Characterization of the association of Electrophorus electricus acetylcholinesterase with sphingomyelin liposomes. Relevance to collagen-sphingomyelin interactions

Electrophorus electricus acetylcholinesterase is a large polymorphic enzyme. Its native forms 18 S, 14 S and 8.5 S possess a tail having a collagen-like structure. It was suggested that this tail is involved in the anchorage of the enzyme at the terminal of the synapse. Watkins et al. [1] showed that all forms of the enzyme having a collagen segment also bind to sphingomyelin liposomes with almost no binding to phosphatidylcholine (PC) liposomes. In agreement with the above results, the binding of acetylcholinesterase reported here was independent of the following liposomal parameters (a) curvature, (b) the physical state of the bilayer, (c) the gel to liquid crystalline phase transition of sphingomyelin, (d) stereospecificity of the sphingomyelin, (e) acyl chain of the sphingomyelin. The binding was reduced with increasing PC content in sphingomyelin vesicles. The binding has no effect on the bilayer integrity. The enzymatic activity can be released from the vesicles by incubation with collagenase. The association of the enzyme with the liposomes had minimal effect on its kinetic parameters (Km, Vmax). The only detectable effect was increasing enzyme stability at low enzyme concentration. This suggested that the binding of the enzyme to sphingomyelin liposomes reduced its surface denaturation. Such association was not unique to acetylcholinesterase since collagen showed similar behavior. Collagen binding to sphingomyelin liposomes was 5-10-times larger than to PC liposomes. The exact details of the interaction of collagen and collagen-like peptides with sphingomyelin bilayers are yet unknown although it differs from the well documented hydrophobic or electrostatic interactions [7]. This work proposes hydrogen bonding as a third mechanism which involves the interface region of sphingolipids molecules and the collagen or collagen-like tail of acetylcholinesterase. This binding is also of interest due to its correlation to the accumulation of sphingomyelin and collagen during aging and the development of atherosclerosis in blood vessels of mammals.     

Acetylcholinesterase: characterization of native and proteolytically derived forms and identification of structural protein components.

The assymmetric 18S and 14S forms of acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus purified by affinity chromatography on N-methylacridinium Sepharose 2B were subjected to trypsin or collagenase proteolysis and changes in the enzyme composition and structure were monitored by sucrose gradient sedimentation, gel chromatography, and sodium dodecyl sulphate - polyacrylamide gel electrophoresis. A distinction between autolytic and tryptic degradation products is described and the generation of two new forms of acetylcholinesterase from the 18S and 14S enzyme by collagenase proteolysis is reported. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.1S and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The proteolytically sensitive component ('tail') of the asymmetric forms of acetylcholinesterase is identified with a subunit of 45 000 daltons on sodium dodecyl sulphate - polyacrylamide electrophoresis gels.     

Regulation of protein synthesis in rabbit reticulocyte lysates: Separation of heme regulated protein kinase into a high and a low molecular weight species

Protein synthesis in rabbit reticulocyte lysates is regulated by heme. In heme deficiency, a heme regulated protein kinase (HRI) is activated that phosphorylates initiation factor eIF-2. Consequently, eIF-2 is inactivated. Results described in this report show that HRI exists in crude and highly purified preparations in two forms; a high molecular weight component which sediments at a sedimentation co-efficient of 14–15S and a previously described 5.8S component (Ranu, R. S. and London, I. M. (1976) $\text{Proc}\text{.}\text{Natl}\text{.}\text{Acad}\text{.}\text{Sci}\text{.}\text{USA}$ 73, 4349–4353). The 14–15S HRI selfphosphorylates poorly and undergoes dissociation into the 5.8S component via an intermediate of 8.5–9S. The 5.8S HRI, on weight basis, is about 5–10 times more active than the 14–15S HRI. In addition, a phosphoprotein phosphatase has been detected in lysates that dephosphorylates selfphosphorylated HRI. This observation suggests that phosphate on HRI turns over. These findings may be relevant ot the mechanism of activation and inactivation of HRI in the absence and presence of heme $\text{in}\text{situ}$.     

