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T Hultin

Publications and source records attributed to T Hultin.

At least 19 recordsLinked to original sources
PubMed1986

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: identification and binding directions of major protein complexes.

Rat liver and mouse ascitic tumour ribosomal proteins are cross-linked selectively in good yield with the newly developed cleavable heterobifunctional reagents 2-(4-hydroxy-2-maleimidophenylazo)benzoic acid N-hydroxysuccinimide ester (reagent A) and 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoic acid N-hydroxysuccinimide ester (reagent B). The primary function of the reagents, an N-aroylated maleimide, binds quantitatively at low pH to accessible cysteine groups. After eliminating the free reagent, the pH is increased to make the secondary function, a juxtanuclear aroyl ester, reactive against neighboring amino groups, essentially lysine. The spacer, 4-phenylazophenol, is readily cleaved by reduction with dithionite. The ranges of cross-linking of the two reagents are approx. 8 and 12 A, respectively. Using the radiolabelled reagent B the secondarily attached protein (and its contact sequence) is made recognizable even in trace amounts. The order of binding of the interacting proteins is thereby established. The two reagents produce similar, but not identical, patterns of selective cross-linking. The following protein complexes are readily observed after conventional staining. With reagent A: S8-S11, L4-L14, L4-L18, L6-L29 and L21-L18a. With the radioactive, longer-range reagent B: L4 ---- L13a, L4 ---- L18, L4 ---- L18a, L4 ---- L26, L6 ---- L29, L14 ---- L13a, L21 ---- L18a and L27 ---- L30 (arrows indicating the direction of binding). Ternary and quaternary complexes are also obtained, especially of the large protein L4. With both reagents a protein designated L6' is cross-linked to L23. The predominant cross-linked complexes can be obtained on a preparative scale for isolation and characterization of contact sequences by optional fragmentation and fractionation methods.

Animals↗
PubMed1986

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: separation and identification of contact sequences of neighboring proteins after CNBr fragmentation.

Mammalian ribosomal proteins were cross-linked in situ with the primarily cysteine-selective heterobifunctional reagents N-succinimidyl 2-(4-hydroxy-2-maleimidophenylazo)benzoate (reagent A, maximum range approx. 8 A) and N-succinimidyl 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoate (reagent B, maximum range approx. 12 A). With reagent B the secondarily attached (N-aryolated) protein becomes labelled specifically at the receptor amino group (lysine). The cross-linked proteins were fragmented with CNBr in attempts to isolate and identify sequences involved in the next-neighbor contacts. Two experimental schemes were adopted. Heavy complexes containing the large protein L4 cross-linked to protein L14 and/or L18 were isolated and treated with CNBr. The split products were submitted to diagonal electrophoresis for separation and identification of the two pairs of contact fragments. Proteins cross-linked with the radiolabelled reagent B were submitted to diagonal electrophoresis. The labelled receptor proteins were excised and treated with CNBr. Fragments carrying the contact sequences were separated by gradient gel electrophoresis and identified by autoradiography. By use of these methods CNBr fragments were isolated containing one or the dual contact sites of the following binary protein complexes: L4-L14, L4-L18, L4-L13a/L18a, L6'-L23, L6-L29, L7-L29, L14-L13a, L21-L18a, and L27-L30 (asterisks indicate the labelled receptor proteins). By varying the site of labelling of the heterobifunctional reagents and the methods of protein fragmentation a complete analysis of the contact sequences of these proteins should be possible.

Animals↗
PubMed1986

A class of cleavable heterobifunctional reagents for thiol-directed high-efficiency protein crosslinking: synthesis and application to the analysis of protein contact sites in mammalian ribosomes.

The synthesis of a new class of cleavable crosslinking reagents is described. The primary function, a ring-substituted maleimide, binds selectively and very efficiently at low pH to cysteine-containing protein sequences. At increased pH the secondary function, an N-hydroxysuccinimide ester of a ring-attached carboxyl group, becomes reactive against adjacent amino groups. The spacer, azobenzene, is readily cleaved by reduction with dithionite provided that a hydroxyl group is included in the ring system. By altering the relative positions of the reactive groups the range of crosslinking can be varied within approximately 8-12 A. After degradation of the crosslinked proteins by optional methods the contact sequences are readily identified by diagonal electrophoresis. By radiolabeling the carboxyl group of the reagent the order of binding of the proteins can be established, and the secondarily attached protein and its contact sequences can be recognized even in trace amounts. The usefulness of the reagents is illustrated by the selective, high-efficiency crosslinking of mammalian ribosomal proteins and the identification of their contact fragments as obtained by CNBr degradation.

Binding Sites↗
PubMed1984

Location of the sulfhydryl groups involved in disulfide interaction between the neighboring proteins L6 and L29 in mammalian ribosomes. S-cleavage of the cyanylated proteins in polyacrylamide gels after separation by dodecylsulfate gel electrophoresis.

