CM-cellulose Column Chromatography Research Articles

Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

Polygalacturonases (PG) represent an important member of pectinases group of enzymes with immense industrial applications. A fungal strain Aspergillus niger MTCC478 was used for the production of polygalacturonase both under submerged and solid-state fermentation condition. Further its production was optimized under solid-state fermentation condition with media comprising of wheat bran and tea extract. Purification of an exo-PG was achieved by acetone precipitation (60–90%) and CM-cellulose column chromatography revealing 15.28-fold purification with a specific activity of 33.47 U/mg protein and 1.2% yield. A relative molecular mass of purified PG was approximately 124.0 kDa. The pH and temperature optimum was found to be 4 and 50 °C, respectively. The kcat and Km value for degradation of PGA by the purified enzyme was found to be 194 s−1 and 2.3 mg/mL, respectively. Cu2+ was found to enhance the PG activity while Ag+ completely inhibited the enzyme activity. The application of the purified PG in orange juice clarification was elucidated.

Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Pigeon Pea (Cajanus cajan) Seeds

Glucose-6-phosphate dehydrogenase has been purified from pigeon pea (Cajanus cajan) seeds and subjected to characterization. The enzyme was purified 123.69 fold with a yield of 21.37% by ammonium sulphate fractionation, PEG-4000 precipitation, CM cellulose column chromatography and DEAE cellulose column chromatography. The catalytically active enzyme is a dimer of 113 KDa with a subunit molecular weight of 55 KDa. Thermal inactivation of enzyme follows first order kinetics at 30&#176C and 40&#176C with half life of 6 and 1.5 min respectively. Km value for glucose-6-phosphate and NADP+ was found to be 2.68 mM and 0.75 mM respectively whereas Vmax value was found to be 0.11 U/mL and 0.13 U/mL respectively. The enzyme shows more affinity towards NADP+ than glucose-6-phosphate. The pKa value was found to be 10.41 indicating that the amino acid residue at active site might be lysine. The enzyme exhibited maximum catalytic activity at pH 8.2. The enzyme was found to be highly thermosensitive with gradual loss of activity above 30&#176C temperature.

A short-chain peptide toxin isolated from Centruroides sculpturatus scorpion venom inhibits ether-à-go-go-related gene K+ channels

From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K+ channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastoma×glioma hybrid cells voltage-clamped in the whole-cell mode. CsEKerg1 contains 43 amino acids and has a molecular weight of 4833. Its amino acid sequence is similar but not identical to that of ergtoxin, a peptide isolated recently from the venom of the Mexican scorpion Centruroides noxius [FASEB J. 13 (1999) 953]. Half inhibition of ERG current occurs at a peptide concentration of 1.12μg/ml.

Characterization of Serine Endopeptidases in Cotyledons of Germinated Vigna mungo Seeds

seeds. SEP activity was separated into two isoforms by CM-cellulose column chromatography. These forms, termed SEP-1 and SEP-II, showed endopeptidase activities even at acidic pH, suggesting that SEPs are unique serine endopeptidases, since most serine proteases are optimum at neutral pH.

An improved, inexpensive method for the large-scale purification of human nerve growth factor.

Human nerve growth factor (hNGF) was purified to near homogeneity on a large scale from human term placenta with an improved and inexpensive method. The purification procedure included tissue homogenization, ultrafiltration and single CM-cellulose column chromatography. The purified hNGF was a 14.4-kDa protein with an isoelectric point of approximately 9.3. The specific activity of the purified hNGF was approximately 38000 units/mg, and the activity was completely inhibited by the monoclonal antibody against recombinant hNGF (rhNGF). Western-blot analysis showed that the purified hNGF could interact with the monoclonal antibody against rhNGF.

Isolation and characterization of chitinase isoforms from the bulbs of four species of the genus Tulipa.

Six chitinase isoforms, designated TBC-1 to TBC-6, were purified to homogenity from the bulbs of four species (Tulipa bakeri, T. tarda, T. turkestanica, and T. praestans) of the genus Tulipa by CM-cellulose column chromatography, Butyl-Toyopearl 650M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S fast protein liquid chromatography (FPLC). The chitinases had molecular weights of 30,000 and isoelectric points of 5.2 to 6.1. These chitinases were found to proteins with similar amino acid compositions and N-terminal sequences. The tulip chitinases all had two half-cystine residues, one more than gladiolus bulb class IIIb chitinase, but many fewer than chitinases of plant class I (15-17 Cys residues/mol), II (5-8 Cys residues/mol), or III (6 Cys residues/mol). The N-terminal sequences of tulip chitinases were similar to the sequence of the gladiolus chitinase, but did not resemble sequence of any class of plant chitinase. The optimal pH of these chitinases toward glycolchitin was pH 5. TBC-1 hydrolyzed (GlcNAc)6 into (GlcNAc)2, (GlcNAc)3, and (GlcNAc)4, and hydrolyzed (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.

