A 46 kDa NTPase common to rat liver nuclear envelope, mitochondria, plasma membrane, and endoplasmic reticulum.

A 46 kDa ATP binding polypeptide of the nuclear envelope, virtually identical to the nuclear envelope NTPase putatively involved in mRNA efflux [6], is present in all rat liver cell membranes. Its presence in nuclear envelope is not the result of cross contamination during isolation.

Negative supercoiling and nucleosome cores. I. The effect of negative supercoiling on the efficiency of nucleosome core formation in vitro.

The efficiency of nucleosome core formation in vitro as a function of DNA topology was investigated. We show that the reconstitution of nucleosome cores by urea/salt dialysis on both negatively supercoiled and linearized plasmid proceed co-operatively, and that negatively supercoiled molecules are reconstituted significantly more efficiently compared with linearized molecules. The free energy of supercoiling, related to the square of the linking deficit, is further shown to be sufficient to account for this difference, which is particularly pronounced at low molar reconstitution ratios of octamer: DNA. At these low molar ratios the average number of cores formed per negatively supercoiled molecule is equal to the input ratio of octamer: DNA, in contrast to linearized molecules, where few if any cores are reconstituted under identical experimental conditions. The possible contribution of supercoil-stabilized non-B-DNA structural transitions to differences in core-DNA interactions on supercoiled and linearized DNA was also investigated. We show that the change in the nuclease susceptibility of a d(A-G).d(C-T) run in the free and reconstituted supercoiled plasmid is consistent with the reversion of the poly(purine).poly(pyrimidine) stretch from an H-DNA form to a B-DNA form following reconstitution of the negatively supercoiled plasmid into nucleosome cores. The biological significance of the supercoil-dependent efficiency of core formation is discussed, and the results related to other work.

Negative supercoiling and nucleosome cores. II. The effect of negative supercoiling on the positioning of nucleosome cores in vitro.

The influence of unrestrained negative superhelical stress on nucleosome core positioning was investigated in vitro for a core located on a section of the early H1-H4 histone gene spacer of Psammechinus miliaris. We show that the position of this core on a reconstituted molecule occupied by 11 nucleosome cores is identical on a linear DNA molecule and a circular DNA molecule in the absence of unrestrained negative superhelical stress. This position is also identical to that previously found on a 337 base-pair fragment of corresponding sequence. We conclude that the core position is determined primarily by the DNA sequence, and is not influenced by core-core interactions or spatial constraints imposed by an altered geometry of the DNA molecule. This finding is supported by the identical positions assumed by the nucleosome core after altering the angular orientation of the DNA molecule, and presumably that of adjacent cores, on either one or both sides of the test core. It is further demonstrated that the core on the histone spacer region assumes identical positions on circular DNA molecules in both the presence and absence of excess negative superhelical stress equivalent to sigma = -0.03. This result indicates that conservative levels of negative supercoiling do not induce a shift in the positions of nucleosome cores. The biological implications of the experimental results are discussed and related to the findings of other workers.

Bitis arietans nerve growth factor is a disulphide-linked homodimer.

1. Nerve growth factor from Bitis arietans venom was isolated in high yield and purified to homogeneity using a rapid two-step procedure involving gel exclusion chromatography and reversed-phase HPLC. 2. On polyacrylamide gel electrophoresis in SDS, the NGF migrates as a 25 kDa homodimer and is thus atypical of other Viperid NGFs. 3. Evidence suggests that, unlike mammalian beta NGFs, the subunits of the Bitis arietans homodimer are covalently linked by a disulphide bond(s). 4. Partial sequence analysis shows that only 6 out of the first 21 amino acids are identical with those of cobra NGF including cys-14 and val-21 which are known to be important for NGF activity.

Stage- and adult tissue-specific expression of a homeobox gene in embryo and adult Parechinus angulosus sea urchins.

We report the isolation of a gene (PaHbox6), encoding a homeobox-containing protein of the South African sea urchin, Parechinus angulosus. Sequencing identified an Antennapedia-class gene encoding a homeobox that is the homologue of the Hawaiian sea urchin Tripneustes gratilla homeobox gene. Extensive restriction-fragment length polymorphism surrounds the gene. RNase-protection analyses revealed expression of PaHbox6 in mesenchyme blastula embryos at maximal levels of 44 +/- 8 transcripts/embryo. Four adult tissues examined (testes, ovary, intestines, Aristotle's lantern) showed expression of PaHbox6, though at greatly differing levels, with testes highest at eleven transcripts/10 pg RNA. Two transcripts of 5.2 and 5.7 kb were identified in adult tissue.

