Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate)

The synthesis and properties of a new cleavable protein cross-linking reagent, [³⁵S]dithiobis(succinimidyl propionate), are detailed. Free primary and secondary aliphatic amino groups are quantitatively acylated by the reagent in either organic or aqueous media within two minutes at 23 °C. By contrast, the half-time for hydrolysis of the active ester termini in buffer at pH 7 is four to five hours, so that protein cross-linkage can be optimized by application of low concentrations of reagent. Accessible amino groups of hemoglobin are acylated with extreme rapidity of 0 °C in pH 7 buffer when [³⁵S]dithiobis(succinimidyl propionate) is applied in 0.4 to 9-fold molar excess. Submicrogram quantities of the cross-linked hemoglobin subunits which result are detectable by monitoring the ³⁵S distribution in sodium dodecyl sulfate-polyacrylamide gels. In addition to amine acylation, two of the six thiol groups in hemoglobin, tentatively located at cysteine 93 of the β chains, are reversibly modified at 0 °C by mercaptan-disul-fide interchange with the reagent or its bis amide analogs. This equilibrium-controlled, pH-dependent reaction occurs at a slower rate than acylation, and is blocked by short preincubation of the protein with N-ethylmaleimide or by addition of 3,3′- dithiodipropionamide (or other disulfides) to the reaction mixture. Disulfides introduced into hemoglobin by acylation and interchange are quantitatively cleaved by reduction for 30 minutes at 37 °C with 10 mm-dithioerythritol buffered at pH 8.5.The properties of high reactivity under mild conditions, long solution half-life, and the radioactive label make [³⁵S]dithiobis(succinimidyl propionate) a particularly useful and versatile probe of extended structures in a variety of biological systems.     

Purification of protein synthesis initiation factor eIF-3 from rat liver microsomes by affinity chromatography on rRNA-cellulose

An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor eIF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25–40% saturation (0°C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation. eIF-3 is eluted from the affinity column at a KCl concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, eIF-3 sediments as a 15 S particle indicating a total mass of 650 000 Da. The purified eIF-3 is highly active in stimulating globin synthesis in a fractionated translation system. Factor eIF-3 contains eight subunits with molecular weights ranging from 40 000 to 110 000. Seven of the subunits are present in one copy per eIF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.     

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K⁺ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5·10⁶ Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (M_r = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.     

Isolation and characterization of three different forms of arylamidase from rat skeletal muscle

An arylamidase hydrolysing L-leucine-4-nitroanilide was extracted from rat skeletal muscle homogenate and furified by means of anion-exchange chromatography on DEAE-Sephadex A-50 followed by gel filtration on Sephadex G-150 and Sepharose 6B. The enzyme was isolated in the form of three different protein complexes that differ in molecular weight, kinetic data, and sensitivity to metal ions. As studied by SDS-gel electrophoresis and repeated gel chromatography on Sepharose 6B these forms are: 1. a stable monomer (A1) of M_r 122 000; 2. a stable dimer (A2) of M_r 244 000; and 3. a stable polymer (A3) of more than M_r 4·10⁶. The arylamidase was optimally active at pH 7.3 and did not require metal ions. Treatment with 1,10-phenanthroline resulted in complete inactivation, the activity could be restored by the addition of manganous chloride. The sulphhydryl-blocking reagent 4-hydroxymercuribenzoate strongly inactivated the arylamidase, this inhibition could be reversed by the addition of 2-mercaptoethanol. Addition of phenylmethylsulfonyl fluoride had no effect on the enzyme activity. Furthermore, the influence of metal ions as well as the substrate specificity were investigated and compared for all three forms of arylamidase.     

Topology of glutathione-insulin transhydrogenase in rat liver microsomes

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent (‘inactive’) state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A₂ and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A₂ resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A₂ is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.     

