The partial purification and properties of pepsin obtained from Turkey proventriculus

In this study, pepsin from turkey proventriculus was purified, and its biochemical properties examined. Initially, the turkey proventriculus (stomach) was mixed with 10% NaCl (1∶2, w/v) and extracted by centrifugation to produce a crude extract. The partial purification of the extract was carried out using Sephadex G-50 resin in gel filtration column chromatography. The fractions obtained by gel filtration were analyzed for milk clotting activity (MCA), protein content, proteolytic activity (PA), purification factor (PF), and SDS-PAGE electrophoresis was also performed. The enzyme was purified 207-fold with a recovery of 36%. The first 4 fractions did not have any activities; fractions 7, 8, and 9 exhibited the highest levels of milk clotting and proteolytic activity. The electrophoretic patterns revealed that further purification steps should be applied for better results.     

Isolation and characterization of two antigenic glycoproteins from the pollen of Prosopis juliflora

Two antigenically active glycoprotein fractions were isolated from crude extract of the pollen of Prosopis juliflora using DEAE-cellulose ion exchange chromatography. The glycoproteins gave single band on polyacrylamide gel electrophoresis. The molecular weight of these two glycoprotein was 20,000 and 10,000 as determined by gel filtration on Sephadex G-75. With the help of crossed immunoelectrophoresis and gel diffusion crude extract exhibited twelve and three precipitating antigens suggesting its heterogeneous nature; and the purified glycoprotein fractions however formed single precipitin band on gel diffusion test and immunoelectrophoresis. As tested by ELISA the polyclonal antisera raised in rabbit showed strong binding affinity with glycoprotein of MW 20,000. These result indicates that the two glycoprotein fractions are not antigenically identical.     

Actin-activated ATPase from human erythrocytes.

A fibrillar protein complex, possessing ouabain-insensitive Ca2+-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg2+-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving bands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca2+-ATPase activity, but was devoid of actin-activated Mg2+-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca2+ and actin-activated Mg2+-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg2+ in the crude extract and Fraction I but not in Fraction II.     

Purification of a Candida albicans germ tube specific antigen

Abstract In a previous work, Marot-Leblond et al. identified a Candida albicans germ tube-specific antigen by the use of a monoclonal antibody (mAb 3D9.3). In the present report, we used a two-step procedure to obtain a purified preparation of this antigen from a Zymolyase extract of Candida albicans germ tubes. The extract was first fractionated by gel filtration chromatography. The immunoreactive fractions were pooled, and the 3D9.3 antigen was further purified by hydrophobic interaction chromatography using a Phenyl-superose column. Analysis by SDS-PAGE, immunoblotting and Concanavalin A staining, revealed a single, polydisperse band ranging from 110 to 170 kDa. The antigen was purified 126-fold by protein content and 16.4-fold by carbohydrate content. Recovery of the antigen was 6.8% following the two-step purification.     

Purification and Properties of Catalasc from a Methanol-utilizing Yeast,Candida sp. N-16

Defatted soybean extract was fractionated into protein fractions and low molecular weight fractions with gel filtration. NAD-dependent aldehyde dehydrogenase from bovine liver mitochondria and from yeast was found to oxidize aldehyde in both fractions. These enzymes, therefore, were used to determine the quantity of aldehyde. When the protein fraction obtained by gel filtration was subjected to gel filtration again, aldehyde was recovered in the protein fractions. The level of aldehyde in the protein fractions was unchanged before and after digestion of the protein with pepsin. When the soybean extract was incubated beforehand with aldehyde dehydrogenase and NAD⁺ and the subjected to gel filtration, no aldehyde was detected in the protein fractions. These results indicate that aldehyde dehydrogenase acts on the soybean protein-bound aldehyde. Alcohol dehydrogenase from horse liver in the presence of NADH did not convert the bound aldehyde to alcohol.

A large portion of the aldehyde in the extract was separated from the protein by acid precipitation of the protein. Aldehyde dehydrogenase acts on the aldehyde remaining in the protein after acid precipitation. Thus acid precipitation helps to save NAD⁺ required for complete removal of aldehyde from the soybean protein by aldehyde dehydrogenase.     

