Rapid one-step purification of ferritin from cell-free translation systems

A method is described for the one-step affinity purification of ferritin from other proteins synthesized in an in vitro wheat germ protein synthesizing system programmed with rat liver mRNA. The peptide products released from the polysomes were chromatographed on an antiferritin-Sepharose 4B affinity column. The ferritin isolated in this manner was judged pure by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The recovery of the purified protein from the antibody column ranged from 83 to 88%. Antibody affinity columns can be arranged sequentially to allow the isolation of numerous proteins from one protein synthesis reaction mixture. This method of isolation is applicable to any protein for which antibody is available.     

A simple procedure for isolating adenosine triphosphatase from mitochondria.

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 mumol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 mumol P/min per mg protein when measured as a release of inorganic phosphate.     

Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

A simple procedure for isolating adenosine triphosphatase from mitochondria

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 μmol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 μmol P/min per mg protein when measured as a release of inorganic phosphate.     

A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage

Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82-99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.     

A rapid method for the isolation of intracytoplasmic membranes from Rhodopseudomonas sphaeroides using an air-driven ultracentrifuge

A method has been developed for the isolation intracytoplasmic (ICM) vesicles (chromatophores) from Rhodopseudomonas sphaeroides using an air-driven ultracentrifuge. Application of conventional techniques used for preparative scale equipment to the air-driven ultracentrifuge allows the rapid isolation of ICM vesicles from reduced quantities of starting material. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis profiles of ICM vesicles isolated in this fashion are essentially indistinguishable from those isolated by conventional means.     

石斛总RNA提取方法的研究

采用TRIZOL法、异硫氰酸胍法、Tris-硼酸法和改良的RNA提取方法提取石斛的总RNA,并通过凝胶电泳、紫外分光光度法检测提取的RNA样品的品质。研究结果表明:改良的RNA提取方法提取的RNA具有28S rRNA和18S rRNA两条清晰的条带,且无降解。OD260nm/OD280nm接近2.0,具有较高的纯度。其它三种方法获得的RNA品质较差,有降解和弥散现象。将改良的RNA提取方法提取的RNA逆转录成cDNA,经RAPD扩增,出现清晰的条带,进一步证明改良的RNA提取方法提取的RNA具有很高的纯度,可以满足进一步分子生物学研究的要求。     

Structure and biochemistry of mouse hepatic gap junctions

A new method for the isolation of gap junctions from mouse liver is described. Particular attention has been directed to minimising the effects of proteolysis during isolation. The purified membrane fragments retain the typical morphological features found in junctions of the intact liver.The junctions show two major polypeptides upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The apparent molecular weights are 26,000 for the more abundant species and 21,000 for the minor component. Preliminary protein chemical characterisation by fingerprint analysis suggests that the two polypeptides are structurally related. While an in vivo origin of the 21,000 molecular weight species cannot be excluded, the sensitivity of the junction proteins to proteolytic degradation in vitro suggests that the 21,000 molecular weight molecule may be a breakdown product of the major component.Image reconstruction methods applied to micrographs of negatively stained isolated junctions show that the membrane contains a close-packed hexagonal lattice of components having marked 6-fold symmetry. It is suggested that these represent hexamers of the 26,000 molecular weight protein.Lipid analysis performed on gap junctions isolated by different procedures shows that the lipid composition is strongly affected by the detergents employed during the isolation. A large amount of phopholipid, but not cholesterol, can be extracted from the structure without affecting its gross morphology. This result suggests that cholesterol is tightly bound to the junction protein and may play a role in determining the structure of the gap junction.     

Isolation of intact mitochondria from the rat liver using vibration

A method for isolating mitochondria from the rat liver is described. In this method the homogenization step is replaced by vibration with the frequency of 50 Hz realized by a simple device. Mitochondria isolated by vibration demonstrate higher indices of the oxidative phosphorylation with succinate and glutamate + malate used as substrates than those isolated by homogenization do. The method described permits decreasing considerably the isolation medium expenditure remaining the mitochondria yield per gram of the liver unchanged.     

Phenol Extraction Op 28 S Ribosomal RNA Prom Rat Liver Cytoplasm

A simple and reproducible phenol method for the isolation of 28 S ribosomal RNA from rat liver cytoplasm, free from poly(A)-RNA is described. The procedure is based on the observation that at lower pH of the homogenate (pH 5.5) 28 S ribosomal RNA is extracted, while 18 S ribosomal RNA remains in the interphase layer.