Effect of membrane fluidity upon binding of Electrophorus acetylcholinesterase to lipid vesicles

A previous report (Watkins, M.S., Hitt, A.S. and Bulger, J.E. (1977) Biochem. Biophys. Res. Commun. 79, 640-647) has indicated that the asymmetric forms of Electrophorus acetylcholinesterase bind exclusively to sphingomyelin vesicles through interaction with the collagen-like 'tail' portion of the enzyme. We report here that acetylcholinesterase also binds to phosphatidylcholine vesicles containing saturated fatty acyl chains and to egg phosphatidylcholine vesicles containing cholesterol. This suggests preferential binding of acetylcholinesterase to membranes of lower fluidity. Surface charge of vesicles and density of zwitterionic lipid headgroups do not significantly affect binding of native acetylcholinesterase. The presence of chondroitin sulfate or hyaluronic acid slightly increases the binding of native acetylcholinesterase to sphingomyelin vesicles, while the presence of 1 M NaCl, bovine serum albumin, or tissue fractions enriched in basement membrane diminish binding. The dissociation constant for native acetylcholinesterase and sphingomyelin vesicles is (1.0-1.5) X 10(-7) M, as measured by a flotation binding assay. The globular, 11S form of acetylcholinesterase also binds to lipid vesicles, although not to the same degree as native acetylcholinesterase. This suggests that the collagen tail of the enzyme enhances binding, but is not essential for binding to occur. These results are consistent with the location of acetylcholinesterase on the surface of the postsynaptic plasma membrane in vivo.     

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.     

Possible interrelationship of multiple alpha-bungarotoxin binding components in the brain of the horseshoe crab, Limulus polyphemus.

Three $\text{[}{}^{125}\text{I]α-bungarotoxin}$ binding components have been detected in solubilized extracts of $\text{Limulus}$ brain tissue. These components have sedimentation coefficients of 9.0S, 15.4S and 17.4S. All three components were degraded by α-chymotrypsin. The addition of butyl alcohol to brain extract suggests an interrelationship of the toxin binding proteins by promoting a simultaneous decrease in the 9S component and increase in the 15.4S and 17.4S components. This transition was also demonstrated by the readdition of isolated fractions of each component to brain extract.     

Immunological cross-reactivity between electric-eel acetylcholinesterase and rat-tail-tendon collagen.

Immunological cross-reactivity between acetylcholinesterase from the electric organ of the electric eel and rat tail tendon collagen was examined both on the cellular and humoral levels. 1. Guinea pigs immunized with rat tail tendon collagen displayed a strong delayed-type skin reaction when tested with the elongated acetylcholinesterase preparation (i.e. 14-S + 18-S molecular forms). However, when the glubular 11-S enzyme was tested, almost no cross-reactivity was obtained. Similarly, guinea pigs immunized with 14-S + 18-S preparation exhibited skin sensitization to rat tail tendon collagen. 2. Using a radioimmunoassay, it was observed that 125I-labeled 14-S + 18-S acetylcholinesterase binds efficiently to rabbit antiserum elicited against rat tail tendon collagen, whereas 125I-labeled 11-S enzyme does not bind at all to this antiserum. Similar results were obtained by passive hemagglutination assay. The experiments suggest that 14-S + 18-S acetylcholinesterase, but not 11-S enzyme, which is devoid of the tail structure, has antigenic determinants in common with collagen from rat tail tendon.     

Pig liver fatty acid synthetase: purification and physicochemical properties.

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 mm dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of S^o_20,w = 13.3 S and D^o_20,w = 2.60 × 10^?7cm²/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10^?7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of $\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{s}\text{D}\text{) = 478,000}$ and sedimentation equilibrium data yielded a value of M_w = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.     

Translation of mouse interferon mRNA in Xenopus laevis oocytes and in rabbit reticulocyte lysates

Mouse interferon mRNA, extracted from NDV (Newcastle disease virus)-induced L-929 cells has been translated with high efficiency in $\text{Xenopus laevis}$ oocytes and rabbit reticulocyte lysates. The translational efficiency of a crude RNA extract was 10 640 interferon units/mg RNA/hour for the $\text{Xenopus}$ oocytes and 4 012 interferon units/mg RNA/hour for the reticulocyte lysates. The translation product fulfilled the usual criteria for mouse interferon, $\text{viz.}$ species specificity and neutralization by specific anti-mouse interferon antiserum. Upon injection of crude interferon mRNA into $\text{Xenopus}$ oocytes, interferon activity appeared both in the oocyte homogenates and the oocyte incubation medium. When analyzed by velocity sedimentation in formamidesucrose, the mouse interferon mRNA showed a rather sharp peak halfway between the 4 S and 18 S RNA markers, as could be expected from a mRNA which codes for a 20,000 dalton protein.     

