RESUMO
Using reverse phase HPLC, we have been able to quantify the protein compositions of reconstituted 30S ribosomal subunits, formed either with the full complement of 30S proteins in the reconstitution mix or with a single protein omitted. We denote particles formed in the latter case as SPORE (single protein omission reconstitution) particles. An important goal in 30S reconstitution studies is the formation of reconstituted subunits having uniform protein composition, preferably corresponding to one copy of each protein per reconstituted particle. Here we describe procedures involving variation of the protein:rRNA ratio that approach this goal. In SPORE particles the omission of one protein often results in the partial loss in uptake of other proteins. We also describe procedures to increase the uptake of such proteins into SPORE particles, thus enhancing the utility of the SPORE approach in defining the role of specific proteins in 30S structure and function. The losses of proteins other than the omitted protein provide a measure of protein:protein interaction within the 30S subunit. Most of these losses are predictable on the basis of other such measures. However, we do find evidence for several long-range protein:protein interactions (S6:S3, S6:S12, S10:S16, and S6:S4) that have not been described previously.
Assuntos
Proteínas Ribossômicas/análise , Ribossomos/química , Cromatografia Líquida de Alta Pressão/métodos , Técnicas In Vitro , Substâncias Macromoleculares , Conformação Proteica , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismoRESUMO
alpha(1) Adrenergic receptors mediate both vascular and lower urinary tract tone, and alpha(1) receptor antagonists such as terazosin (1b) are used to treat both hypertension and benign prostatic hyperplasia (BPH). Recently, three different subtypes of this receptor have been identified, with the alpha(1A) receptor being most prevalent in lower urinary tract tissue. This paper explores 4-aryldihydropyrimidinones attached to an aminopropyl-4-arylpiperidine via a C-5 amide as selective alpha(1A) receptor subtype antagonists. In receptor binding assays, these types of compounds generally display K(i) values for the alpha(1a) receptor subtype <1 nM while being greater than 100-fold selective versus the alpha(1b) and alpha(1d) receptor subtypes. Many of these compounds were also evaluated in vivo and found to be more potent than terazosin in both a rat model of prostate tone and a dog model of intra-urethral pressure without significantly affecting blood pressure. While many of the compounds tested displayed poor pharmacokinetics, compound 48 was found to have adequate bioavailability (>20%) and half-life (>6 h) in both rats and dogs. Due to its selectivity for the alpha(1a) over the alpha(1b) and alpha(1d) receptors as well as its favorable pharmacokinetic profile, 48 has the potential to relieve the symptoms of BPH without eliciting effects on the cardiovascular system.
Assuntos
Antagonistas Adrenérgicos alfa/síntese química , Pirimidinonas/síntese química , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/farmacocinética , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Disponibilidade Biológica , Células CACO-2 , Cristalografia por Raios X , Cães , Humanos , Masculino , Hiperplasia Prostática/tratamento farmacológico , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 1/metabolismo , Relação Estrutura-AtividadeRESUMO
[reaction: see text] Synthesis of the 8-amino-5,6,7,8-tetrahydroimidazo[1,5-a]pyridine ring system was accomplished by intramolecular cyclization of an iminium ion, derived from condensation of an amine and a substituted gamma-(1-imidazolyl)butyraldehyde. The reaction was used to produce conformationally restricted farnesyltransferase inhibitor analogues which exhibit improved in vivo metabolic stability.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Imidazóis/síntese química , Piridinas/síntese química , Administração Oral , Animais , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase , Imidazóis/química , Imidazóis/farmacocinética , Imidazóis/farmacologia , Indicadores e Reagentes , Modelos Moleculares , Conformação Molecular , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Rizatriptan is a novel 5-HT1D/1B agonist for relief of migraine headache. The pharmacokinetics, metabolite profiles, and tolerability of rizatriptan were examined in a multiple-dose study in healthy subjects. Rizatriptan (N = 24) (or placebo, N = 12) was administered as a single 10 mg dose, followed 48 hours later by administration of one 10 mg dose every 2 hours for three doses on 4 consecutive days, corresponding to the maximum daily dose for a migraine attack. The AUC of rizatriptan and its active N-monodesmethyl metabolite after three 10 mg doses was approximately threefold greater than the plasma concentrations following a single 10 mg dose. Metabolite profiles were similar after single and multiple doses. Adverse events during rizatriptan were mild and transient; similar events occurred during placebo, with a somewhat reduced incidence. Diastolic blood pressure tended to increase compared with placebo (approximately 5 mmHg), particularly on the first multiple-dose day (p < .01 vs. placebo). In conclusion, rizatriptan is well tolerated by healthy subjects during multiple-dose administration, with no unexpected accumulation of drug in plasma.
