High molecular weight complexes of folic acid in mammalian tissues

Twenty-four hours after injection of tritiated folic acid into normal rats, a large amount of labeled folates were found to be associated with the high molecular weight fraction of liver, kidney and intestine. The bound folate was associated with three fractions in the liver cell supernatant having approximate molecular weights of ? 350,000, 150,000 and 25,000 daltons, respectively. A fourth fraction which had an approximate molecular weight of 90,000 daltons was isolated from the liver nuclear fractions. The bound folates associated with these fractions were almost exclusively polyglutamate forms.     

In situ determination of the molecular weight of hepatic gamma-glutamyl transferase and gamma-glutamyl hydrolase activities

The molecular weight of gamma-glutamyl transferase from normal rat liver and hepatoma tissue was determined by radiation-inactivation and found to be approx 100000 in each case. The molecular weight previously reported for the subunit containing the gamma-glutamyl binding site (22000) is considerably less than that of the holoenzyme, suggesting that in situ the large subunit is implicated in both transferase and hydrolase activities.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

Isolation of a sodium dodecyl sulfate-insoluble transglutaminase substrate from liver plasma membranes

Rat liver plasma membranes contain transglutaminase activity and a large molecular weight protein complex which serves as a substrate for this enzyme. When plasma membranes were solubilized in sodium dodecyl sulfate and disulfide-reducing agents the transglutaminase substrate was recovered in the detergent-insoluble fraction. The insolubility of the complex suggested that it might be further studied by adsorbing membranes onto glass slides, then extracting with the detergent and reducing agent. After extraction, dark field light microscopy revealed numerous flattened sheets which varied in size from 4 to 12 micrometers. To confirm that these structures were the large molecular weight transglutaminase substrate, the plasma membranes were solubilized in sodium dodecyl sulfate and dithiothreitol and sedimented through a sucrose gradient containing the agent. The large molecular weight substrate was the only material found at the 1.11/1.23 g/cm3 interface. Microscopic examination showed the same structures previously observed on the glass slides. We conclude that the large molecular weight transglutaminase substrate is a sodium dodecyl sulfate-insoluble, morphologically distinct, protein complex. Due to its considerable size, nondissociable nature, and association with the lateral membrane, the sodium dodecyl sulfate-insoluble transglutaminase substrate may serve as a type of skeleton or scaffolding for this plasma membrane domain.     

Chicken liver amidophosphoribosyltransferase. Ligand-induced alterations in molecular properties.

A homogeneous amidophosphoribosyltransferase (EC 2.4.2.14) preparation, which was sensitive to purine nucleotide inhibitors, was obtained from chicken liver. From the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the subunit weight was estimated to be approximately 58 000. In Tris-HCl buffer, the predominant form of the enzyme had an S20,w of 6.5, Strokes radius of 40 A, and estimated molecular weight of 110 000. Incubation with 5-phosphoribosyl 1-pyrophosphate or Pi resulted in an increase in the S20,w to 9.1--9.5, Strokes radius 50 A, and estimated molecular weight to 200 000. Incubation of the large form with AMP led to a decrease in the molecular wight of the enzyme. It is concluded that chicken liver amidophosphoribosyltransferase is an allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.     

Fractionation of Soluble Copper- and Zinc-Binding Proteins From Cattle Liver

The distribution of copper and zinc among soluble proteins in liver from normal slaughter cattle was examined after gel filtration of the proteins. Gopper- and zinc-binding proteins were mainly separated into three fractions. Varying amounts of zinc were eluted in a fourth fraction of molecular weight less than 2,000. A clear relationship was noted between the amount of copper bound to the low molecular weight fraction (m.w. ~ 10,000) and the total liver zinc concentration. The high molecular weight protein fraction (m.w. > 65,000) dominated in liver with zinc concentrations below 40 µg/g wet weight and total copper concentrations from 16 to 240 µg/g, while in liver with zinc concentrations above 40 µg/g and copper concentrations ranging from 20 to 107 µg/g, the low molecular weight metallothionein-like fraction dominated.     