The uptake of R-type cobalamin-binding protein by isolated rat liver cells

The uptake of R-type cobalamin-binding protein from human granulocytes and plasma by isolated parenchymal rat liver cells has been studied. When [⁵⁷Co]cyanocobalamin-saturated granulocyte-binding protein or transcobalamin III was incubated with the liver cells in a concentration of 500 pM, more than 80% of the vitamin was taken up in 1 h. Vitamin B-12 bound to plasma transcobalamin I, however, was not taken up unless the protein was desialylated by neuraminidase from Vibrio cholerae. The uptake of iodinated pure granulocyte-binding protein, saturated with cobalamin, reached 100% and was accompanied by increasing intracellular proteolytic degradation of the binding protein. EGTA and asialo-orosomucoid completely inhibited this process of uptake and degradation, whereas partial inhibition was caused by chloroquine and colchicine. These observations provide evidence that these (asialo)-R-type cobalamin-binding proteins are taken up by the cell through the plasma membrane receptor for asialoglycoproteins by means of endocytosis followed by proteolysis of the binding protein in the lysosomes.     

Resolutions and identification of the core deoxynucleoproteins of the simian virus 40

Low molecular weight basic core proteins of SV₄₀ are resolved by Tris-Acetate-SDS polyacrylamide gel electrophoresis into a minimum of four polypeptides. These are the electrophoretic counterparts of the evolutionarily conserved histones F3, F2b, F2a2, and F2a1. Host African green monkey kidney cells contain an active protease activity which readily cleaves histone F3 during nuclear isolation in hypotonic buffers.     

Regulation of protein phosphorylation by polyamines in hepatocytes

The effects of natural aliphatic polyamines on basal and hormone-stimulated protein phosphorylations in hepatocytes were studied. Cells isolated from adult rats were incubated in suspension with [³²P]orthophosphate, in the absence or presence of polyamines at varying concentrations and for different times; hepatocytes were then exposed to various hormones for 10 min. Phosphoproteins contained in total cell lysates were analyzed by one- and two-dimensional gel electrophoresis and autoradiography. Spermine, the most effective amine, decreased the basal level of phosphorylation of proteins with 46, 34 and 22 kDa, and increased that of a 18 kDa protein. These effects, maximal with an external concentration of 7.5–10 mM, were detectable after a lag period of about 10 min and reached a plateau after 45 min. Prereatment of cells with the polyamine almost completely prevented stimulation of the phosphorylation of the 46 and 34 kDa proteins by insulin; in contrast, the effects of phenylephrine on the same proteins were only partly inhibited, whereas those of glucagon appeared largely unaffected. The major polyamine effect observed in intact cells (i.e., decreased phophorylation) could be reproduced in a cell-free system where no kinase activity persisted. Indeed, spermine added directly to cell extracts strongly accelerated dephosphorylation of the 46 kDa protein and also of the 61 kDa protein identified as pyruvate kinase; furthermore, restoration of the activity of this enzyme occurred concomitantly with dephosphorylation of the 61 kDa protein in the presence of spermine.     

Changes in the reactivity of proteins of rat liver ribosomes against [C]iodoacetamide depending on their organization in ribosomal subparticles, 80S ribosomes and on the attachment of poly-(U)

(1) The isolated mixtures of ribosomal proteins can be substituted by [¹⁴C]-iodoacetamide up to an average of about 2 equivalents per 20 000 dalton. The extent of substitution of single proteins measured after two-dimensional polyacrylamide gel electrophoresis shows that all proteins are reactive.

(2) Also in the subunits, all proteins are accessible to substitution. Compared with isolated proteins, however, the reactivity is decreased and the amount of labelling for most proteins ranges as low as 5 to 20%.

(3) Reassociation of ribosomal subunits decreases the reactivity of 12 proteins of the small subunit and that of 20 proteins of the large subunit.

(4) The presence of messenger inhibits the substitution of 10 proteins of the small subunit and of 6 proteins of the large one.

(5) Seven proteins of the small subunit and 3 proteins of the large one are influenced both by the other subunit and by messenger-RNA.     

Erythrocyte membrane cholesterol levels and their effects on membrane proteins

The susceptibility of the band 3 protein of the erythrocyte membrane to proteolytic digestion at either surface of the membrane was not altered when the membrane cholesterol level was increased by 65–103%. Cross-linking of the major membrane proteins by o-phenanthroline · Cu, glutaraldehyde, dimethylsuberimidate and dimethyladipimidate was also unaffected.     