Molecular heterogeneity of the SHAP-hyaluronan complex. Isolation and characterization of the complex in synovial fluid from patients with rheumatoid arthritis

We previously found that a covalent complex of SHAPs (serum-derived hyaluronan-associated proteins), the heavy chains of inter-alpha-trypsin inhibitor family molecules, with hyaluronan (HA) is accumulated in synovial fluid of patients with rheumatoid arthritis, and the complex is circulated in patient plasma at high concentrations. How the SHAP-HA complex participates in this disease is unknown. To address this question, it is essential to clarify the structural features of this macromolecule. The SHAP-HA complex purified from synovial fluid of the patients by three sequential CsCl isopycnic centrifugations was heterogeneous in density, and the fractions with different densities had distinct SHAP-to-HA ratios. Agarose gel electrophoresis and column chromatography revealed that there was no apparent difference in the size distribution of HA to which SHAPs were bound between the fractions with different densities. The SHAP-HA complex in the higher density fraction had fewer SHAP molecules per HA chain. Therefore, the difference between the fractions with different densities was due to a heterogeneous population of the SHAP-HA complex, namely the different number of SHAP molecules bound to an HA chain. Based on the SHAP and HA contents of the purified preparations, we estimated that an HA chain with a molecular weight of 2 x 106 has as many as five covalently bound SHAPs, which could give a proteinaceous multivalency to HA. Furthermore, we also found that the SHAP-HA complex tends to form aggregates, judging from the migration and elution profiles in agarose gel electrophoresis and gel filtration, respectively. The multivalent feature of the SHAP-HA complex was also confirmed by the negative staining electron micrographic images of the purified fractions. Taken together, those structural characteristics may underlie the aggregate-forming and extracellular matrix-stabilizing ability of the SHAP-HA complex.     

Novel preparation of cardiolipin from beef heart

A new method is described for the isolation of beef heart cardiolipin. A lipid-protein complex, rich in cardiolipin, is obtained by a one-step solvent fractionation of the tissue total lipid extract. Cardiolipin in the complex is largely freed of protein by salt denaturation and is further purified by gel filtration on Sephadex LH-20 followed by column chromatography on bicarbonate-treated silicic acid. The highly purified product is obtained as the sodium salt in a yield of 85-100 mg/100 g of fresh tissue.     

The formylpeptide chemoattractant receptor copurifies with a GTP-binding protein containing a distinct 40-kDa pertussis toxin substrate

Detergent extraction of plasma membranes from differentiated HL60 cells, specifically labeled with the chemoattractant, formyl-Nle-Leu-Phe-Nle-[125I-Tyr] Lys, resulted in the solubilization of a receptor-radioligand complex. GTP-binding activity coeluted with the radioligand when the sodium cholate extract was purified by chromatography on wheat germ agglutinin-Sepharose 6MB. A molecular size of approximately 59 A was estimated for the lectin-Sepharose-purified receptor complex by gel filtration chromatography on Ultrogel AcA 34. The isolated complex eluted from the gel filtration column exhibited an enhanced rate of ligand dissociation in response to GTP gamma S. Approximately 0.65 mol of pertussis toxin substrate/mol of receptor was estimated following partial purification of the receptor-ligand complex by sequential chromatography on wheat germ agglutinin-Sepharose, DEAE-Fractogel, and Ultrogel AcA 34. The pertussis toxin substrate which copurified with the receptor was compared with two distinct G proteins, containing alpha-subunits of 40 and 41 kDa, previously purified from HL60 cell plasma membranes. Approximately 86% of the pertussis toxin substrate identified in the receptor preparation consisted of the 40-kDa polypeptide. Differences in the peptide maps indicate that the predominant G protein which coelutes with the receptor is distinct from the purified G protein with an alpha-subunit of 41 kDa but homologous to the purified G protein with an alpha-subunit of 40 kDa.     

