A majority of Aspergillus induced diseases are reported to be caused by Aspergillus fumigatus. In immunocompromized and post transplant cases it can lead to invasive aspergillosis. Due to this the molecular fingerprinting of aspergillus isolates by RFLP analysis and development of DNA diagnostic probes are gaining importance. Different methodologies are being adopted for extraction of the genomic DNA from fungus. The existing procedures for isolation of DNA are time consuming and range from several hours to few days. The most difficult step in the isolation of DNA from aspergillus species is to disrupt the tough chitin rich cell wall without causing damage to genomic DNA. We report here a rapid method for extraction of genomic DNA based on the cleavage of chitin with chitinase. The subsequent modification steps included are lysis and microwave treatment. The chromosomal DNA obtained by this procedure is 1.5-2.0 micrograms per mg of wet weight of mycelia and is observed to be minimally sheared. It is pure enough for restriction analysis and for use in the PCR to detect the gene coding for 18 kDa allergen which has been identified in our laboratory using western blot analysis with human patient sera.
The presented purification procedure for the muscarinic receptor from calf striatum includes the extraction of lipids with hexane in the first step and the removal of 39% of non-receptor proteins with 2 M NaCl in the second step. The simplicity of such an approach to the purification of the receptor warrants its use in the routine practice for quantitative purposes. The high affinity binding of tertiary 3H-dexetimide (3H-DEX) and quaternary 3H-N-methylscopolamine (3H-NMS) is preserved after the removal of irrelevant lipids and proteins from the P2-pellet. The overall yield of receptors - 80%, when labelled with 3H-NMS, was satisfactory. Moreover, the final product, the NaCl-pellet, exerts a higher density of 3H-NMS binding sites per mg proteins by a factor of about 1.7. The overall yield of receptors and purification factor were lower, when measured with 3H-DEX. The total yield of 3H-DEX binding sites amounted to about 40% and the receptor density per mg protein decreased by a factor of 0.85. We did not succeed in the improvement of the ratio specific/non-specific binding, neither for 3H-DEX nor for 3H-NMS for the purified receptor preparations. The use of 3H-NMS is preferable to 3H-DEX in plasma sample assays because of a negligible effect of plasma on ligand binding when compared with 3H-DEX.
Electroporation was used as a powerful and simple method to probe to the intracellular distribution and trafficking of signal sequences. By coupling synthetic peptides to carrier reporter groups, specific amino acid sequences responsible for nucleolar targeting of Semliki Forest virus (SFV) Core (C) protein were found out. In the N-terminal part of the C protein the sequences (66)KPKKKKTTKPKPKTQPKK(83) and (92)KKKDKQADKKKKP(105) are able to situate BSA or KLH as reporter proteins in the nucleolus, suggesting that SFV C protein contains at least two independent nucleolar targeting sequences.
Peroxidase (E.C. 1.11.1.7., hydrogen donor oxidoreductase) is widely distributed and has been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance. However the role that it plays in metabolism is not clear due to the large number of reactions it catalyzes and the considerable number of isozymic species (2). In tomato plants, Evans and Aldridge (3) separated out six isoperoxidases and in a later paper Evans reported 12 isoperoxidases from tomato shoots (4). A homogeneous tomato fruit peroxidase isozyme was obtained by Jen et al. (5) using hydrophobic chromatography. Isozymes were not detected in Euphorbia characias peroxidase (6), in Ipomoea batatas peroxidase (7) and in Hordeum vulgare peroxidase (8). The simultaneous presence of Cu (II) amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (6,8,9). In the graminea Oryza sativa, widely distributed, an FAD amine oxidase is present that oxidizes diamines (10). In this plant we also found two isoperoxidases called perox I and II. Only perox I was purified to homogeneity and its enzymatic, physical and chemical properties have been studied.
The crude extracellular extract of Aspergillus niger (syn A. ficuum) NRRL 3135 contains glucoamylase (exo-1,4-alpha-D-glucanohydrolase, EC 3.2.1.2). The enzyme, a glycoprotein, was purified 7-fold by ion-exchange chromatography, chromatofocusing, and conconavalin A affinity chromatography. The molecular weight of the enzyme was estimated to be 90 kDa by SDS-PAGE and gel permeation chromatography. The pI of the enzyme was 3.4. The temperature optimum of the enzyme was 60 degrees C and the pH optimum was 5.0. The Vmax values for soluble starch, maltose, maltotriose, maltotretraose, maltopentaose, and isomaltose were 55.2, 11.7, 32.3, 47.8, 59.2, 12.5 nKat glucose/sec, respectively and the Km values for the same substrates were 0.09%, 0.67 mM, 0.76 mM, 0.76 mM, 0.68 mM, and 122.01 mM, respectively.