Multiple forms of human gamma-glutamyltransferase: preparation and characterization of different molecular weight fractions

Eight fractions of human gamma-glutamyltransferase were prepared from liver tissue, serum and bile by gel filtration. Bile, pooled serum from patients with high gamma-glutamyltransferase activities and serum in which liver tissue had been incubated, each contained an enzyme fraction with molecular weight greater than 10(6). A fraction of about 80,000 molecular weight was obtained from bile, and by incubation of liver tissue in serum or sodium chloride solution, but not from the serum pool. The main enzyme fraction in native serum had a molecular weight of about 300,000, and the molecular weight of gamma-glutamyltransferase partially purified from liver was initially 160,000. The fractions had similar Km and Ki values, and differences in heat stability and binding to concanavalin A were not marked.     

Intrahepatic assembly of very low density lipoproteins. Phosphorylation of small molecular weight apolipoprotein B

The possibility that apo-B is phosphorylated was examined using cultured rat hepatocytes. Rabbit antiserum prepared against rat apo-B was found to specifically react with both large and small molecular weight apo-B (by electroblotting assay and by immunoprecipitation of [35S]methionine-labeled proteins synthesized and secreted by hepatocytes). Following a 4-h incubation with [35P]orthophosphate, immunoprecipitation, and sodium dodecyl sulfate electrophoresis, an autoradiographic band corresponding to small molecular weight apo-B was obtained from cells and medium. Compared to the relative abundance of 32P which was associated with secreted small molecular weight apo-B, there was little (if any) detected in large molecular weight apo-B. Addition of excess unlabeled apo-B (obtained from rat serum) totally competed with the specific antiserum for this radioactive protein, indicating it was antigenically related to apo-B. Moreover, isolation of the 32P-labeled apo-B electrophoretic band, followed by acid hydrolysis and phosphoamino acid analysis, showed that at least 20% of the 32P originally associated with small molecular weight apo-B was in the form of phosphoserine. Control experiments ruled out the possible contamination of apo-B with phospholipid as well as the possibility that the phosphoserine produced by acid hydrolysis could have been derived from phosphatidylserine. To examine the relevance of these data to the in vivo state, rats were injected with [32P]orthophosphate. Immunoprecipitation of their livers followed by autoradiographic analysis showed the presence of 32P in small molecular weight apo-B. These data show for the first time that small molecular weight apo-B is synthesized as a phosphoserine containing protein.     

汞与硒在不同汞暴露水平地区猪肝脏和肾脏蛋白质中的分布

通过对贵州万山汞污染地区及北京地区猪肝脏和肾脏组织上清液进行凝胶过滤色谱分离(SephadexG 10 0 ) ,随后用原子荧光法测定它们蛋白质组分中汞和硒的含量 ,研究在汞暴露水平不同状态下微量元素汞和硒在动物体蛋白质分子水平上的分布 .发现这两个地区猪肝脏和肾脏组织上清液蛋白质组分中汞和硒的分布模式有明显差异 .贵州万山汞污染地区猪肝脏上清液中汞浓度比北京地区高 ,硒浓度也相应高 ,且前者与高分子量和低分子量蛋白结合的硒均明显高于后者 ;而北京地区猪肝脏上清液中的硒主要以与高分子量蛋白结合的形式存在 .贵州汞污染地区猪肝脏上清液中汞主要与高分子量蛋白结合 ,而北京地区猪肝脏上清液中汞则分布较为均匀 .贵州万山地区猪肾脏上清液中 ,含硒峰在高分子量蛋白区和低分子量区都有分布 ;而北京地区猪肾脏上清液中 ,硒则主要集中分布于高分子量蛋白范围 .这两个地区猪肾脏上清液中都有分子量约为 11kD的金属硫蛋白 (MT)存在 ,北京地区猪肾脏上清液中汞主要以与金属硫蛋白结合的形式出现 ,而贵州万山地区猪肾脏上清液中的汞除与金属硫蛋白结合外 ,尚有相当大部分是以与高分子量蛋白结合的形式存在 .研究结果表明 ,由于这两个地区汞暴露水平的差异 ,不仅使这两地区猪肝、肾上清液中的汞与硒含量     

Molecular weight distribution of ribosomal proteins from several vertebrate species.