Photosensitized cross-linking of erythrocyte membrane proteins. Evidence against participation of amino groups in the reaction

Exposure of human erythrocyte ghosts (pH 8, 10°C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent.Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with $\text{N-}\text{ethylmaleimide}$, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH₂ groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141–151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH₂ groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH₂ groups are not involved in the reaction.     

Variant forms of matrix protein in Escherichia coli B/r bearing N plasmids

Plasmids of the N incompatibility group have been found to decrease or virtually eliminate the synthesis of the 36 500 dalton outer membrane matrix protein of their Escherichia coli B/r hosts (Iyer, R. (1977) Biochim. Biophys. Acta 470, 258–272 and Iyer, R., Darby, V. and Holland, I.B. (1978) FEBS Lett. 85, 127–132) or modify its composition. Although the 34 000 dalton tol G protein is slightly increased in some strains, it is identical in composition to the homologous protein from the plasmidless host. In three of five N⁺ strains, the synthesis of the modified matrix proteins depends on the temperature of cultivation of the strains in which they occur. The alterations to the matrix proteins are non-identical and do not affect the expression of several plasmid-coded functions including those of sensitivity to the N plasmid-specific filamentous bacteriophage IKe (Khatoon, H. and Iyer, R. (1971) Can. J. Microbiol. 17, 669–675), or their interbacterial transfer via conjugation to appropriate recipient strains. Thus, although the significance of the variant matrix proteins in N⁺ strains with respect to plasmid-mediated functions remains unclear, N plasmids nevertheless provide a convenient system which might be used to elucidate the events that precede the insertion of this protein into the outer membrane of E. coli B/r hosts.     

Catecholamine-induced phosphorylation of cardiac muscle proteins

The catecholamine-induced phosphorylation of cardiac muscle protein was investigated using a rat ventricular muscle slice preparation. Slices 0.5 mm thick and weighing 40–50 mg were incubated for 40 min in oxygenated bathing medium containing ³²P to partially label intracellular ATP. Subsequent addition of 10^?5 M isoproterenol for 10 min resulted in a 44–63% (based on protein) or a 63–70% (based on inorganic phosphate) increase in ³²P incorporation into 100 000 × g particulate and 100 000 × g supernatant (soluble) fractions without an increase into homogenates, 1000 and 29 000 × g particulate fractions prepared from the slices. The catecholamines also produced a 93% increase in ³²P incorporation ans a 27% increase in inorganic phosphate in trichloroacetic acid-insoluble protein that was obtained from ventricular slice homogenates. A significant increase in the incorporation of ³²P occurred in the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein within 2 and 1 min, respectively. While the β-adrenergic blocking agent propanolol had no effect by itself on ³²P incorporation, it prevented the isoproterenol-induced incorporation of ³²P into the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein. Removal of isoproterenol from the bathing medium eliminated the differences in ³²P incorporation, indicating that the effects of the catecholamine were reversible. Norepinephrine and ipinephrine at 10^?5 M caused phosphorylation effects similar to that of isoproterenol. When the slices were bathed under anoxic conditions isoproterenol failed to enhance the incorporation of ³²P into proteins of the 100 000 ×g particulate and supernatant fractions or acid-insoluble protein. SDS gel eloectrophoresis of ventricular slice homogenates revealed that isoproterenol enhanced the ³²P incorporation into several myocardial proteins having molecular weights of 155, 94 (glycogen phosphorylase), 79, 68–77, and 54–59 · 10³ and decreased the incorporation into a 30 · 10³ dalton protein(s). These results are consistent with the notion that catecholamines may increase the phosphorylation of myocardial proteins in the intact myocardium which in turn may play a role in catecholamine-induced glycogenolysis and augmentation of contractility.     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

The synthesis of freezing-point-depressing protein of the winter flounder Pseudopleuronectusamericanus in Xenpuslaevis oocytes

