The folding of ovalbumin. Renaturation in vitro versus biosynthesis in vitro.

Hen ovalbumin, the major secretory product of oviduct cells, is a 43 000-dalton glycoprotein. Many studies have led to controversy over the question of whether ovalbumin (OA) can be fully renatured after chemical denaturation. We have studied the renaturation of OA after denaturation with guanidinium chloride, urea or alkaline pH. Denatured OA displays an intrinsic viscosity consistent with nearly complete unfolding of the protein. Removal of the denaturant results in a complete reversal of the changes in intrinsic viscosity. However, closer examination of the renatured protein reveals major differences from the native form. Renatured OA (OAR) can be completely separated from the native form (OAN) by affinity chromatography on phenyl-Sepharose. OAR displays altered tryptophan fluorescence, u.v.-absorption and c.d. spectra. Only OAR binds anilinonaphthalenesulphonate (as measured by fluorescence enhancement). OAR, but not OAN, binds about 2 mol of the covalent hydrophobic affinity probe phenyl isothiocyanate/mol. Renaturation, and the production of OAR, occurs regardless of the oxidation state of the disulphide bonds, of phosphorylation of the protein, and of the presence or the absence of the single carbohydrate chain. OAR may be either monomeric or an irreversible aggregate. Which of these two states is formed depends on the protein concentration during renaturation. Monomeric and aggregated OAR can be distinguished on the basis of some spectroscopic characteristics, but they share the essential hydrophobic characteristics that distinguish them from OAN. OAN and OAR do not spontaneously interconvert. Antibodies raised to each can be made monospecific by immunoabsorption. Thus two stable forms of OA can be obtained, one of which, OAR, displays hydrophobic characteristics. OAN, but not OAR, is formed when OA is synthesized in vitro in a translation system.     

In vitro genetics.

Understanding the basis of specificity in an intermolecular interaction is a common if difficult task; designing a specific intermolecular interaction is much more challenging. A new technique is described that has applications to both problems, at least with regard to nucleic acids. The power of this method lies in its ability to isolate extremely rare sequences with precisely specified properties from very large pools of random sequences.     

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.     

In vitro secondary MLR. I. Kinetics of proliferation and specificity of in vitro primed responder cells.

We have examined the kinetics and specificity of secondary in vitro mixed lymphocyte reactions (MLR). With limited numbers of primed responder cells (PRC) in the presence of "excess antigen" it was possible to obtain proliferative responses that were proportional to the number of PRC initially placed in culture. The responding cells, after an initial lag period, seem to grow exponentially until day 3 of culture. The responses of PRC (with the strain combinations and culture conditions described in this report) seemed to be directed toward stimulator cell determinants whose expression was determined by genes in the I region of the MHC. In one case, the relevant incompatibilities could be further restricted to the I-A region. Although PRC responded best to stimulator cells sharing the I region with the priming stimulator cell, apparent cross-reactivity could be observed by restimulating PRC with stimulator cells that did not carry the MHC haplotype of the priming stimulator cell. The rate of proliferation (measured as 3H-thymidine incorporation) in these apparent cross-reactions was reproducible and comparable to the rate observed in response to the priming stimulator cell. It was possible, therefore, to estimate the proportion of PRC that reacted in the presence of third party stimulator cells compared to the response of these PRC to the priming stimulator cells. We have estimated that the response of A (B6) PRC against H-2d and H-2s haplotype stimulator cells is about half of the response of these PRC to H-2b, the priming stimulator cell.     

In vitro growth of cytotoxic human lymphocytes. I. Growth of cells sensitized in vitro to alloantigens.

Human lymphocytes sensitized to allogeneic determinants in vitro were continuously cultured on medium prepared from phytohemagglutinin-(PHA-P) stimulated human mixed lymphocyte cultures. With such human-conditioned medium (HCM), human peripheral lymphoid cells could be grown in culture for over 90 days with a doulbing time of about 54 hr. Cytotoxic lymphocytes could be grown in culture for this time with no loss of cytotoxicity or change in cytotoxic specificity.     

Labelling of cardiolipin in vitro.

A method for labelling the polar head groups of cardiolipin is described. Labelling was carried out on sonicated cardiolipin/water suspensions. The free hydroxyl group of cardiolipin was oxidised with an excess of p-(diazonium) benzenesulfonic acid (DABS) and then reduced with NaB3H4. Isopropanol was oxidised in the presence of DABS to test the reactivity of the diazonium salts, and the reaction product was analysed by means of gas-chromatography. Labelled cardiolipin, identified by thin-layer chromatography (TLC), was chromatographically pure and identical to untreated cardiolipin. The hydrolysis of cardiolipin confirmed that the labelling was at the level of polar head groups.     

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.     

In vitro growth of murine T cells. II. Growth of in vitro sensitized cells cytotoxic for alloantigens.

Growth factors (GM), produced by murine lymphoid cells incubated with Concanavalin A, have been used to grow cytotoxic lymphoid cells in culture. C57BL/6 and DBA/2 lymphoid cells were sensitized against each other in primary, secondary, and tertiary in vitro cultures. These sensitized cells were grown in vitro in GM and retained their cytotoxic properties. Cells grew in culture about 10-fold every 5 to 7 days for over 2 months. Initial growth of cytotoxic cells in GM resulted in marked enhancement of specific cytotoxicity that returned to original levels after subsequent subcultures. After five 10-fold cell culture generations some nonspecific cytotoxicity directed against the responding target cell strain appeared in continuous cultures. This technique for growing large numbers of cytotoxic cells may be of value in the development of adoptive immunotherapies.     

