gel filtration. The resulting enzyme was shown homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated as 48, 000 by SDS-gel electrophoresis. The enzyme contained 453 amino acid residues including 40 moles of alanine and 16 moles of tryptophan. The enzyme was specific to 3β-hydroxysteroids and its relative activity (RA) was 37. 5 for β-sitosterol, 25 for 5α-cholestan-3β-ol, 25 for pregnenolone and 0. 1 for stigmasterol on the basis of 100 for cholesterol. The Km value for the oxidation of cholesterol was 0.33 mM. The enzyme was inhibited by CuSO4, AgNO3 and HgCl2, and partially inhibited by p-chloromercuribenzoic acid, but unaffected by metal chelating reagents.

Primary Structure of a Ribonuclease from Bullfrog (Rana catesbeiana) Liver1

A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.

The isolation of acid phosphatase isoenzymes of Oncopeltus fasciatus, (Dallas) (Hemiptera: Lygaeidae)

1. 1. Acid phosphatase isoenzymes of selected ages of fourth, fifth, and adult stage milkweed bugs were isolated by CM-cellulose and DEAE-cellulose chromatography and gel electrophoresis. 2. 2. Acid phosphatase-I (AP-I) and acid phosphatase-II (AP-II) were isolated by CM-cellulose chromatography and were found in all examined bugs. 3. 3. Day 14 adult females gave a third isoenzyme, AP-III, from the CM-cellulose column. When acid phosphatase-III (AP-III) was applied to the DEAE-cellulose column, three additional isoenzymes, acid phosphatase-III-1 (AP-III-1), acid phosphatase-III-2 (AP-III-2), and acid phosphatase-III-3 (AP-III-3), were isolated. 4. 4. Gel electrophoresis separated acid phosphatase-I (AP-I) into two isoenzymes, AP-I-1 and AP-I-2, in samples from all groups except the Day 14 adult male which had only AP-I-1 on gels run at pH 8.3. 5. 5. Acid phosphatase-II (AP-II) was a single diffuse band in all groups on gels run at pH 4.5. 6. 6. Acid phosphatase-III-1 (AP-III-1), acid phosphatase-III-2 (AP-III-2), and acid phosphatase-III-3 (AP-III-3) from Day 14 adult females gave each a single diffuse band on gels run at pH 4.5. 7. 7. Juvenile hormone application to the fifth instar bugs had no effect on acid phosphatase activity, total protein, or the electrophoretic separation of acid phosphatase isoenzymes of the fifth instar bugs when compared to the acetone controls.

Isolation and partial characterization of human low molecular weight protein associated with pulmonary surfactant.

A low molecular weight (MW) protein was isolated from the bronchoalveolar lavage fluid of a patient with alveolar proteinosis. The protein was isolated on DEAE-cellulose and CM-cellulose columns by a cross-reaction with the monoclonal antibody against pig low MW protein (15 kDa) used as a marker. Acidic ethanol-soluble proteins obtained from the fractions eluted by 0.09 to 0.16 M NaCl concentration from the CM-cellulose column migrated mainly as a 15-kDa band in the SDS-PAGE system without urea but mainly as a 5-kDa band in a system with 8 M urea. The isoelectric point of the protein was pH 10 to 11, and it contained a large proportion of hydrophobic amino acids (72%), especially leucine (17%). The arginine content was also high (9%). Two monoclonal antibodies were raised against this low MW protein, and immunohistochemical studies revealed that the antigen was located in the inclusions of alveolar wall cells in normal lungs and in lungs from a patient with alveolar proteinosis. These results indicate that the low MW protein originates from lamellar inclusions of alveolar wall cells (possibly type II epithelial cells) and is secreted into alveolar spaces.

On the activities of parathyroid hormone-like factor and transforming growth factors in extract of pancreatic cancer associated with humoral hypercalcemia of malignancy.