Two-dimensional polyacrylamide gel electrophoresis of proteins from the separated ribosomal subunits of rabbit reticulocytes, rabbit liver, mouse liver, rat liver, chicken liver, and toad liver was performed using the "pH 4.5/SDS" system previously described (Martini and Gould, 1975), with internal standards to measure the molecular weight distributions. With few exceptions, the patterns were remarkably similar, indicating a high degree of conservation during evolution of both net charge (largely determining mobility in the first dimension) and size (determining mobility in the second dimension). The aggregate mass (sum of molecular weights) of both small and large subunit proteins, about 0.65 X 10(6) and 0.95 X 10(6) daltons respectively, were invariant. These figures are significantly smaller than the hydrodynamically determined mass of protein in the subunits. The implications of this discrepancy, which is opposite that found in the prokaryotes, is discussed.     

Electron microscopy of 26 S complex containing 20 S proteasome.

A high molecular weight protease complex (26 S complex) involved in the intracellular protein degradation of ubiquitinated proteins was purified from rat liver and studied by electron microscopy. The most prevalent molecular species with best preserved symmetrical morphology had two large rectangular terminal structures attached to a thinner central one having four protein layers. We concluded that they were the closest representation of the 26 S complex so far reported. The central structure was identified as 20 S proteasome and the terminal one as recognition units for ubiquitinated proteins.     

Subunit structures of AMP deaminase isozymes in rat

AMP deaminases A and B have been purified to apparent homogeneity from rat muscle and liver, respectively. The molecular weights of 286,000 and 351,000 were obtained for the native muscle and liver enzymes, respectively, by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the muscle preparation exhibited a single polypeptide band with a molecular weight of 72,000; the liver preparation, a molecular weight of 85,000. The data indicate that each enzyme has a tetrameric structure.     

Carboxylesterases (EC 3.1.1). The molecular sizes of chicken and pig liver carboxylesterases.

The molecular size of pig liver carboxylesterase has been investigated under a variety of conditions of pH and ionic strength. From equilibrium and velocity sedimentation at pH 4.0 and pH 7.5, and from chromatography on Sephadex G-200,we conclude that the monomeric molecular weight is similar to 65,000 daltons and that the enzyme associates to form trimers. Association equilibrium constants for the monomer-trimer system were estimated to be 0.02 1-2 g-2 at pH 4 (concentration-dependent molecular weight data) and 2 times 10-5 1-2g-2 at pH 7.5 (frontal gel chromatographic results). These studies were aided by comparisons of the properties of the pig liver enzyme with those of chicken liver carboxylesterase, which is shown to exhibit the velocity and equilibrium sedimentation characteristics of a homogeneous protein with molecular weight similar to 65,000. Studies of pig and chicken liver carboxylesterases in 6 M guanidinium chloride, 0.1 M in beta-mercaptoethanol, support the proposition that the monomeric species of these enzymes have molecular weights of similar to 65,000. On polyacrylamide gel electrophoresis in SDS, there is no evidence for a major species of molecular weight less than similar to 65,000 for the pig enzyme, but ca. 50 percent of the chicken esterase is dissociated into two species of molecular weight similar to 30,000.     

Subcellular localization of high- and low-molecular weight acid phosphatases from chicken liver

The subcellular localization of high and low molecular weight acid phosphatases in chicken liver was studied. The high molecular weight acid phosphatase is mainly associated with the particulate fraction, particularly with the mitochondrial-lysosomal fraction, whereas the low molecular weight form seems to be a soluble cytoplasmic enzyme. Biochemical properties including optimal pH, molecular weight determination and the effect of some modifier substances indicate that mitochondria-lysosomes and microsomes contain the same high molecular weight acid phosphatase form.     