Protein folding in vitro.

It is becoming increasingly evident that intermediates observed in protein folding in vitro may be closely related to conformational states that are important in various intracellular processes. This review focuses on recent advances in in vitro protein-folding studies with particular reference to the molten globule state, which is purported to be a common and distinct intermediate of protein folding.     

Disulfide-bonded dimerization of fibronectin in vitro.

Human plasma fibronectin was denatured with 8 M urea and reduced with dithiothreitol. Dialysis or dilution of the solution led to formation of fibronectin dimers which migrated in non-reducing SDS/PAGE similarly to untreated control protein. When the redimerized fibronectin was reduced and re-electrophoresed it formed a doublet of alpha and beta chains of equal intensity indicating that it was a heterodimer. Low concentrations (less than 1 mM) of Fe3+ enhanced the redimerization of fibronectin, suggesting that metal ions may mediate oxidative reactions in the formation of the disulfides. Consequently, redimerization of fibronectin was completely prevented by deferoxamine, an iron chelator. Dimerization of fibronectin took place most effectively at pH greater than or equal to 8.8 but decreased strongly at lower pH, representing more unfavourable conditions for the action of the thiolate anion in the thiol/disulfide exchange reaction. Redimerized fibronectin, however, lost many of its binding properties to macromolecular ligands, suggesting that the disulfide bonding did not entirely regenerate the proper conformation of the protein. Pulse/chase experiments of fibroblast cultures showed that the initially monomeric fibronectin was rapidly and quantitatively dimerized under conditions representing natural pH and environment. SDS/PAGE analysis of the dialyzed urea-denatured/reduced thrombin and plasmin digests of fibronectin revealed that the NH2-terminal 30-kDa fragment and other fragments that contained intrachain disulfides quantitatively regained their non-reduced electrophoretic mobility. The results show that the dimerization and formation of intrachain disulfides of fibronectin may occur, in part, spontaneously, based on the amino acid sequence information of the protein. However, complete disulfide formation may also need other factors, present only in living cells, as suggested by pulse/chase experiments in fibroblasts.     

Comparative cytotoxicity of phenols in vitro.

Two melanotic human melanoma cell lines, IRE 1 and IRE 2, and the lymphoma- and leukaemia-derived cell lines Raji and K 562, were exposed to different concentrations (from 5 X 10(-3) M to 10(-5) M) of phenols, both substrates (s) and non-substrates (ns) of tyrosinase, in the presence or absence of the oxygen-radical-scavenger enzymes superoxide dismutase, catalase and peroxidase. Monophenols were tyrosine (s), 4-hydroxyanisole (s) and butylated hydroxyanisole (ns); diphenols were L-3,4-dihydroxyphenylalanine (s), dopamine (3,4-dihydroxyphenethylamine) (s), terbutylcatechol (s), hydroquinone (s) and resorcinol (ns); triphenols were 6-hydroxydopa (3,4,6-trihydroxyphenylalanine) (s) and methyl gallate (s). Triphenols and o- and p-diphenols underwent complete oxidation in culture medium within 24 h of incubation and were significantly more toxic than monophenols and the m-diphenol resorcinol, which, under the same cultural conditions, were much more stable. No significant differences in percentage survival were found among the different cell lines for each drug tested. The major component of toxicity up to 24 h of di- and tri-phenols is due to toxic oxygen species acting outside the cells and not to cellular uptake of these phenols as such. In fact the addition of oxygen-radical-scavenger enzymes significantly (P less than 0.01) decreased the adverse effect of these drugs on all cell lines. The lower toxicity of monophenols and resorcinol as compared with that of di- and tri-phenols is due, in our opinion, to the fact that they are less oxidized under the conditions existing in the culture medium, and therefore do not produce sufficient levels of oxygen radicals. For these compounds, a primary intracellular action has to be taken into account to explain their cytotoxicity.     

Cloning of RNA molecules in vitro.

A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purified Q beta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized medium results in the formation of separate 'colonies', each comprising the progeny of a single RNA molecule (a clone). The colonies were visualized by staining with ethidium bromide, by utilizing radioactive substrates, and by hybridization with sequence-specific labeled probes. The number and identity of the RNA colonies corresponded to that of the RNAs seeded. When a mixture of different RNA species was seeded, these species were found in different colonies. Possible implementations of this technique include a search for recombinant RNAs, very sensitive nucleic acid diagnostics, and gene cloning in vitro.     

In vitro synthesis of ornithine transcarbamylase.

An in vitro system for the synthesis of ornithine transcarbamylase (OTCase) was established using iS-30 extract from E. coli MDS6-2(lambda) and DNA of a lambda transducing phage carrying argI and argF genes. This in vitro synthesis was completely dependent on the additon of DNA, and was sensitive to chloramphenicol and rifampicin. Radioisotopic analysis confirmed that the synthesized enzyme catalyzes the carbamylation of ornithine to citrulline. In the in vitro system the repression and derepression of OTCase synthesis could be observed by mixing iS-30 extracts prepared from argR+ and argR- cells. A remarkable maturation effect could be observed for the FFF enzyme, but not for the III enzyme. This system is considered to reflect the in vivo situation, and should therefore be useful for investigations on the regulation of OTCase synthesis in vivo.     

In vitro assembly of gap junctions.

Gap junction structures were assembled in vitro from octyl-beta-D-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.