Ricin B-chain promotes the internalisation of liposomal contents into rat hepatoma cells.

Ricin B-chain covalently attached to liposomes has been shown to promote the binding of the liposomes to rat hepatoma cells through its galactosyl binding site. Internalisation of the bound liposomes is demonstrated by the cytotoxicity of methotrexate-containing liposomes, the transfection of cells with targeted liposomes containing pSV2-neo DNA and the intracellular activity of an enzyme encapsulated in liposomes targeted with the ricin B-chain.

Aberrant DNase I digestion kinetics of nucleosomal core particles from sea urchin sperm.

The pancreatic deoxyribonuclease (DNase I) digestion rates at the susceptible sites on nucleosomal core particles from blastula, gastrula and sperm cells of the sea urchin, Parechinus angulosus, have been determined. Although there are differences in their isohistone composition, the rates of digestion are similar for both embryonic stages. The rates of digestion for sperm core particles are 3-5 times lower than for embryo core particles at the more, and up to 2.5 times lower at the less susceptible sites. An explanation for these differences could be sought in the sperm isohistones H2B which are characterized by N-terminal extensions of 20-25 amino acid residues.

The amino acid sequence of wheat histone H2B(2). A core histone with a novel repetitive N-terminal extension.

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphylococcus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1) and bovine H1.

The primary structure of the histone H2A(2) type from wheat germ. A core histone type with both, N-terminal and C-terminal extensions.

The histone H2A(2) type from wheat germ comprises at least two highly homologous isohistones with 151 amino acid residues. Microheterogeneity occurs mainly at the N-terminal and C-terminal regions. These isohistones have both N-terminal (7 amino acid residues) and C-terminal (15 amino acid residues) extensions relative to calf thymus histone H2A.

Isolation of nuclei from melittin-destabilized cells.

Melittin, the hydrophobic polypeptide from bee venom, sufficiently destabilizes the plasma membrane of cultured cells to allow cell disruption in the absence of detergents with minimal homogenization. Nuclei are thus isolated in high yield with intact nuclear membranes and high transcriptional activity.

The primary structure of histone H3 from wheat.

Wheat embryo histone H3 has been isolated and purified and the elucidation of the complete amino-acid sequence is described. Peptides were generated by cleavages with CNBr, S. aureus V8 proteinase, endoproteinase Lys-C and trypsin. The peptides were purified by HPLC and the sequence determined by solid-state and gas-phase sequencing methodology. The amino-acid sequence of the protein is identical to pea embryo histone H3 and the sequence deduced from the nucleotide sequence of a wheat embryo histone gene (Tabata T. et al. (1984) Mol. Gen. Genet. 196, 397-400).

The reconstitution of a hybrid histone octamer containing avian 110Cys-des-thio-histone H3 and sea-urchin 73Cys-histone H4.

A hybrid histone octamer was reconstituted from erythrocyte H2A and H2B, avian [110 Cys-des-thio]histone H3 and the sea-urchin sperm [73Cys]H4 variant. [110Cys-Des-thio]histone H3 was prepared by reaction of natural H3 with Raney nickel. The ability of the hybrid octamer to crystallize to the same form as the natural octamer demonstrated that the chemical modification of cysteine to alanine in H3 and the mutation from threonine to cysteine in sperm H4 do not alter histone-histone interactions in the octamer. Since the sulfhydryl groups of both H4 molecules are fully accessible to 5,5'-dithiobis(2-nitrobenzoate) these residues provide suitable sites for the introduction of a single cysteine-specific label per H4 molecule in the octamer.

The use of sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate as a crosslinking reagent to identify cell surface receptors.

Conditions for solubilizing and iodinating the heterobifunctional thiol-cleavable photoreactive crosslinking reagent sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate which leave the ester moiety, disulfide bond, and azido group reactive are described. Iodination was performed in a mixture of dimethyl sulfoxide and bicarbonate, pH 9.0 (1:20, v/v), as solubilizing agent and Iodogen as oxidant. The lectin phytohemagglutinin was derivatized with the iodinated crosslinker and the interaction between phytohemagglutinin and mononuclear cells was chosen as the model system to monitor the efficiency of sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate as a crosslinking reagent. Transfer of 125I to the biologically significant T11 lymphocyte receptor in addition to 125I labeling of other membrane proteins to which the lectin binds was detected by polyacrylamide gel electrophoresis under reducing conditions.