Isolation of pure 28 S or 18 S ribosomal RNA in preparative amounts requires density gradient cen-trifugation or preparative gel electrophoresis. In this communication a rapid and reproducible method for the isolation of 28 S ribosomal RNA is proposed.     

Are Alzheimer neurofibrillary tangles insoluble polymers?

The two methods currently available for the bulk isolation of Alzheimer tangles of paired helical filaments (PHF) are based on a brief treatment of a neuronal-enriched preparation with sodium dodecyl sulfate (SDS) (Method I) and on heating of whole brain homogenate with SDS and beta-mercaptoethanol (Method II). PHF were isolated from the same Alzheimer brain by these two methods, subjected to SDS-polyacrylamide gel electrophoresis and immuno-labelled with monoclonal antibodies to PHF after transferring from the gel to nitrocellulose paper. The PHF isolated by method I revealed the presence of 45 kilodalton to 62 kilodalton PHF polypeptides, whereas the PHF isolated by method II were excluded from the gel. However, PHF isolated by both methods were digested with proteinase-K, though the degradation of PHF of method I was considerably more rapid than that of PHF isolated by method II. These findings should establish that the solubility of PHF might depend on the methods employed for their isolation and that they might not be insoluble polymers of covalently crosslinked polypeptides which accumulate irreversibly in the brain of patients with Alzheimer disease.     

Immunoprecipitation is inappropriate for the isolation of radiochemically pure albumin from tissues

Rats were given intravenous injections of l-[1-¹⁴C]leucine. Twelve minutes after injection, testes, kidneys, livers, and hepatomas were excised rapidly from one group of animals bearing Morris hepatoma 5123tc. From a second group of rats, the blood was removed 90 min after injection. Tissue extracts and serum were divided into three portions each, and albumin was isolated from each of the three portions by one of three different methods.The three different isolation procedures were the following: (A) pretreatment of the tissue extracts and serum with bovine serum albumin and its specific antiserum and subsequent immunoprecipitation of the rat serum albumin, (B) direct immunoprecipitation followed by dissolving the precipitated rat serum albumin in acid/ethanol, precipitation with ether, and ion-exchange chromatography of the redissolved albumin on CM-cellulose, and (C) a modification of a procedure published previously including fractionation with trichloroacetic acid, ethanol, ether, and ammonium sulfate, chromatography on Sephadex G-100 and DEAE-cellulose, and preparative disc electrophoresis in polyacrylamide gel at pH 10.3 and pH 2.7.With method (A), radiochemically pure albumin can be obtained only from serum. Even though testis and kidney do not synthesize albumin, albumin preparations isolated by this procedure from these organs contain significant amounts of radioactivity. Specific radioactivities measured in albumin prepared by method (A) from the four tissues examined are 5–19 times larger than those in preparations isolated by method (C). Thus, method (A) is inappropriate for the isolation of radiochemically pure albumin from the tissues studied.Procedure (B) is sufficient to obtain radiochemically pure albumin from the serum as well as from the other tissues examined except liver. With liver, this method yields an albumin preparation containing 53% more radioactivity than does albumin isolated with method (C).Method (C) is the only procedure yielding radiochemically pure albumin from all sources, including liver. In liver and hepatoma, the properties of the radioactive impurities are very similar to the properties of albumin itself. Therefore, several purification steps and a careful analysis of the distribution of radioactivity among the albumin fractions after chromatographies and electrophoreses are necessary to separate radioactive impurities from the albumin from homogenates of these two sources.     

Immunoprecipitation of a cytoplasmic precursor of rat-liver cytochrome oxidase.

A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm. 1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular weight of 50000. 2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes. 3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic microsomal fraction. 4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts; these again were immunologically related. The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.     

Isolation and immunoenzyme determination of human beta 2-microglobulin

Two procedures for isolation of homogeneous beta 2-microglobulin from urine of patients with systemic lupus erythematosus were developed: a procedure for isolation of beta 2-microglobulin with two-stage gel chromatography on Sephacryl S-200 and a procedure for isolation of homogeneous beta 2-microglobulin with affinity chromatography on rabbit polyclonal antibodies. The microglobulins isolated with the two procedures showed identical physicochemical properties and were used in development of a competitive immunoenzymatic assay method for determination of beta 2-microglobulin in the blood. The method was approved for determination of beta 2-microglobulin in blood serum of patients.     