Inhibitor of pyrimidine metabolism from tumor tissues

Inhibitors of normal rat liver 5′-nucleotidase and dUMP kinase $\text{in vitro}$ were found in rapidly proliferating tissues, such as Yoshida sarcoma. Two inhibitors were separated from Yoshida sarcoma by zone electrophoresis, gel filtration on Sephadex G-200 and DEAE-cellulose column chromatography. One inhibited both 5′-nucleotidase and dUMP kinase, while the other inhibited only dUMP kinase. These inhibitors were not detectable in normal rat liver. They were induced in regenerating rat liver and present in rapidly proliferating tissues, such as Yoshida sarcoma and Ehrlich ascites tumor and rat marrow cells. These inhibitors were heat labile. One had a large molecular weight (500,000>) and the other a small molecular weight ($\text{Ca}\text{.}$ 50,000).     

Characteristics of the fractional composition of membranes of membrane-bound ribosomes

Electrophoresis in PAAG was used to study the fractional composition of proteins from free and membrane-bound ribosomes isolated from the rat liver. The membrane-bound ribosomes are shown to differ in the protein composition from the free ones in the presence of two fractions with a molecular weight of 35 and 50 kDa which are localized in a large subunit of the organelle. The possible role of these fractions in ribosome-membrane interaction is discussed.     

Activity staining for glutamate and alanine dehydrogenases in polyacrylamide gels of varying porosity. A study of bovine placental enzyme forms.

A procedure has been developed for characterizing the various molecular forms of placental and liver glutamate dehydrogenases through a combination of activity staining and varying gel pore size in electrophoresis. At a concentration of 2 mg/ml, the bovine liver GDH remained associated in a very high molecular weight form, while the placental enzyme was substantially dissociated to a molecular species of near 240,000 molecular weight and several charge isomeric species of near 160,000 molecular weight. The general approach outlined here provides a means of definitely correlating the electrophoretic behavior of various dehydrogenase isozymes with both glutamate and alanine dehydrogenase activities and molecular size and should be applicable, even in crude extracts to other dehydrogenase enzymes which exhibit multiple forms or states of association.     

Purification and properties of a membrane-bound aldehyde dehydrogenase from rat liver microsomes

A membrane-bound aldehyde dehydrogenase was solubilized from rat liver microsomes and purified about 150-fold by chromatography on ω-aminohexyl- and 5′-AMP-Sepharose columns with a recovery of about 40%. The purified enzyme was homogeneous upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its monomeric molecular weight was estimated to be 51,000. In aqueous solution, it existed as large, polymeric aggregates. Its activity towards straight-chain aliphatic aldehydes increased as their carbon chain length was increased at least up to dodecanal, whereas aldehyde dehydrogenase in the cytosolic fraction of rat liver was most active with hexanal as substrate.     

Identification of an Mr 77,000 - 80,000 product of in vitro translation of rat liver mRNA using antibody to glycogen synthase

Rabbit antibody to rat liver glycogen synthase has been used to identify a product of Mr 77,000 - 80,000 from in vitro translation of rat liver mRNA. A comparison of various protease inhibitors on the relative molecular weight of rat liver glycogen synthase suggest that higher molecular weight enzyme forms could arise from incomplete hydrolysis of glycogen before enzyme isolation and enzyme subunit Mr determinations.     

Demonstration of cross-linked cytokeratin polypeptides in transplantable rat hepatoma cells.

Covalently cross-linked multimers of cytokeratins were shown to be present in transplantable Morris hepatoma 7777 cells. These high molecular weight antigens were not detectable in normal rat liver cells. However, identical high molecular weight antigens were also demonstrated in rat liver cells when the cells were homogenized in solutions containing Ca2+. The cross-linking reaction was suggested to be mediated by the action of tissue transglutaminases.