Steroid-affinity purification of the rat liver glucocorticoid hormone receptor complex.

The molybdate-stabilized GHRC was isolated from rat liver cytosol with a 9000-fold purification and 46% yield. The major purification step was achieved using an affinity matrix consisting of an agarose support coupled to a dexamethasone ligand via an aliphatic spacer arm. Spacer arms containing disulfide bridges were found to be unsuitable due to their instability in cytosol. To reduce the non-specific binding properties of the affinity matrix, underivatized amino groups were acetylated, since the receptor was found to bind avidly to such groups thus evading elution by the ligand. Sodium molybdate present during biospecific elution from the gel stabilized the steroid-binding activity of the receptor. The use of denaturing and sulfhydryl modifying reagents (NaSCN, DMSO, Mersalyl) during elution led to partial or complete irreversible loss of steroid-binding activity of the unoccupied receptor. Efficient biospecific elution occurred at competing concentration of high affinity steroid in the presence of sodium molybdate. The ligand specific eluate was further purified by DEAE-Sephacel chromatography resulting in additional purification of 3.2-fold. The GHRC eluted from the DEAE-Sephacel column at a salt concentration characteristic of the untransformed GHRC. Molybdate was removed from the purified untransformed GHRC in the ligand eluate by DEAE-Sephacel chromatography in the absence of molybdate, for subsequent heat transformation.

Identification of nuclear envelope proteins and glycoproteins which co-isolate with the nuclear protein matrix.

Nuclear envelopes and nuclear matrices were isolated from rat liver nuclei. Although differences in polypeptide composition of the structures are evident on SDS gel electrophoresis, they have an almost identical distribution of concanavalin A-binding glycoproteins. These matrix-associated concanavalin A-binding glycoproteins derive entirely from the nuclear envelope and are recovered almost quantitatively in the matrix. They constitute easily identifiable markers for nuclear envelope association with matrix or other nuclear subfractions. Surface labelling of nuclei with 125I using solid-phase lactoperoxidase further confirmed that a large number of envelope-associated nuclear surface proteins co-isolate with the matrix. Protein kinase activity, as well as endogenous substrates for the kinase(s) are shown to be the same in both envelopes and matrix. Envelope-derived proteins and glycoproteins may comprise a substantial proportion of total matrix protein.

Carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate.

We found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed us to synthesize in a single-step procedure carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[gamma-32P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue.

Transformation in vitro and covalent modification with biotin of steroid-affinity-purified rat-liver glucocorticoid-hormone-receptor complex.

The molybdate-stabilized rat liver glucocorticoid receptor complex was purified 9000-fold with a 46% yield by steroid-affinity chromatography and DEAE-Sephacel ion-exchange chromatography. The purified glucocorticoid receptor was identified as a 90-92-kDa protein by SDS/polyacrylamide gel electrophoresis. Raising the temperature to 25 degrees C in the absence of molybdate resulted in increased binding of the receptor complex to DNA-cellulose or nuclei, similar to the effect on the cytosolic complex. The purified complex has a sedimentation coefficient of 9-10 S before and after heat treatment in the absence of molybdate. The appearance of smaller 3-4-S species was unrelated to the extent of DNA-cellulose binding of the complex. The process termed 'transformation', i.e. increasing the affinity for DNA, is not concomitant with subunit dissociation or loss of RNA. Highly purified glucocorticoid receptor could be covalently modified with biotin to retain its steroid-binding activity but with a 50% decrease in nuclear binding capacity. The biotin-modified complex reacts with streptavidin in solution without losing its steroid.

The quantitation of biotinylated compounds by a solid-phase assay using a 125I-labelled biotin derivative.

The biotin analogue biotinylglycyltyrosine has been synthesized and labelled to a specific activity of 2000 Ci/mmol with 125I. This analogue has been used in conjunction with immobilized streptavidin in an assay which detects as little as 1 fmol biotin or biotinylated molecules in solution. The determination of biotinylated insulin in a tissue extract and the quantitation of a transcription assay are given as examples.