Tribolium castaneum and T. confusum were washed in HPLC-grade methanol, and the methanolic washes were analyzed by UV spectroscopy, reversed phase HPLC, and GC/MS. The methanolic washes from both species contained methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ). The amounts of MBQ recovered from the two species were not significantly different, but the amounts of EBQ and total benzoquinones (MBQ+EBQ) recovered from T. castaneum were significantly greater than for those recovered from T. confusum. The methods described are superior to previous methods for isolating, identifying, and quantifying the benzoquinones in these beetles, since they are relatively simple, fast, do not require handling of the beetles, and are sensitive enough to quantify the benzoquinones of a single beetle.
An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.
Resolution of a wide range of polypeptides, 3.5-200kDa, on a single low acrylamide and cross linkage gel of 7.7% T, 2.6% C is described here. Laemmli's (4) original discontinuous SDS polyacrylamide gel electrophoresis (SDS-PAGE) system is modified by increasing the ionic strength of both stacking and resolving gels, and replacing the usual glycine buffer with a tricine cathode buffer as described by Schagger and von Jagow (7). This system offers the advantage of a wide range of protein fractionation, with sufficient band resolution, on a single, low acrylamide concentration and cross linkage gel. Moreover, increased gel ionic concentration allows higher protein and salt load, and renders this system suitable for preparative work.
The chlorpromazine-sensitive GTPase from the cell membrane of rat cerebral cortex was purified to homogenity by using DEAE Bio-Gel A agarose, hydroxyapatite and heparin agarose chromatography. The purified chlorpromazine-sensitive GTPase was purified 370-fold to obtain a final specific activity of 40 mumol GTP hydrolyzed2min/mg protein. The purified enzyme was inhibited by chlorpromazine but not by compound 48/80. Magnesium was required for its activity instead of calcium. The purified enzyme had an apparent pH optimum of 8.0, and molecular weight was estimated to be 58,000.
Isolation of cyanobacterial ferredoxin is normally carried out using nucleases in order to degrade the nucleic acids that accompany this protein during the purification procedure. However, this practice presents the inconvenience that these proteins remain in trace amounts in the purified ferredoxin preparations, although they are not visible by electrophoretical techniques. Evidence of that fact is shown in this report and an alternative procedure is described for the rapid preparation of ferredoxin from crude extracts of Anabaena PCC 7119. The method involves a treatment of the crude extract with streptomycin sulphate, a high molecular weight polication that precipitates the nucleic acids in the beginning of the purification.
A unique family of proline-rich proteins (PRPs) is induced in rats following prolonged isoproterenol treatment. PRPs can be divided into glycosylated (GPRP), basic (BPRP) and acidic (APRP) proline-rich proteins based on their physicochemical characteristics. Inducible rat parotid PRPs were isolated from aqueous extracts of parotid glands of isoproterenol-treated animals by sequential chromatography on columns of DEAE-Sepharose CL-6B, Sephadex G-100 and FPLC on Suprose-12 column. The GPRP showed a single homogeneous band on sodium dodecylpolyacrylamide gel electrophoresis with an estimated molecular weight of approximately 220,000. Compositional analysis of GPRP revealed that this protein contained 19.7% glutamic acid/glutamine, 28.2% proline and 9.5% glycine, and 44% carbohydrate, consisting of fucose (2.81g/100g), mannose (9.78g/100g), galactose (9.29g/100g), N-acetylglucosamine (18.03g/100g) and N-acetylgalactosamine (3.90g/100g). Basic PRPs consisted of a family of proteins with estimated molecular masses ranging from 14-45 kDa. These proteins contained 42.6% proline, 20.65% glutamic acid/glutamine and 21.33% glycine. Acidic PRPs also comprised of a family of metachromatically stained ladder of 40-60 kDa containing 29.1% proline, 21.5% glutamic acid/glutamine and 17.8% glycine. APRP were heavily glycosylated containing N-acetylglucosamine (6.34g/100g), N-acetylgalactosamine (19.04g/100g) and glucuronic acid (38.08g/100g).
Four gelatin cleaving proteases were partially purified from culture media of Porphyromonas gingivalis (FAY-19M-1) by sequential chromatography on columns of DEAE-Sepharose, Sephadex G-100 and chromatofocusing on PBE-94. The molecular mass of each of these proteases, estimated by relative mobility on gelatin-containing SDS-PAGE, was 50 kDa (Pool D1b), 120 kDa (Pool E1a), approximately 160 kDa (Pool E1b) and > 300 kDa (Pool A1a), respectively. These proteases also differed with respect to charge characteristics, inhibition profile and cleavage specificity. Protease pools A1a and E1a were inhibited by thiol modifying reagents. Protease pool A1a was also inhibited by N-tosyl-L-lysine chloromethyl ketone, and E1a was inhibited by antipain. Protease pool D1b was inhibited by E-64, leupeptin and antipain, and protease E1b was not inhibited by either of these inhibitors. The detailed substrate specificity of these proteases was checked by using chromogenic substrates, synthetic peptides and native proteins. Protease E1b was very active in degrading collagen, fibrinogen, fibronectin, IgG, IgA, third component of complement (C3), serum albumin, transferrin and varies; is directly proportional to 1-acid glycoprotein as substrates. Fibrinogen, fibronectin and complement C3 component were also cleaved by A1a, D1b and E1a. Synthetic peptides insulin B chain, cecropin P-1 and magainin were cleaved by E1b. Based on FAB analysis E1b showed preferential cleavage at hydrophobic or neutral residues. Protease A1a was active towards chromogenic substrates with either lys or arg in P1 position. Protease D1b cleaved chromogenic substrates with arg in P1 position and cleaved synthetic peptides magainin and (KIAGKIA)3-NH2 at lys residues also. Protease E1a showed glycyl-prolyl peptidase activity.