Reversible phosphorylation of fructose 1,6-bisphosphatase mediates enzyme role in glycerol metabolism in the freeze-avoiding gall moth Epiblema scudderiana

Fructose-1,6-bisphosphatse (FBPase) from larvae of the freeze-avoiding gall moth Epiblema scudderiana occurs in two forms, which are interconverted by reversible phosphorylation and separable by CM-cellulose column chromatography. The phosphoenzyme has properties that would make it the more active form in vivo. Compared with the dephosphorylated form, the phosphoenzyme had three-fold lower values for K m fructose-1,6-bisphosphate and K a Mg 2+ and lower sensitivities to allosteric inhibitors (I 50 values for fructose-2,6-bisphosphate and AMP were 50% and 10-fold higher, respectively). The proportions of the two enzyme forms in the larvae changed with the seasons and with acclimation to warm (15°C) vs cold (4°C) temperatures. The phosphorylated enzyme predominated (70% of total activity) in early autumn and during the spring, as well as in warm acclimated larvae, all situations where gluconeogenesis via FBPase would be favoured. During the autumn cold-hardening period when the larvae are actively synthesizing the antifreeze, glycerol, the ratio of the two enzyme forms changed to about 50:50. This, plus allosteric inhibition and low temperature effects on enzyme kinetics, would effectively suppress FBPase activity and prevent futile recycling of glycerol carbon back into glycogen during the winter months when the 2 M pool of polyol must be sustained for antifreeze protection. Acclimation studies suggested that low temperature itself might be the signal that triggers enzyme dephosphorylation, and this could integrate control over FBPase with the well-known phosphorylation-mediated activation of glycogen phosphorylase by low temperature in cold-hardy insects.

Purification of a bacteriolytic enzyme from Bacillus sp. active against Streptococcus mutans

Bacillus sp. strain YU-1006, which was isolated from soil, produced a bacteriolytic enzyme which was active against Streptococcus mutans KCTC 3283. The enzyme was purified 60 fold with a 6% yield by CM-cellulose column chromatography and CM-Sephadex G-50 column chromatography. Lytic activity was stable in the range of pH 6.0~9.0 up to 42°e active enzyme is a 24,000 dalton monomer as estimated by SDS-PAGE.

Purification and characterization of two chitinase isoforms from the bulbs of gladiolus (Gladiolus gandavensis).

Two chitinase isoforms, designated GBC-a and GBC-b, were purified from the bulbs of gladiolus (Gladiolus gandavensis) using CM-cellulose column chromatography followed by Butyl-Toyopearl 650 M hydrophobic column chromatography, gel filtration on Sephadex G-75, and Mono-S FPLC. GBC-a and GBC-b are weakly acidic and weakly basic proteins with molecular masses of 30 kDa, and isoelectric points of 6.0 and 7.5, respectively. GBC-a and GBC-b were found to be homologous proteins with similar amino acid compositions and N-terminal sequences. The number of half-cystine residues in GBC-a and GBC-b was only one each, which is much lower than those of plant class I (15-17 Cys residues/mol), class II (5-8 Cys residues/mol), and class III (6 Cys residues/mol) chitinases. The N-terminal sequences of GBC-a and GBC-b were completely different from those of plant three classes of chitinases. The optimal pHs of these chitinases toward glycolchitin were pH 5. GBC-a hydrolyzed (GlcNAc)5 into (GlcNAc)2, (GlcNAc)3 and (GlcNAc)4, and (GlcNAc)5 into (GlcNAc)2 and (GlcNAc)3.