Purification of native forms of eel acetylcholinesterase: active site determination

The 14 and 18 S forms of acetylcholinesterase from the electric organ of Electrophorus electricus were purified by chromatography on an N-methyl-3-aminopyridinium derivative of Affi-Gel 202. a further increase in purity was seen when these forms were separated by density gradient sedimentation subsequent to the affinity step. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated that the 14 and 18 S forms were highly purified following these procedures. Using [³H]diisopropyl fluorophosphate labeling and separation of labeled enzyme from unreacted [³H]diisopropyl fluorophosphate by gel filtration, active site numbers of 8.3 and 11.4 were determined for the 14 and 18 S forms, respectively. These numbers compare to 4.2 active sites determined for the 11.8 S globular form of acetylcholinesterase. These results are in accord with a proposed model of two and three tetrameric structures comprising the head groups of the 14 and 18 S forms of electric tissue acetylcholinesterase.     

Muscle and nerve in muscular dysgenesis in the mouse at birth: Sprouting and multiple innervation

Muscular dysgenesis (mdg) in the mouse is a recessive autosomal mutation affecting the striated musculature: during the whole gestation period, the muscles never show any sign of contractile activity. They are cytologically immature at birth, although the diaphragm is more mature than limb muscles, as confirmed by the levels of creatine phosphokinase. In both limb muscles and diaphragm the cytochemical localization of acetylcholinesterase demonstrates focal accumulations on the entire surface of $\text{mdg}\text{mdg}$ muscles, whereas such foci of acetylcholinesterase activity are restricted to a narrow end plate-rich region in $\text{+}\text{mdg}$? diaphragms. Teased single $\text{mdg}\text{mdg}$ myofiber preparations show that one myofiber can possess several foci of acetylcholinesterase, generally presenting aspects of very immature motor end plates. A study of the motor innervation, after silver nitrate impregnation, provides evidence for a spectacular overgrowth and a generalized sprouting of $\text{mdg}\text{mdg}$ nerves and axons. The $\text{mdg}\text{mdg}$ nerve terminals are generally very immature-looking, with an intense ultraterminal sprouting. Aspects suggesting a denser multiple innervation of $\text{mdg}\text{mdg}$ than $\text{+}\text{mdg}$? myofibers have been observed and choline acetyltransferase activity is increased in $\text{mdg}\text{mdg}$ tissues. Acetylcholinesterase specific activity and the number of α-bungarotoxin binding sites per milligram protein increased in $\text{mdg}\text{mdg}$ compared to $\text{+}\text{mdg}$? diaphragms. The very low amount of 16 S (and 12 S) acetylcholinesterase is probably related to $\text{mdg}\text{mdg}$ muscle inactivity. If the cytological and biochemical data are compared, it seems possible to propose that $\text{mdg}\text{mdg}$ myofibers and axons are in contact in several regions of the same myofiber, in variably mature appositions, and with a very dense multi-innervation.     

A general and rational approach to the optimal recovery of mitochondria by differential centrifugation in homogenous media.

The sedimentation coefficients ($\text{S}$-values) of mitochondria have been determined in isotonic sucrose media for different mammalian tissues. Since the values show a relatively great variation, a general and rational approach to the optimal recovery of mitochondria from any tissue by differential centrifugation is presented. The method is primarily based on the determination of the average sedimentation coefficient at the actual isolation conditions. From the $\text{S}$-value thus determined and known centrifugation parameters, a specific procedure can be designed in each case.     