Extract of exocrine pancreatic cancer associated with humoral hypercalcemia of malignancy was examined for biological activities of PTH-like factor and transforming growth factor (TGFA and TGFB), both of which are possible causes of hypercalcemia. The crude extract had both PTH-like and TGF activities. On Bio Gel P-60 column chromatography, PTH-like and TGFA activities were eluted at around 10 kD, whereas TGFB activity was eluted at around void fractions, 10 kD and 6 kD. Liver extract, used as a control material, exhibited only TGFB activity at around 6 kD. CM-cellulose column chromatography of 10 kD fractions resulted in a subtle distinction between PTH-like activity and TGF activities. Further fractionation of the peak with PTH-like activity on reverse-phase high performance liquid chromatography separated PTH-like activity distinctly from TGFB activity. TGFA activity was lost through the procedure. It was concluded that the exocrine pancreatic cancer associated with hypercalcemia produced not only PTH-like activity but also TGFA and TGFB activities. Several chromatographic analyses suggested that PTH-like activity and at least TGFB activity stem from distinct molecules and that the PTH-like factor has no significant TGFB activity intrinsically.

Induction and Partial Characterization of Antibacterial Proteins in the Hemolymph of the Silkworm,Bombyx mori

The injection of live or heat-killed bacteria into larvae of the silkworm, Bombyx mori, induced antibacterial activity in the hemolymph. A wide variety of gram-negative and gram-positive bacteria were effective as inducing agents, but saline alone, yeast cells and fungal spores were not effective. The antibacterial activities were separated into six bands on native polyacrylamide gel electrophoresis, which were sensitive to trypsin. Some of these antibacterial proteins were partially purified by CM-cellulose column chromatography. The proteins were heat-stable and showed no lysozyme activity. The proteins repressed the growth of various gram-positive and gram-negative bacteria.

An isomalto-dextranase accompanied by isopullulanase activity from Arthrobacter globiformis T6.

An isomalto-dextranase was highly purified from the cell-free culture broth of Arthrobacter globiformis T6 by consecutive column chromatographies on CM-cellulose. The final purified enzyme was judged to be homogeneous on polyacrylamide gel, SDS-polyacrylamide gel and ampholine electrophoresis.The isopullulanase to G2-dextranase activity ratio was almost the same (ca. 0.18%) at each purification step. The isopullulanase activity appeared at exactly the same position as the G2-dextranase activity on CM-cellulose column chromatography, analytical polyacrylamide gel electrophoresis and isoelectric focusing. The stabilities of the isopullulanase as to pH and temperature well coincided with those of the G2-dextranase. Furthermore, the inactivation profiles of the isopullulanase with various metal ions and inhibitors were almost the same as those of the G2-dextranase. From these results, it was strongly suggested that a single enzyme molecule was responsible for both the G2-dextranase and isopullulanase activities.

Isolation and Characterization of two luffins, protein-biosynthesis inhibitory proteins from the seeds of Luffa cylindrica.

Two protein-biosynthesis inhibitory proteins, luffin-a and -b, were isolated from the seeds of the sponge gourd (Luffa cylindricd). Luffins were extracted from defatted meal of seeds of the sponge gourd with water at pH 4.0 and purified by gel filtration through a Sephadex G-75 column followed by CM-cellulose column chromatography and FPLC with Mono S column. Luffin-a and -b were basic proteins with isoelectric points of about 10, with molecular masses of 28, 000 Da and 29, 000 Da, respectively, as judged by SDS-PAGE. Luffin-a and -b were found to be homologous proteins having similar amino acid compositions (except threonine and proline), and having N-terminal sequences of Asp-Val-Arg-Phe-Ser-Leu-Ser-Gly- and Ala-Asn-Val-Ser-Phe-Ser-Leu-Ser-Gly-, respectively. Inhibitory activities of luffin-a and -b on cell-free protein synthesis were approximately 5.5 and 1.5 fold, respectively, stronger than that of ricin A-chain.

Improved purification and further characterization of an isomalto-dextranase from Arthrobacter globiformis T6.

A highly purified isomalto-dextranase (EC 3.2.1.94) was simply separated from the cell-free culture broth of Arthrobacter globiformis T6 by CM-cellulose column chromatography and characterized further. The purified enzyme was essentially homogeneous on PAGE and SDS-PAGE as well as isoelectric focusing. The enzyme was an acidic protein with pI 5.15. E1%1cm at 280 nm was 24.6. The molecular weight of the enzyme was estimated to be about 69, 000 by SDS-PAGE. No carbohydrate moiety seemed to be associated with the enzyme protein. The optimum pH and temperature of the enzyme was pH 5.3 and 65°C, respectively. The enzyme was completely stable over the range of pH 3.0-8.0 at 4°C for 24 hr, and retained about 90% of its original activity after heating at 60°C for 10 min. Inactivation of the enzyme was found to be partial with 5mM Ag+, and complete with 5 HIM Hg2+, Fe3+, KMnO4, and NBS. The enzyme split dextran, retaining the α-configuration of the anomeric carbon atoms in the hydrolysis products. The Km value of the enzyme for dextran was 0.038%. Isomaltitol was a potent competitive inhibitor (Ki=0.79 mM)

Production of raw cassava starch-digestive glucoamylase by a 2-deoxyglucose-resistant mutant of Rhizopus sp.