Characterization and amino-terminal sequence of phospholipase A 2-II from the venom of Agkistrodon bilineatus (common cantil)

1. 1. Phospholipase A 2 was isolated from the venom of Agkistrodon bilineatus by Sephadex G-75 and CM-Cellulose column chromatographies. 2. 2. The purified phospholipase A 2 gave a single band on disc polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ODS-HPLC. 3. 3. The enzyme preparation had a mol. wt of 14,000, isoelectric point of pH 10.12 and possessed 121 amino acid residues. 4. 4. The enzyme hydrolyzed the phospholipids phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol and phosphatidyl serine. 5. 5. The contraction of mouse diaphragm was inhibited by phospholipase A 2-II. 6. 6. Phospholipase A 2 activity of this preparation was inhibited by ethylenediamine tetraacetic acid, ethyleneglycol (β-aminoethyl) N, N, N′, N′-tetraacetic acid, p-bromophenacyl bromide or N-bromosuccinimide, but not by iodoacetic acid or diisopropyl fluorophosphate. 7. 7. The amino-terminal sequence of the PLA 2-II was determined.

Isolation and characterization of phospholipase A 2, from Agkistrodon bilineatus (common cantil) venom

1. 1. Phospholipase A 2 was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies. 2. 2. The purified phospholipase A 2-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. 3. 3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues. 4. 4. The purified phospholipase A 2 possessed lethal, indirect hemolytic and anticoagulant activities. 5. 5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS). 6. 6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A 2-I. 7. 7. Phospholipase A 2 activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

Purification and some properties of three chitinases from the seeds of rye (Secale cereale).

Three chitinases, designated RSC-a, -b, and -c, were purified from the seeds of rye (Secale cereal) using ammonium sulfate precipitation, CM-cellulose column chromatography, gel filtration on Sephadex G-75, and S-Sepharose column chromatography. RSC-a, -b, and -c are basic proteins having molecular masses of 33 kDa, 26 kDa, and 26 kDa, and isoelectric points of 9.7, 10, and > 10, respectively. RSC-b and -c were found to be homologous proteins having similar amino acid compositions and N-terminal sequences. RSC-a contains more Thr, Ser, Glu, Pro, Gly, and Cys than RSC-b and -c and has a different N-terminal sequence from them. They hydrolyze glycolchitin and colloidal chitin, but not cell walls of Micrococcus lysodeikticus. These enzymes are stable at pH 4-8 and their optimum pHs toward glycolchitin are 5.

Serum B12 Binding Proteins Versus Seminal Plasma Binder

It was well confirmed that B12 binding protein in human serum and body fluids largely consists of two kinds of binding proteins, physiologically B12 transporting transcobalamin and haptocorrins which does not involve in B12 transport. In seminal plasma, very potent B12 binding protein, in quantity as much as 23 times of normal serum, was identified. Separation characteristics, gel filtration, CM cellulose column chromatography and column IEF all agreed as one of the haptocorrins. One peculiar feature of seminal plasma binder is marked heterogeneity of much lower pI regions suggesting the presence of increased amount of sialic residues on the molecule. The preliminary data from this laboratory support the view, however, physiological role such as the relationship to the spermatogenesis is not known. The most recent findings in clinical observation on B12 is the treatment of male infertility using methycobalamin. The detailed analysis of seminal binders may open up new arena of B12 bioeffect.

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca 2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca 2+ (0.1 mM concentration) and differentiated cells grown in high Ca 2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin α (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.

Presence of another species of myelin basic protein (MBP) in the developing chick brain

It has been observed in the chick myelin that a protein with a molecular weight of 19.5K, in addition to the major species of myelin basic protein (MBP) (MBP(1), mol. wt 18.5K), reacts with anti-MBP serum directed against MBP(1). The larger MBP (MBP(2) mol. wt 19.5K), which seemingly does not correspond to any of the MBP species from other animal sources, is detected at high level in chick myelin from the initial stage of myelination. MBP(1) is easily extracted from the fluffy layer of Folch's upper layer, while MBP(2) is extracted by sonication of the acidified fluffy layer. Both extracted MBPs can be purified by using high performance liquid chromatography (HPLC) with a reverse phased column after Sephadex G-100 and CM cellulose column chromatographies. The amino acid compositions of both MBPs are similar to each other. Further, both MBPs are phosphorylated using protein kinase C with 5.3 and 3.6 mol of incorporated phosphates to 1 mol of MBP(1) and MBP(2), respectively. One dimensional peptide mapping of both MBPs with Staphylococcus aureus V8 protease, carboxypeptidase Y and 2-(2-nitrophenylsulfenyl)-3-methyl-3?-bromoindolenine reveals different degradation patterns. This difference is also demonstrated by cyanogen bromide fragmentation. The MBP(2) cleavage has a low degradation rate in contrast to the complete cleavage of MBP(1). The two fragments (mol. wt 8 and 9K) from MBP(1) and one fragment (mol. wt 7K) from MBP(2), which are purified by HPLC, can be sequenced from the N-terminus. It is suggested that the methionine site is different in both MBP(1) and MBP(2), and there are a small number of amino acid substitutions between both MBPs.