石榴皮多酚提取物及纯化物对α-葡萄糖苷酶的抑制作用研究

采用紫外分光光度法研究了石榴皮多酚提取物及其2种纯化物(P-1和P-2)对α-葡萄糖苷酶体外抑制作用以及纯化物对该酶的抑制作用类型.结果显示,石榴皮多酚提取物和纯化物对α-葡萄糖苷酶活性均表现出较强的抑制作用,且其作用大小与浓度呈明显的剂量-效应关系;3种多酚样品中,纯化物P-2的抑酶活性最强,纯化物P-1次之,提取物最弱,它们对α-葡萄糖苷酶的半数抑制浓度(IC50,mg/mL)分别为0.045、0.185和0.278.石榴皮多酚纯化物P-2对α-葡萄糖苷酶抑制作用类型为反竞争性抑制;浓度为0.01 mg/mL时该纯化物对α-葡萄糖苷酶的抑制常数Ki为1.22 μg/mL.     

Characterization and partial purification of a neurotrophic factor for cerebrocortical neurons from chicken cerebral extract

The survival of cerebrocortical neurons from 6 to 8-day-old chick embryos was investigated in a serum-free hormone-supplemented medium. The addition of cerebral extract promoted the survival of cortical neurons in a dose-dependent manner, but induced almost no neurite outgrowth. The trophic activity was higher in the adult cerebrum than in the embryonic cerebrum. The tropic factor was partially purified from adult chicken cerebrum, and the molecular weight of the factor was estimated to be about 60 kDa. The activity for survival factor was fairly resistant to heat or trypsin treatment. When the partially purified sample was, however, treated with trypsin (1 mg/ml) for 20 h and applied to a TSK G2000 SW gel filtration column, the activity moved from 60 to 70 kDa untreated active fractions to fractions with about 10 kDa. These physicochemical properties of the survival factor suggest a new class of macromolecular trophic factors in the brain.     

Actin-activated ATPase from human erythrocytes

A fibrillar protein complex, possessing ouabain-insensitive Ca²⁺-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg²⁺-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving hands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca²⁺-ATPase activity, but was devoid of actin-activated Mg²⁺-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca²⁺ and actin-activated Mg²⁺-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg²⁺ in the crude extract and Fraction I but not in Fraction II.     

Identification of polysaccharides from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their antioxidant activities

A large number of polysaccharides are present in the pericarp tissues of harvested litchi fruits. A DEAE Sepharose fast-flow anion-exchange column and a Sephadex G-50 gel-permeation column were used to isolate and purify the major polysaccharides from litchi fruit pericarp tissues. Antioxidant activities of these major polysaccharide components were also evaluated. An aqueous extract of the polysaccharides from litchi fruit pericarp tissues was chromatographed on a DEAE anion-exchange column to yield two fractions. The largest amount of the polysaccharide fraction was subjected to further purification by gel filtration on Sephadex G-50. The purified product was a neutral polysaccharide, with a molecular weight of 14 kDa, comprised mainly of 65.6% mannose, 33.0% galactose and 1.4% arabinose. Analysis by Smith degradation indicated that there were 8.7% of (1-->2)-glycosidic linkages, 83.3% of (1-->3)-glycosidic linkages and 8.0% of (1-->6)-glycosidic linkages in the polysaccharide. Furthermore, different polysaccharide fractions extracted and purified from litchi fruit pericarp tissues exhibited strong antioxidant activities. Among these fractions, the purified polysaccharide had the highest antioxidant activity and should be explored as a novel potential antioxidant.     

A physiological requirement of Na+ for the regulation of cAMP levels in intact NG108-15 cells.

An antigonadotropic compound present in extracts of bovine pineal gland which reduces compensatory ovarian hypertrophy in adult mice was partially purified by gel filtration and further characterized by ion-exchange chromatography and high voltage paper electrophoresis.An acetic acid extract of bovine pineal glands was gel-filtered on Sephadex G-25, from which two antigonadotropic fraction were obtained and designated as F4 and F5. Each of these fractions was further purified by high voltage paper electrophoresis. The antigonadotropic activity of F4 was found in the neutral and acid regions. The F4 fraction was also further purified by cation exchange chromatography. The fraction eluted at pH 4.4 from the cation exchange chromatogram was found to be antigonadotropic. This fraction (pH 4.4) was then further purified by high voltage paper electrophoresis. Antigonadotropic activity was found in the area of the neutral region of the electrophoretogram.The antigonadotropic material is thought not to be melation or arginine vasotocin based on the antigonadotropin being eluted from Sephadex G-25 in a fraction distinct from these two compounds.     