Proteins L6 and L29 occupy closely adjacent sites in mammalian 60-S ribosomal subparticles and are easily cross-linked by intermolecular disulfide bond formation. For locating the interacting thiols within the polypeptide chains the dissociated proteins L6 and L29 obtained from the isolated disulfide complex were subjected to S-cleavage following [14C]cyanylation of the two cysteine residues. Four split products of the [14C]cyanylated proteins were isolated by dodecylsulfate gel electrophoresis. Two of these could be identified by autoradiography as the selectively labeled C-terminal fragments. For unequivocal assignment of the fragments to the parent proteins, a simple and generally applicable method of cleaving cyanylated proteins in polyacrylamide gel for subsequent diagonal analysis was developed. The experiments indicated that the sulfhydryl group of L6 interacting with L29 is located at a distance of approximately 80 amino acid residues from the N-terminus. In the intact ribosome this sequence contains a clostripain-sensitive and trypsin-sensitive portion of the protein more or less exposed at the ribosomal surface. In the case of protein L29, the interacting sulfhydryl group was located at a distance of approximately 40 amino acid residues from the C-terminal.

Amino Acid Sequence↗
PubMed1984

Isolation of the contact regions of the neighboring mammalian ribosomal proteins L6 and L29. Cyanogen bromide cleavage of the disulfide complex after preparative electrophoresis in non-oxidative polyacrylamide gels.

Proteins L6 and L29 in the 60-S subparticle of mammalian ribosomes interact in situ under gentle conditions by intermolecular disulfide bond formation. For identifying the contact regions of the two proteins the disulfide complex was isolated from whole ribosomes by preparative polyacrylamide gel electrophoresis and subjected to cyanogen bromide cleavage. For effective cleavage non-oxidizing conditions had to be maintained throughout the preparation. A simple and generally applicable method of high-efficiency gel pre-electrophoresis with anionic and cationic thiols was developed. Under these conditions reversibly cross-linked CNBr fragments of L6 and L29 could be isolated in high yield (Mr approx. 13 000 and 7000, respectively). After [14C]carboxymethylation of the reduced disulfide links smaller contact sequences were obtained by pepsin digestion and characterized by two-dimensional peptide mapping. These smaller contact peptides were contained within the corresponding CNBr fragments. Both contact peptides were hydrophilic and relatively basic.

Animals↗
PubMed1982

Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization. Evidence for a nuclear origin.

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm3, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)+RNA was therefore investigated. In gel electrophoresis poly(A)+RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3 . 10(5) and 5 . 10(5) Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)+RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)+RNA component of approx. 5 . 10(5) Da. 125I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)+RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)+RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)+RNA from developing embryos. The hybridization of labelled, nuclear poly(A)+RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)+RNA in dormant cysts is essentially of nuclear origin. The 5 . 10(5)-Da component is largely homologous with the corresponding component of nuclear poly(A)+RNA at later stages.

Animals↗
PubMed1981

Early effects of dimethylnitrosamine on the initiation of protein synthesis in mouse liver.

1. Dimethylnitrosamine (37.5 mg/kg body wt.) was administered to mice by a single intraperitoneal injection, and the early effects on protein synthesis and related functions were studied in a liver S-30 system. 2. The incorporation of [14C]leucine into protein decreased rapidly after dimethylnitrosamine administration. The effect was associated with a decreased ability of the system to utilize methionyl-tRNAfMet and formyl-methionyl-tRNAfMet for 80 S ribosomal initiation-complex formation (primary initiation), and a loss of poly(A)-containing RNA from the postmicrosomal fraction. All the three effects developed simultaneously, and were clearly demonstrable within 15 min. 3. Initiation-complex formation in the polyribosomal fraction (re-initiation) was decreased to the same extent as the primary initiation, indicating that the initiation defect was not a result of the decrease in free mRNA. 4. The inhibition of initiation was only manifest at the joining of the 40 S pre-initiation complex to 60 S ribosomal subunits. It was not a result of methionyl-tRNAfMet deacylation. The functions between the formation of the methionyl-tRNAfMet-containing 80 S ribosomal complex and the first translocation on the ribosome were not involved, since the incorporation of formylmethionine into N-terminal polypeptides decreased to the same extent as the 80 S initiation-complex formation. 5. Inhibitors of protein synthesis (cycloheximide and pactamycin) decreased poly(A)-containing RNA in the postmicrosomal fraction in a similar way to dimethylnitrosamine.

Animals↗
PubMed1981

Non-involvement of nucleolytic activities in the early effect of dimethylnitrosamine on the content of free mRNA in mouse liver.