Spermatozoa were initially separated from fresh boar ejaculates using a 1.0 M sucrose density gradient. Spermatozoa (1 x 10(8) cells/ml) were subjected to gas cavitation (650 psi, 10 minutes), followed by a 4-step centrifugation technique to yield the final plasma membrane preparation. Purity of the plasma membrane isolate was determined using microscopic techniques (i.e. differential interference contrast and transmission electron microscopy) and marker enzymes for biochemical characterization. Plasma membranes were found to be removed primarily from the periacrosomal region of the sperm. Acrosomes appeared to remain intact on the cavitated spermatozoa. Transmission electron microscopy yielded a homogenous population of 100-200 microns unilamellar vesicles. Enzyme markers specific for plasma, acrosome and mitochondrial membranes substantial the purity observed under visual examination.
A rapid simplified method was developed to obtain highly pure bovine thrombin. Prothrombin was directly activated when it was enriched from bovine plasma without prior purification. The activated thrombin was isolated by a single Heparin-Sepharose affinity chromatography step. About 87% of activated thrombin was recovered and the yield was 25.1 mg of thrombin per liter of starting plasma. Specific activity of the final preparation was 4018 NIH units/mg, representing a 402 fold purification over the starting material. Comparative experiments showed that the simplified method was about six times as effective as previous two-step methods.
A new purification method for rat liver glucokinase was developed. Glucokinase was purified to homogeneity in a yield of 70% in 5 days. The procedure consists of DEAE-cellulose ion-exchange chromatography, QAE-Toyopearl ion-exchange chromatography, glucosamine-Sepharose affinity chromatography, and HiLoad Superdex 200 gel filtration. Purified glucokinase had a specific activity of 200 units/mg protein and was highly stable in the presence of 100 mM glucose, 300 mM KCl, and 20% glycerol. We found that some of the methionine residues of glucokinase were oxidized to methionine sulfoxide residues during dialysis in the presence of glucose. It would appear that this oxidation is caused by formation of hydroxyl radicals in the presence of glucose and contaminating transition metal(s).
Different amounts of wheat germ agglutinin were immobilized to agarose gel, previously activated by different amounts of the coupling agent divinyl sulfone. The effectiveness of these affinity sorbents was characterized specific binding of ovomucoid with the gel. These studies revealed the formation of clear optima in binding capacity of the affinity gel, depending on conditions of its synthesis. The ratio between the concentration of the coupling agent and immobilized lectin were found to be crucial for optimization of binding properties of the affinity sorbent.
Fresh normal human feces were fractionated using Percoll gradients and phosphatidylinositol-specific phospholipase C. Studies concerning how cells or cell membrane fragments are fractionated with CEA activity indicate that fecal associated CEA activity is not primarily associated with soluble antigens but with whole cells or fragmented cell membranes.
A recombinant human erythropoietin (rH-EPO) was obtained from the culture supernatants of human B-lymphoblastoid cells transfected by the human EPO gene. rH-EPO was purified by a two-step method based on immunoaffinity and ion exchange chromatography. The first step was achieved by an anti-EPO monoclonal antibody (Mab). This Mab, immobilized on Sepharose 4B, allowed a 410-fold purification of the protein. The second step consisted of ion exchange chromatography on DEAE Sephacel. The combination of these two steps results in a highly purified rH-EPO with a global yield of about 50%; the specific activity of the protein was 176,000 IU/A280. The NMR spectrum was characteristic for a well structured, single-conformation protein. The purified protein was analyzed by SDS-PAGE and isoelectric focusing. The biological activity of purified rH-EPO was measured in vivo, by the incorporation of 59Fe into red blood cells (RBC) of polycythemic mice and in vitro by the proliferative response of an EPO-dependent cell line. The purified protein expressed in lymphoblastoid cells of human origin had the same biological activity as that of urinary EPO and rH-EPO produced in other mammalian cells.
A crude lysozyme preparation was recovered in waste from the scallop processing industry. Lysozyme was then purified 229-fold in preparative scale by chromatography on S Sepharose and Blue Sepharose. Further purification on Sephacryl S-200 resulted in a lysozyme preparation with a specific activity of 64,000 units/mg protein. The apparent molecular mass of the partially purified lysozyme was 10 kDa as judged by gel filtration. Optimum pH for lysis of Micrococus luteus under the present conditions was 5.2. The enzyme was very active at low temperatures. At 4 degrees C the scallop viscera lysozyme exhibits about 55% of the activity measured at 37 degrees C.
