Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-order fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1.5 M KCl) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of Mr 55,000, 48,000, and 40,000 was noted. On two- dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (Mr 48,000, and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the Mr 55,000 protein which focused at pH values higher than 6.4. The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins. Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell. The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue- specific patterns of their protein subunits.     

Rapid purification of intact tonofilaments from newborn rats : Comparison with glial filaments and neurofilaments

A rapid, new procedure for the isolation of intact tonofilaments from newborn rat skins is described. The filament preparations show two major protein subunits on SDS-PAGE with molecular weights of 58000 and 66000 D. An antiserum prepared against the 58000 D protein reacted specifically with the tonofilament preparation, but not with the protein subunits of neurofilaments, glial filaments, tubulin or actin. This specificity is confirmed by indirect immunofluorescence: anti-P58 reacts with the epidermis, whereas antisera against the neurofilament or glial filament proteins and anti-tubulin do not. These data suggest that epidermal filaments represent a class of intermediate filaments distinct from either glial filaments or neurofilaments.     

Isolation of rat hepatocyte plasma membranes. II. Identification of membrane-associated cytoskeletal proteins

Rat liver plasma membranes were isolated as presented in the preceding paper (Hubbard, A. L., D. A. Wall, and A. Ma., 1983, J. Cell Biol., 96: 217-229) and found to contain many filaments associated both with desmosomes along the lateral surface and with the cytoplasmic aspects of membranes comprising each of the three domains (lateral [LS], bile canalicular [BC] and sinusoidal [SF] ). Exposure of the plasma membranes to alkaline media (up to pH 11) resulted in loss of recognizable filaments without loss of domain morphology or membrane enzyme activities. Electrophoretic analysis of solubilized components from control and alkaline-extracted plasma membranes revealed that three major polypeptides present at 43, 52, and 56 kdaltons in the control had been released by alkaline treatment (pH 11) and could be quantitatively recovered in the supernate. The 43-kdalton component was identified as cytoplasmic actin by comparison of its tryptic 125I- peptide map to those of muscle (alpha) and brush border (beta, gamma) actins. The 52- and 56-kdalton polypeptides were identified as tonofilament components by their solubility properties and their ability to reassemble into 9.5-nm filaments from monomers present in an alkaline extract.     

Studies on muscle differentiation. I. Isolation and purification of structural proteins from leg muscles of the frog, Rana nigromaculata

A procedure for isolating sequentially structural proteins from leg muscles of adult frog, Rana nigromaculata, was described. The procedure consisted of a novel method of isolating frog myosin in combination with established methods of isolating actin and tropomyosin. All the purified preparations of structural proteins isolated by our procedure were found to be almost homogeneous as examined by electrophoresis and ultracentrifugal sedimentation. All the preparations also showed characteristic physico-chemical properties expected to the respective authentic proteins. Frog myosin preparations isolated by two different procedures were not significantly contaminated with actin or actomyosin, but were contaminated with RNA or RNA-protein and with a slowly sedimenting minor component which was released upon denaturation of the preparation.     

Structure and regulation of the assembly of bacteriophage T4 fibrillar proteins. II. Isolation and initial structural characteristics of whiskers

A procedure for purification of bacteriophage T4 whiskers and it's monomeric subunits--gene product wac--has been developed. We have shown, that the whiskers are composed of two identical copies of gene product wac with molecular weight of 56 kDa each. The dimer of gene product wac is a highly ordered structure and it's length is about 70.0 +/- 10.0 nm, as revealed by electron microscopy. The amino acid composition of whiskers is very similar to that of watersoluble keratins. We have proposed a new term for the definition of the whiskers--the fibritin.     

Isolation and purification of histone-specific acetyltransferases from the rat liver

There forms of histone-specific acetyltransferases--A, B and C are obtained from the rat liver nuclei. The isolation process included nuclei generation, ammonium sulphate salting-out of proteins, DEAE-cellulose, hydroxyl-apatite, phosphocellulose chromatography and Sephadex C-200 gel-filtration. Acetyltransferases A, B and C from the nuclei were purified 56.8, 144.1 and 42.3 times, respectively. Histones were preferential substrates of the obtained enzymes. Molecular mass of acetyltransferases was determined by Sephadex G-150 and G-200 gel-filtration. It was 120 for enzyme A, about 90 for B and above 200 kDa for C.     

Isolation and structural characterization of cap-binding proteins from poliovirus-infected HeLa cells.