In order to improve the productivity of raw cassava starch-digestive glucoamylase of Rhizopus sp. MB46 in a liquid culture, a mutant strain, AF-1, which is resistant to 2-deoxyglucose, was derived. The mutant strain produced glucoamylase in the presence of 0.5% glucose though the parent strain did not. With a rice bran liquid medium the productivity was over 2-times that of the wild type strain. A rice bran liquid medium supplemented with β-cyclodextrin was also effective for glucoamylase production. Other maceration enzymes were also produced at a higher level with mutant strain AF-1 than with the wild type strain in a liquid culture as well as in a solid culture. The elution patterns of these enzymes on CM-cellulose column chromatography were principally the same with both strains except for glucoamylase. When 10% of raw cassava starch and cassava waste were digested with the culture filtrate of mutant strain AF-1, glucose was produced in 7% after 60-h incubation and 3.2% after 48-h incubation, respectively.

The isolation of a fetal rat liver glutathione S-tranferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Y c ( M r 28 000) and a subunit ( M r 25 500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit ‘Y fetus’. The enzyme which we term glutathione S-transferase Y cY fetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.

Partial sequence of hemoglobin from cobra (Naja naja naja).

Hemoglobin from the cobra snake, Naja naja naja, was isolated and its chains separated on a CM-cellulose column. The separation profile revealed an alpha and two beta chains having the molar proportions of [alpha]2[beta 1]1[beta 2]1. The N-terminal amino acid sequence of the intact chains and of the CNBr peptides were carried out. The beta 2 chain was found to be heterogeneous comprising a minor component amounting to 11%. This later showed changes at two positions 9 and 14 in the first 30 residues sequenced.

Effect of avidin binding to SH1 on the interface between subfragment-1 and F-actin.

The subfragment-1-avidin complex, in which avidin is attached to a well defined thiol group called SH1, was purified by CM cellulose column chromatography or affinity chromatography using lipoic acid agarose. The interaction of the purified complex with F-actin was compared to that of normal subfragment-1 using chemical cross-linking and limited tryptic digestion techniques. It was found that the binding of avidin to SH1 lowered the extent of cross-linking between the subfragment-1 heavy chain and actin. The amount of the 175K product decreased to about 50% of the normal level and that of the 165K product decreased to about 35%. It was also found that the binding of avidin abolished the protective effect of F-actin on the 50K-22K junction of the S-1 heavy chain against tryptic attack. Since more than 95% of the S-1-avidin complex was attached to F-actin under our experimental conditions, these changes are due to an alteration of the S-1-actin interface. Considering the facts that SH1 is located on the side of S-1 facing the F-actin, in the tertiary structure, and is close to the cross-linked site and to the 50K-22K junction, in the primary structure, it is quite likely that avidin bound to SH1 causes these effects by sterically preventing the close contact of S-1 and actin.

Purification of human leukocyte interferon A derived from Escherichia coli: An aging process to prevent the formation of its oligomers

Recombinant human leukocyte interferon A (rIFN-αA) was purified from Escherichia coli cells by monoclonal antibody affinity chromatography (Ab) and CM-cellulose column chromatography (CM). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CM eluate was found to contain a large amount of rIFN-αA oligomers, in addition to the monomer, linked by intermolecular disulfide bonds. In contrast, the Ab eluate was found to contain a large amount of rIFN-αA monomers. These results show that the oligomers were formed at the CM step. Free SH groups were detected at a significant level in the Ab eluate, but were detected barely in the CM eluate, indicating that the Ab eluate contained some amount of the unstable monomer with free SH groups. To prevent the formation of the oligomers we established a simple and easily scaled-up process, called an aging process, during which the Ab eluate was incubated at pH 3.3 at 37°C for several days. This process is effective for changing the unstable monomer to the stable monomer and thus preventing the formation of the oligomers.