Molecular weight and symmetry of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The molecular weight and polypeptide chain stoichiometry of the native pyruvate dehydrogenase multienzyme complex from Escherichia coli were determined by independent techniques. The translational diffusion coefficient (D^o_20,w) of the complex was measured by laser light intensity fluctuation spectroscopy and found to be 0.90 (±0.02) × 10^?11m²/s. When this was combined in the Svedberg equation with the measured sedimentation coefficient (s^o_20,w = 60.2 (±0.4) S) and partial specific volume ($\text{v}\text{?}\text{= 0.735 (±0.01)}\text{ml/g}$), the molecular weight of the intact native complex was calculated to be 6.1 (±0.3) × 10⁶. The polypeptide chain stoichiometry (pyruvate decarboxylase: lipoate acetyltransferase: lipoamide dehydrogenase) of the same sample of pyruvate dehydrogenase complex was measured by the radioamidination technique of Bates et al. (1975) and found to be 1.56:1.0:0.78.From this stoichiometry and the published polypeptide chain molecular weights estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, a minimum chemical molecular weight of 283,000 was calculated. This structure must therefore be repeated approximately 22 times to make up the native complex, a number which is in good agreement with the expected repeat of 24 times if the lipoate acetyltransferase core component has octahedral symmetry. It is consistent with what appears in the electron microscope to be trimer-clustering of the lipoate acetyltransferase chains at the corners of a cube. It rules out any structure based on 16 lipoate acetyltransferase chains comprising the enzyme core.The preparation of pyruvate dehydrogenase complex was polydisperse: in addition to the major component, two minor components with sedimentation coefficients (s^o_20,w) of 90.3 (±0.9) S and 19.8 (±0.3) S were observed. Together they comprised about 17% of the total protein in the enzyme sample. Both were in slowly reversible equilibrium with the major 60.2 S component but appeared to be enzymically active in the whole complex reaction. The faster-sedimenting species is probably a dimer of the complex, whereas the slower-sedimenting species has the properties of an incomplete aggregate of the component enzymes of the complex based on a trimer of the lipoate acetyltransferase chain.     

Organophosphate toxicity: Kinetic differences between acetylcholinesterase of the housefly thorax and head?

Acetylcholinesterases from the thoraces and heads of the same houseflies were solubilized by autolysis and purified by affinity chromatography. The highest specific activities obtained were 812 units/mg for thoracic and 890 units/mg for head acetylcholinesterase. The enzymes from both sources consist of a single 7.4S form and have identical subunit structures, Michaelis constants, and kinetic constants for inhibition by the organophosphate paraoxon. $\text{In vivo}$ it has previously been shown that organophosphates preferentially inhibit the thoracic acetylcholinesterase. We conclude this is unlikely the consequence of biochemical differences between the enzyme from the two regions.     

Identification of discrete disulfide-linked oligomers which distinguish 18 S from 14 S acetylcholinesterase

Acetylcholinesterase (EC 3.1.1.7) purified by affinity chromatography from 1.0 m ionic strength extracts of electric organ from the eel Electrophorus electricus consists of a mixture of 18 and 14 S enzyme forms. When examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate without exposure to disulfide reducing agents, these purified preparations show two major high molecular weight bands (>300,000), labeled oligomers A and B, in addition to a major band corresponding to catalytic subunit dimers (150,000 M_r). All these major bands reflect intersubunit disulfide bonding. The 18 and 14 S forms in purified preparations were separated by extensive sucrose gradient centrifugation. Gel analyses of the isolated 18 and 14 S pools indicated that the larger oligomer A derives from the 18 S pool, while oligomer B is found primarily in the 14 S pool. These observations support a previous model for 18 S acetylcholinesterase (T. L. Rosenberry and J. M. Richardson (1977) Biochemistry, in press) which considers this molecule to consist of one oligomer A unit, composed of three pairs of catalytic subunits disulfide-bonded to a collagen-like tail structure, and three catalytic subunit dimers. Proteolytic cleavage of the tail structure in the 18 S form can occur to release an 11 S enzyme tetramer containing a residual tail fragment and to leave a 14 S form. We propose this 14 S form to consist of one oligomer B unit, composed of two pairs of catalytic subunits disulfide-bonded to the remaining tail structure, and two catalytic subunit dimers.     

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-¹⁴C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the $\text{in situ}$ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in $\text{vitro}$ by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.     

Isolation of a mitochondrial DNA topoisomerase from human leukemia cells

Mitochondria from human acute lymphoblastic leukemia cells contain an ATP-independent DNA topoisomerase which can relax negative and positive supercoils. This enzyme has been purified 200-fold by carboxymethyl-cellulose or double stranded DNA-cellulose chromatography. In contrast to the molecular weights reported for mitochondrial topoisomerases in other systems, the native leukemia enzyme has a molecular weight of 132,000 daltons as determined by gel permeation chromatography in buffer containing 0.4 M KCl. It also exhibits a sedimentation coefficient of 7.1 S when centrifuged through a 10–30% glycerol gradient in this high salt buffer. The enzyme is presumably a type I topoisomerase analogous to those found in rat liver and $\text{Xenopus}$$\text{laevis}$ mitochondria.     