Isolation of the porcine ileal intrinsic factor receptor by sequential affinity chromatography

An extract containing solubilised receptor was passed through four columns containing Sepharose to which had been covalently coupled anti-cobalophilin IgG, vitamin B-12-intrinsic factor, vitamin B-12, and free intrinsic factor, respectively. Following a wash the receptor was eluted with EDTA, then residual Triton X-100 micelles and vitamin B-12-intrinsic factor were removed by Sephadex G-200 filtration. The receptor was purified 84 000-fold, sodium dodecyl sulphate electrophoresis indicated two subunits and gel filtration of its vitamin B-12-inttrinsic factor complex resolved it into two molecular species.     

Inhibition of DNA polymerase alpha activity by proteins from rat liver

We determined that there is a protein in rat liver capable of inhibiting DNA polymerase alpha. To assay for this inhibitor, DNA polymerase alpha was purified from R3230AC rat mammary tumor, a rich source of this enzyme. Protein fractions from Sephacryl S200 gel filtration of total soluble liver extract showing inhibition of DNA polymerase alpha were further chromatographed on DEAE-cellulose. This step revealed two inhibitor protein populations with the major form corresponding to a molecular weight of 143,000 dalton. Soluble extract from isolated rat liver nuclei also showed the presence of at least two inhibitors; the major form was 200,000+ dalton in molecular weight. Both the 143,000 and 200,000+ dalton inhibitor proteins were capable of inhibiting the R3230AC tumor DNA polymerase alpha in a dose-dependent manner. These inhibitors exhibited similar inhibition of nuclear matrix-associated DNA polymerase alpha from either the R3230AC tumor or from regenerating rat liver.     

Influence of detergents and pH on the isolation, purification and molecular properties of alpha-galactosidase-C2 from germinating guar (Cyamopsis tetragonolobus)

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.     

A heat-stable nicotinamide–adenine dinucleotide glycohydrolase from Pseudomonas putida KB1. Partial purification and some properties of the enzyme and an inhibitory protein

A thermostable NAD(P)⁺ glycohydrolase (EC 3.2.2.6) detected in cell-free extracts of Pseudomonas putida KB1 was purified to a single component on polyacrylamide-gel electrophoresis. A heat-labile inhibitor of the enzyme was also partially purified. Enzyme free of inhibitor is present in culture supernatants. After an ultrasonic treatment enzyme–inhibitor complex and excess of inhibitor are present in both the cell-debris and soluble fractions. The general properties of the enzyme and inhibitor are described. The molecular weights of enzyme, inhibitor and enzyme–inhibitor complex, determined by gel filtration are about 23500, 15000 and 35000 respectively. The binding of inhibitor and enzyme is inhibited by the presence of substrate.     

Gel-filtrated fractions of alveolar bone extract contain factors promoting cell attachment and a mitogenic effect on periodontal ligament fibroblasts

The purpose of this study was to investigate the effect of acetic acid-extracted bone proteins on human periodontal ligament fibroblasts (hPF) with respect to mitogenic and cell attachment promoting activity. Alveolar bone was harvested from healthy donors and subjected to 0.5 M acetic acid extraction, dialysis and lyophilization, and gel filtration. Promotion of cell attachment and stimulation of DNA synthesis by the crude extract and gel-filtrated fractions were studied in cultured hPE Many protein components, varying in molecular weight from 10-14 to 120 kDa, were detectable in 10% SDS-PAGE of the extract. Gel filtration of bone extract disclosed four fractions with molecular weights of 55, 34, 29 and 19-20 kDa. Both the 34 and 55 kDa fractions at a concentration of 5 microg/ml, but not the 29- or 19-20 kDa fractions, were found to promote cell attachment while only the 55 kDa fraction (5 microg/ml) stimulated DNA synthesis of hPF, Both mitogenic activity and the promotion of the cell attachment by gel-filtrated active fractions were resistant to thermal treatment (70 degrees C) and pH (4 to approximately 8) changes. These findings suggest that acetic acid extract of alveolar bone may contain components which are capable of modulating cell attachment and mitogenesis of hPF.