Purification and properties of wall-bound .ALPHA.-glucosidase from suspension-cultured rice cells.

Wall-bound α-glucosidase (EC 3.2.1.20) has been solubilized from suspension-cultured rice cells with Sumyzyme C and Pectolyase Y-23 and isolated by a procedure including fractionation with ammonium sulfate, Sephadex G-100 column chromatography, CM-cellulose column chroraatography, Sephadex G-200 column chromatography, and preparative disc gel electrophoresis. The molecular weight of the enzyme was 64, 000. The enzyme readily hydrolyzed maltose, maltotriose, and amylose, but hydrolyzed isomaltose and soluble starch more slowly. The Michaelis constant for maltose of the enzyme was estimated to be 0.272 mM. The enzyme produced panose as the main α-glucosyltransferred product from maltose.

Glutathione S -transferases: Of rats and men

Rat liver glutathione S-transferases have been purified to apparent electrophoretic homogeneity by S-hexylglutathione-linked Sepharose 6B affinity chromatography and CM-cellulose column chromatography. At least 11 transferase activity peaks can be resolved including five Yb size homodimeric isozymes, two Yc size homodimeric isozymes, one Ya homodimeric isozyme, one Yα homodimeric isozyme, and two Ya-Yc heterodimeric isozymes. Distribution of the GSH peroxidase activity among the CM-cellulose column fractions suggests the existence of further multiplicity in this isozyme family. Substrate specificity patterns of the Yb subunit isozymes revealed a possibility that each of the five Yb-containing isozymes is composed of a different homodimeric Yb size subunit composition. Our findings on the increasing multiplicity of glutathione S-transferase isozymes are consistent with the notion that multiple isozymes of overlapping substrate specificities are required to detoxify a multitude of xenobiotics in addition to serving other important physiological functions.

Chemical form of cadmium (and other heavy metals) in rice and wheat plants.

Chemical forms of heavy metals such as Cd, Cu, Ni, and Pb in rice and wheat plants grown in nutrient solution containing a heavy metal were investigated. Fractionation of an extract of Cd-treated rice plants on Sephadex G-75 showed cadmium to be associated with organic compounds of high (fraction A), intermediary (fraction B), and low molecular weight (fraction C). Material A, whose molecular weight was greater than 440,000, is probably nonspecific binding of Cd to normal cell components. Materials B and C can be classified as types of metallothionein. The molecular weight of B was 33,100. This material contains 12 mg Cd/g protein. The UV-absorption spectrum of B showed absorptions at 280 and 250 nm. Material B was not eluted even at a very high ionic strength from the DEAE-cellulose column, but it was eluted at a very low ionic strength from a CM-cellulose column, indicating a highly anionic molecule which differs from metallothionein in animals. Fraction C contains two materials: one a Cd-containing material whose molecular weight was estimated to be approximately 7000 and the other an inorganic Cd salt. In addition to cadmium, copper, lead, and nickel in rice and wheat have been studied. As a result, heavy metal-containing materials whose molecular weights were estimated to be approximately 16,000 and 8900 (Ni-treated rice plants), 7000 (Pb-treated rice plants), 5000 (Cd-treated wheat plants), and 21,000 (Cu-treated wheat plants) were isolated.

Studies on glutathione S-transferase in molluscs

1. 1. Glutathione S-transferase can be detected in both terrestrial and aquatic molluscs. The enzyme is ubiquitiously distributed in the tissues of the mollusc, Achatina fulica. 2. 2. The kidney has the highest amount of activity on per gram basis when compared to other organs. 3. 3. The enzyme was found to be present not only in the soluble fraction but also in the microsomes and mitochondria. 4. 4. 100,000 g supernatants from kidney homogenates contain isoenzymes that bind to both DEAE-Sephadex and CM-Cellulose columns. 5. 5. Such supematants can be resolved into four acidic and one basic form by isoelectric focussing. 6. 6. The enzyme is able to conjugate GSH on to DNCB, methyl iodide and p-nitrobenzyl chloride while it is unable to do the same with 1,2-epoxy-3-(p-nitrophenoxy) propane and ethacrynic acid. 7. 7. The molecular weight of the enzyme is about 40,000 daltons.

Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction.

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.