In poliovirus-infected HeLa cells, poliovirus RNA is translated at times when cellular mRNA translation is strongly inhibited. It is thought that this translational control mechanism is mediated by inactivation of a cap-binding protein complex (comprising polypeptides of 24 [24-kilodalton cap-binding protein], 50, and approximately 220 kilodaltons). This complex can restore the translation of capped mRNAs in extracts from poliovirus-infected cells. We have previously shown that the virally induced defect prevents interaction between cap recognition factors and mRNA. Here, we show that the cap-binding protein complex (and not the 24-kilodalton cap-binding protein) has activity that restores the cap-specific mRNA-protein interaction when added to initiation factors from poliovirus-infected cells. Thus, the activity that restores the cap-specific mRNA-protein interaction and that which restores the translation of capped mRNAs in extracts from poliovirus-infected cells, copurify. The results also indicate, by an alternative assay, that the cap-binding protein complex is the only factor inactivated by poliovirus. We also purified cap-binding proteins from uninfected and poliovirus-infected HeLa cells. By various criteria, the 24-kilodalton cap-binding protein is not structurally modified as a result of infection. However, the 220-kilodalton polypeptide of the cap-binding protein complex is apparently cleaved by a putative viral (or induced) protease. By in vivo labeling and m7GDP affinity chromatography, we isolated a modified cap-binding protein complex from poliovirus-infected cells, containing proteolytic cleavage fragments of the 220-kilodalton polypeptide.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

Kinetic properties of hexose-monophosphate dehydrogenases. II. Isolation and partial purification of 6-phosphogluconate dehydrogenase from rat liver and kidney cortex

6-Phosphogluconate dehydrogenase (6PGDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely isolated (more than 95%) from glucose-6-phosphate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 60–75% ammonium-sulphate fractionation, by which both hexose-monophosphate dehydrogenases activities were separated, and finally the protein fraction was applied to a chromatographic column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of the isolated partially purified liver and renal 6PGDH were examined. The saturation curves of this enzyme in both rat tissues showed a typical Michaelis-Menten kinetic, with no evidence of co-operativity. The optimum pH for both liver and kidney-cortex 6PGDH was 8.0. The Km values of liver 6PGDH for 6-phosphogluconate (6PG) and for NADP were 157 M and 258 M respectively, while the specific activity measured at optimum conditions (pH 8.0 and 37°C) was 424.2 mU/mg of protein. NADPH caused a competitive inhibition against NADP with an inhibition constant (K_i) of 21 M. The Km values for 6PG and NADP from kidney-cortex 6PGDH were 49 M and 56 M respectively. The specific activity at pH 8.0 and 37°C was 120.7 mU/mg of protein. NADPH also competitively inhibited 6PGDH activity, with a K_i of 41 M. This paper describes a quick, easy and reliable method for the separation of the two dehydrogenases present in the oxidative segment of the pentose-phosphate pathway in animal tissues, eliminating interference in the measurements of their activities.Publication No 170 from Drugs, Environmental Toxics and Cell Metabolism research group. Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain     

Sequence homology between the structural proteins of Kilham rat virus.

The capsid proteins of the autonomous parvovirus Kilham rat virus were purified and analyzed for peptide composition. Partial proteolysis mapping and two-dimensional thin-layer chromatography of tryptic peptide digests revealed extensive amino acid sequence homology between the two major Kilham rat virus capsid proteins.     

Isolation and purification of rat acute-phase alpha 2-macroglobulin

Acute-phase alpha 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase alpha 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase alpha 2-macroglobulin, after which the protein by mass determination and by two different protein assays. Pure rat acute-phase alpha 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing rocket immunoelectrophoresis assay.     

大鼠大脑微血管片段的分离纯化及鉴定

目的:分离大鼠大脑微血管并纯化去除完整的神经细胞,用于克隆血脑屏障上的特异表达基因.方法:采用液相合成法制备粒径200～500 nm的铁氧体磁珠,经两侧颈内动脉插管注入大鼠大脑半球.采用机械分离和酶消化相结合的方法解离脑组织,用筛网滤去组织块和大血管,再在磁场下分选标记磁珠的微血管片段,并从形态学、分子生物学和生物活性角度鉴定获得的脑微血管片段.结果:扫描电镜下没有发现微血管周围存在完整的神经细胞,但在部分区域有胶质的终足包裹.全脑组织微管相关蛋白2a、谷氨酰胺合成酶和CD31的RT-PCR产物均在相应位置出现阳性条带,分离纯化的脑微血管仅CD31阳性.微血管片段内皮细胞摄取的Rh123荧光强度显著低于传代培养的微血管内皮细胞荧光强度.结论:采用本方法可以获得高纯度的、不附带完整神经细胞的脑微血管片段.