Purification and properties of α-glucosidase from suspension-cultured sugar-beet cells

The main α-glucosidase from sugar-beet cells has been isolated by a procedure including fractionation with ammonium sulphate, Sephacryl S-200 HR column chromatography, CM-cellulose column chromatography, Mono Q column chromatography, and preparative disc gel electrophoresis. The M r of the enzyme was 53 000. The enzyme hydrolysed maltooligosaccharides at a faster rate than maltose. The K m values for maltose and maltohexaose were 3.5 and 0.35 mM.

Hb Isehara (or Hb Redondo) [β92(F8)HIS→SN]: An Unstable Variant with a Proximal Histidine Substitution at the Heme Contact

A 50-year-old Japanese female patient was found to have hemolytic anemia. Isoelectrofocusing of her hemolysate revealed two abnormal hemoglobin bands, one of which was very close to the Hb A2 band, and the other between the Hb A2 and Hb F bands. CM-cellulose column chromatography of the globin prepared from the abnormal hemoglobin showed that the abnormal chain eluted faster than the normal beta and delta chains; the beta X chain, however, did not separate from the normal beta chain in urea cellulose acetate electrophoresis. An instability test of the patient's hemolysate revealed the presence of an unstable component. Structural analysis of the abnormal beta chain indicated that the histidine residue at beta 92(F8) was replaced by an asparagine or aspartic acid residue. DNA amplified by polymerase chain reaction was sequenced by the dideoxy method. The nucleotide sequence of the beta 92 codon was AAC instead of CAC, suggesting that the amino acid substitution corresponded to His----Asn, which is the same as is found in Hb Redondo or beta 92(F8)His----Asn----Asp.

Evidence for Identifying Fatty Acids in Rat Liver Glutathione S-Transferase and Its Possible Involvement in the Secondary Structure1

A rat liver glutathione S-transferase isozyme (GST 3-3) has been purified to apparent electrophoretic homogeneity by procedures including Sephadex G-100, S-hexylglutathione-linked Sepharose, and CM-cellulose column chromatographies. The present study provides evidence for the existence of endogenous fatty acids in the purified GST 3-3 by gas-liquid chromatographic and mass-spectrometric analyses. The liver GST isozyme associated long chain fatty acids such as 14:0, 16:0, 18:0, and 18:1 and the molar ratio of fatty acids to the protein was estimated to be around 3.2. When the enzyme preparation was freed of fatty acids by a mild delipidization technique using Lipidex, the GST activity was significantly decreased. Computer analysis of the circular dichroism spectra revealed that rat liver GST 3-3 contained approximately 49% alpha-helix and 24% random coil. By delipidizing, the enzyme's alpha-helix was decreased to 4% and the random coil was in turn increased to 62%. The enzymatic and structural properties of the delipidized enzyme, however, could be restored to nearly the original levels by recombining with the fatty acids. These findings suggest that weakly bound fatty acids are responsible for the functional capacity of the GST by virtue of changing the protein conformation.

A single nucleotide deletion in codon 123 of the β‐globin gene causes an inclusion body β‐thalassaemia trait: a novel elongated globin chain βMakabe

The beta-globin gene from a Japanese individual with an inclusion body beta-thalassaemia trait has been characterized by gene cloning and DNA sequencing. An adenine deletion was detected at the first position of codon 123 (ACCCC) of one allele whereas the other allele had a normal sequence. Heterozygosity for this mutation in the patient was confirmed by Southern blots of the genomic DNA digested with HphI, the recognition site of which is eliminated by this deletion. This one base deletion results in the shift of a reading frame in such a manner that the normal termination codon is out of phase. This frameshift mutation results in the synthesis of an elongated beta-globin chain with 10 extra amino acid residues and with an altered C-terminus. Analysis of labelled globin chains using CM-cellulose column chromatography failed to demonstrate any abnormal protein, thereby suggesting that the beta-globin chain variant is highly unstable and probably degrades rapidly after synthesis. This event will lead to an accumulation of free alpha-chains precipitating in the red blood cells and an inclusion body beta-thalassaemia phenotype would ensue.

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.