The serum of the winter flounder $\text{Pseudopleuronectus}$$\text{americanus}$ contains a freezing-point-depressing protein of a molecular weight approximately 10,000 with 60% alanine in its composition. When injected into Xenopus o?cyte, a 6–10 S, poly A-rich RNA preparation isolated from the fish liver polysomes stimulated 3–4 fold the incorporation of [³H] alanine into 10% trichloroacetic acid-soluble, non-dialysable proteins. Analysis of the protein fractions showed a translation product similar in molecular weight and electrophoretic mobility to flounder freezing-point-depressing protein. These observations indicated that the 6–10 S RNA from the flounder contained mRNA for the synthesis of flounder's freezing-point-depressing protein.     

Glyphosine,a plant growth regulator,affects chloroplast membrane proteins

Glyphosine (N,N-bis(phosphonomethyl)glycine) is known to increase sucrose levels in sugarcane and to cause chlorosis in maize and other plants. It has been suggested (Crofts, S.M., Arntzen, C.J., Vanderhoef, L.N. and Zettinger, C.S. (1974) Biochim. Biophys. Acta 335, 211–217) that its primary mode of action is to inhibit the synthesis of plastid rRNA. Growth of Lemna gibba L. G-3 on 5 · 10^?4M glyphosine causes the plants to produce fronds lacking chlorophyll. The plastids in these white fronds contain only a few internal membrane structures, some of which are stacked. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows an accumulation of substantial amounts of both the large and small subunits of ribulosebisphosphate carboxylase by the white fronds. The membrane fraction from these fronds contains only traces of the light-harvesting chlorophyll $\text{a}\text{b}$ apoprotein in comparison to control plants. In vivo labeling and immunoprecipitation show that the large subunit of ribulose-bisphosphate carboxylase is actively synthesized by the white fronds. However, labeling of the chlorophyll $\text{a}\text{b}$ apoprotein and a 32000 dalton protein in the membrane fraction is extremely low compared to control plants. We conclude that in Lemna, glyphosine differentially affects the synthesis and/or processing of soluble proteins and some membrane chloroplast proteins, and could be useful in understanding the biogenesis of chloroplast membranes.     

Characterization of acetylcholine receptor isolated from Torpedo californica electroplax through the use of an easily removable detergent, β-d-octylglucopyranoside

Non-ionic detergents used for the solubilization and purification of acetylcholine receptor from Torpedo californica electroplax may remain tightly bound to this protein. The presence of detergent greatly hinders spectrophotometric and hydrodynamic studies of the receptor protein. β-d-Octylglucopyranoside, however, is found to be effective in solubilizing the receptor from electroplax membranes with minimal interference in the characterization of the protein. The acetylcholine receptor purified from either octylglucopyranoside or Triton X-100-solubilized extracts exhibits identical amino acid compositions, α-Bungarotoxin and (+)-tubocurarine binding parameters, and subunit distributions in SDS-polyacrylamide gels. The use of octylglucopyranoside allows for the assignment of a molar absorptivity for the purified receptor at 280 nm of approx. 530 000 $\text{M}{}^{\mathrm{?1}}\text{·}\text{cm}{}^{\mathrm{?1}}$. Additionally, successful reconstitution of octylglucopyranoside-extracted acetylcholine receptor into functional membrane vesicles has recently been achieved (Gonzales-Ros, J.M., Paraschos, A. and Martinez-Carrion, M. (1980) Proc.Natl. Acad. Sci. U.S.A. 77, 1796–1799).Removal of octylglucopyranoside by dialysis does not alter the specific toxin and antagonist binding ability of the receptor or its solubility at low protein concentrations. Sedimentation profiles of the purified acetylcholine receptor in sucrose density gradients reveal several components. Sedimentation coefficients obtained for the slowest sedimenting species agree with previously reported molecular weight values. Additionally, the different sedimenting forms exhibit distinctive behavior in isoelectric focusing gels. Our results suggest that both the concentration and type of detergent greatly influence the physicochemical behavior of the receptor protein.