Interaction of A-nor A.19-dinor,and A-homo-5α-androstane derivatives with the androgen receptor and the epididymal androgen-binding protein

The equilibrium affinity constant for rat prostate androgen receptor and epididymal androgen binding protein (ABP) has been determined for thirty-four potential progestogens. Three A-nor-, four A,19-dinor-, and one A-homo-5α-androstane derivative bind to the androgen receptor (K_D<0.5 μM). Five of these compounds also bind to ABP with an affinity of the same order of magnitude. “Anordrin” (compound $\text{24}$) and “Dinordrins” (compounds $\text{10, 14, 15, 16, 17}$), which are potential female contraceptives, do not bind with high affinity to the androgen receptor or to ABP. The following modifications in A-nor derivatives favour binding to the receptor as compared to ABP: 19-nor substitution (compound $\text{1}$), C-18 methyl homologation (compound $\text{5}$), 2α-ethinylation (compound $\text{22}$). One 2α-allenyl A-nor derivative (compound $\text{25}$) and one A-homo derivative (compound $\text{34}$) bind almost exclusively to ABP. The interaction with either binding protein is decreased by oxidation or esterification of the hydroxyl group at C-17, and by addition of a 17 α-ethinyl group. The latter modifications are likely to increase the specificity of androstane derivatives for receptors other than androgen binding proteins, such as the progesterone receptor.     

Evidence of the presence of a latent active site in the large subunit of rat kidney gamma-glutamyl transpeptidase.

γ-Glutamyl transpeptidase (EC 2.3.2.2) of rat kidney is composed of two nonidentical polypeptide chains, the small and large subunits. The active site of this enzyme has previously been shown to be located in the small subunit [Inoue, M., Horiuchi, S. &; Morino, Y. (1977) Eur, J. Biochem. $\text{73}$, 335–342; Tate, S. S. &; Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. $\text{74}$, 931–935] The denaturation of this oligomeric enzyme in 6 M urea, followed by chromatography on a Sephadex G-150, resulted in the separation of the large and small subunits. The removal of urea gave rise to an enzymatically active preparation from the denatured large subunit. Under several renaturation conditions, the small subunit polypeptide chain did not exhibit the enzymatic activity. Upon incubation with 6-diazo-5-oxo-L-[1,2,3,4,5-¹⁴C]norleucine, an affinity label for γ-glutamyl transpeptidase, the renatured preparation of the large subunit was covalently labeled with the affinity label with concomitant loss of the enzymatic activity. When the native enzyme was inactivated by the ¹⁴C-affinity label, radioactivity was selectively incorporated into the small subunit. These findings indicate that the isolated large subunit possesses an active site which is masked in the native state of the enzyme.     

RNA-protein interactions in the ribosome. II. Binding of ribosomal proteins to isolated fragments of the 16 S RNA

Specific fragments of the 16 S ribosomal RNA of Escherichia coli have been isolated and tested for their ability to interact with proteins of the 30 S ribosomal subunit. The 12 S RNA, a 900-nucleotide fragment derived from the 5′-terminal portion of the 16 S RNA, was shown to form specific complexes with proteins S4, S8, S15, and S20. The stoichiometry of binding at saturation was determined in each case. Interaction between the 12 S RNA and protein fraction $\text{S}\text{16}\text{S}\text{17}$ was detected in the presence of S4, S8, S15 and S20; only these proteins were able to bind to this fragment, even when all 21 proteins of the 30 S subunit were added to the reaction mixture. Protein S4 also interacted specifically with the 9 S RNA, a fragment of 500 nucleotides that corresponds to the 5′-terminal third of the 16 S RNA, and protein S15 bound independently to the 4 S RNA, a fragment containing 140 nucleotides situated toward the middle of the RNA molecule. None of the proteins interacted with the 600-nucleotide 8 S fragment that arose from the 3′-end of the 16 S RNA.When the 16 S RNA was incubated with an unfractionated mixture of 30 S subunit proteins at 0 °C, 10 to 12 of the proteins interacted with the ribosomal RNA to form the reconstitution intermediate (RI) particle. Limited hydrolysis of this particle with T₁ ribonuclease yielded 14 S and 8 S subparticles whose RNA components were indistinguishable from the 12 S and 8 S RNAs isolated from digests of free 16 S RNA. The 14 S subparticle contained proteins S6 and S18 in addition to the RNA-binding proteins S4, S8, S15, S20 and $\text{S}\text{16}\text{S}\text{17}$. The 8 S subparticle contained proteins S7, S9, S13 and S19. These findings serve to localize the sites at which proteins incapable of independent interaction with 16 S RNA are fixed during the early stages of 30 S subunit assembly.