Purification and properties of gamma-glutamyl transferase from normal rat liver

gamma-Glutamyl transferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, ED 2.3.2.2) has been partially purified from both whole rat liver (600-fold) and from isolated biliary tract (1200-fold). The most highly purified fraction gave two protein bands on polyacrylamide gel electrophoresis, the major band alone having enzyme activity. The enzyme purified from biliary tract appears identical to that from whole liver preparation according to molecular weight, kinetic parameters and the effects of various inhibitors. Three liver cell-types; parenchymal, Kupffer and biliary tract were isolated by perfusion of the rat liver in situ with collagenase, followed by selective cell isolation. Approx. 80-90% of the total recovered enzyme activity was found in the biliary tract. Nearly 50% of the apparent enzyme activity in the parenchymal cell was attributable to a nonspecific hydrolase.     

A Rapid and Efficient Method for the Isolation of Proteins from Polyacrylamide Gels

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

Structural characterization of human liver heparan sulfate

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.     

A rapid and effecient method for the isolation of proteins from polyacrylamide cells.

A procedure is described for the rapid and efficient electrophoretic elution of protein from polyacrylamide gels which is then collected in a dialysis bag tied to the end of a tube containing the gel slices. To illustrate the method a heterogeneous preparation of alkaline phosphatase was used from which a single homogeneous component was isolated in six hours with a recovery of 86%. The eluted protein is collected in a volume which can easily be kept below 1.5 ml, thus eliminating the need for subsequent concentration. The method has also been used successfully in two other systems in which a human lung tumor-associated antigen and glycogen synthetase from yeast were isolated. Since the method utilizes a standard analytical gel electrophoresis apparatus with no modifications or accessories, it should be immediately applicable for the isolation of many different proteins from polyacrylamide gels.     

A detergent-independent procedure for the isolation of gap junctions from rat liver

In this paper, the isolation of rat liver gap junctions from alkali-extracted rat liver plasma membranes is described. The purification is significantly more rapid than the commonly used detergent-based approaches and is subject to less variability. The gap junctions isolated by this method are comprised of a 27,000-Da polypeptide previously identified as the major gap junction polypeptide. The isolated gap junctions have the characteristic double-membrane organization and subunit structure observed in vivo. The protein yield is from 8 to 10 micrograms/g of liver (wet weight), about a 10-fold increase in recovery over that of earlier isolation procedures. With the availability of increased amounts of material, antibodies were raised to the liver gap junction polypeptide. Immunofluorescence localization of these antibodies on rat liver sections revealed a distribution consistent with that expected from electron microscopic analysis of liver thin sections. Double diffusion of antibody against solubilized gap junctions in detergent-containing gels resulted in the formation of precipitin arcs, suggesting response to multiple determinants. Antibody binding to the 27,000-Da gap junction polypeptide was demonstrated by immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels containing rat liver plasma membranes and isolated gap junctions. These results confirm the identification of the 27,000-Da polypeptide as the major protein component of gap junctions.     

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: II. nitric oxide synthesis and related changes

Liver cells isolated from intact tissue can reaggregate to form three-dimensional, multicellular spheroids in vitro. During this process, cells undergo a histological and environmental change. How cells respond biochemically to this change has not been studied in detail previously. We have investigated some biochemical changes in rat liver cells during the formation and maintenance of spheroids. Liver cells were isolated from male Sprague rats and spheroids cultured by a gyrotatory-mediated method. Liver cells were shown to respond to the isolation procedure and the formation of spheroids triggered histological environmental changes that increased arginine uptake, nitric oxide (NO) and urea syntheses, as well as raised levels of GSH, GSSG, glutamic acid and aspartic acid secretion within the first couple of days after cell isolation. Levels were maintained at a relatively stable level in the mature spheroids (>5 days) over the 3 week period of observation. P450 1A1 activity was lost in the first 2 days and gradually recovered thereafter. This study, for the first time, shows that liver cells after isolation and during spheroid formation actively uptake arginine and increase NO and urea syntheses. A high level of NO is likely to play an important role in modulating a series of biochemical changes in liver cells. It is considered that liver cells actively respond to the 'challenge' induced by the isolation procedure and subsequent histological environmental changes, and biochemical modulation and instability result. The stable cell-cell contacts and histological environment in mature spheroids permit and support functional recovery and maintenance in vitro. This period of stability permits the use of spheroids in toxicity studies to establish acute and chronic paradigms.