1. After dimethylnitrosamine (DMNA) administration to mice, the content of poly(A)-containing RNA decreases rapidly in the postmicrosomal fraction of the liver. We report here that the loss of free mRNA is not a result of increased nucleolytic activity. On the contrary, a decreased activity of microsomal endonuclease, assayed by its effect on polyribosomal mRNA, was demonstrated already 15 min after the administration of DMNA at 37.5 mg/kg body wt. The loss of activity was more pronounced in the rough than in the smooth membranes. Total detergent-released microsomal nucleases, as assayed by use of labelled poly(U) as substrate, showed a less rapid decline. No corresponding increase in enzyme activities was observed in the postmicrosomal fraction. 2. The dimethylnitrosamine effect on the microsomal endonuclease was not accounted for by altered lysosomal contamination of the microsomal fraction. 3. No early effect of dimethylnitrosamine administration was found on the cytoplasmic ribonuclease inhibitor.

Animals↗
PubMed1981

Selectivity of interaction of univalent cations with mammalian ribosomes studied by equilibrium dialysis in the presence of the K+ analogue, 204Tl+.

The interaction of K+ with mammalian ribosomes was studied by equilibrium dialysis and compared with that of other univalent cations. The heavy K+ analogue, Tl+, binds more firmly than K+ to ribosomes and, unlike K+, has a practically useful isotope. With 204Tl+ as a marker of K+-selective binding the ribosome-cation interaction could be followed down to levels below 0.1 average Tl+-occupied site per ribosome. The Tl+/ribosome ratio varied with the free Tl+ concentration in a multiple way. At high Tl+ saturation Tl+ was easily displaced by Mg2+. With decreasing Tl+ saturation the competitive activity of Mg++ was strikingly reduced, indicating that Tl+ and Mg++ compete with different efficiency for different classes of sites. The experiments on univalent cations were performed at 1.5 mM Mg2+ under two complementary conditions: (1) Ribosomes were pretreated with 5 x 10(-2), 5 x 10(-3), and 5 x 10(-4) M LiNO3, NaNO3, KNO3, and CsNO3, and then equilibrated with different concentrations of 204TlNO3 in the same buffers. (2) Ribosomes were pretreated with 10(-2), 10(-4), and 10(-6) M 204 TlNO3, and then equilibrated with different concentrations of LiNO3, NaNO3, KNO3, and CsNO3 (displacement experiments). At high Tl+ saturation Na+ and Li+ were about as active as K+ and Cs+ in competing with 204Tl+. With decreasing Tl+ saturation a differentiation occurred in favor of K+ and Cs+, with some preference for K+. It is concluded that ribosomes contain a limited number of sites with pronounced ion specificity. Of physiological cations K+ is most firmly bound to these sites.

Animals↗
PubMed1980

Characterization of a poly(A)+RNA-containing structural component directly associated with cytoplasmic membranes in dormant Artemia cysts.

Poly(A)+RNA-containing material was extracted from the purified cytoplasmic membranes of dormant Artemia cysts by treatment with mild detergents. Sedimentation analysis of the extracts showed a predominant poly(A)-containing fraction at 40 S, associated with about 6% of the extracted proteins. Only limited amounts of poly(A)-containing material were found in the heavier fractions. Poly(A)+RNA extracted from the 40-S fraction sedimented around 14 S. The poly(A)-containing 40-S structures could be purified by treatment with non-ionic or zwitterionic detergents followed by resedimentation in sucrose gradients in the presence or absence of detergent. When the 40-S fraction was analyzed by isopycnic centrifugation in Cs2SO4 gradients, the main part of the poly(A)-containing material banded at a density of 1.27 g/ml. Electron-microscopic examination of this fraction revealed circular or slightly bullet-shaped profiles measuring 17-26 nm. When the 40-S fraction had been submitted to mild RNAase treatment prior to density gradient centrifugation, the material was displaced towards lower density and became less distinct. Purified 40-S particles showed a complex protein pattern not very similar to that of polyribosomal poly(A)+RNA-containing particles from developing embryos, but with components in common with unfractionated membranes. The particles also contained some lipids. The experiments indicate that a major part of the membrane-bound, latent poly(A)+RNA in dormant Artemia cysts occurs in the form of relatively uniform, detergent- and Cs2SO4-resistant structures, independent of ribosomes, but intimately associated with membrane components.

Animals↗
PubMed1980

The use of rRNA-cellulose chromatography in the rapid isolation of homogeneous protein synthesis initiation factor eIF-2 from rat liver microsomes.

A rapid and efficient procedure is described for preparing homogeneous polypeptide chain initiation factor eIF-2 from rat liver microsomal salt wash. The method involves ammonium sulfate fractionation, affinity chromatography on rRNA-cellulose and DEAE-cellulose chromatography. The preparation is highly active in initiation complex formation.

Animals↗