Assuntos
Agonistas do Receptor de Serotonina/efeitos adversos , Agonistas do Receptor de Serotonina/farmacocinética , Triazóis/efeitos adversos , Triazóis/farmacocinética , Adulto , Pressão Sanguínea/efeitos dos fármacos , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Placebos , Agonistas do Receptor de Serotonina/administração & dosagem , Fatores de Tempo , Triazóis/administração & dosagem , TriptaminasRESUMO
The 5HT1D agonist sumatriptan is efficacious in the treatment of migraines. MK-462 is a drug of the same class which is under development in our laboratories. Bioanalytical methods of high efficiency, specificity and sensitivity were required to support the preclinical and clinical programs. These assays were based on HPLC with tandem MS-MS detection. MK-462 and sumatriptan were extracted using an automated solid-phase extraction technique on a C2 Varian Bond-Elut cartridge. The n-diethyl analogues of MK-462 and sumatriptan were used as internal standards. The analytes were chromatographed using reversed-phase (nitrile) columns coupled via a heated nebulizer interface to an atmospheric pressure chemical ionization source. The chromatographic run times were less than 7 min. Both methods were precise, accurate and selective down to plasma concentrations of 0.5 ng/ml. The assay for MK-462 was adapted to separately monitor the unlabeled and 14C-labeled species of the drug following intravenous administration of radiolabeled material to man.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Agonistas do Receptor de Serotonina/sangue , Sumatriptana/sangue , Triazóis/sangue , Humanos , Pressão , Valores de Referência , Reprodutibilidade dos Testes , Agonistas do Receptor de Serotonina/farmacologia , Sumatriptana/farmacologia , Triazóis/farmacologia , TriptaminasRESUMO
A method based on LC-MS-MS has been developed for the determination of timolol in plasma using the (CD3)3-labelled species as the internal standard. Timolol is isolated from plasma by a simple solid-phase extraction and converted to its oxazolidin-2-one prior to analysis on a 50 x 4.6 mm reversed-phase high-performance liquid chromatography column packed with SynChropak, C18, 5 microns. The column eluate is passed by means of a heated nebulizer interface into a corona discharge atmospheric pressure chemical ionization source where the analyte and its internal standard are detected using multiple reaction monitoring (MRM). The very high specificity of this technique permits chromatographic run times of less than 2 min. The method has a lower quantifiable limit of 0.5 ng ml-1, with intra- and inter-day relative standard deviations less than 10%, and enables the determination of timolol in plasma after ocular administration to volunteers.
Assuntos
Cromatografia Líquida , Espectrometria de Massas , Timolol/sangue , Calibragem , Cromatografia Líquida de Alta Pressão , Humanos , Fosgênio/química , Reprodutibilidade dos TestesRESUMO
New drug candidates are being synthesized at an ever increasing rate and, until recently, the pharmacokinetics of only a few of these could be evaluated. Our laboratory is taking a novel approach to rapid multiple pharmacokinetic screening of potential drug candidates in which mixtures of new substances are co-administered to animals and analyzed simultaneously in plasma using liquid chromatography with tandem MS/MS detection in conjunction with a Prospekt automated on-line solid-phase extraction system. Plasma is sampled via an autosampler and extracted by the Prospekt with the eluent being introduced directly via a reverse phase HPLC column and a heated nebulizer interface to the mass spectrometer. Generic extraction and chromatographic conditions generally give good recoveries. The chromatographic run-times are less than 8 min. The accuracy and precision of these assays are carefully controlled with recoveries generally in the range 80-120% and coefficients of variation less than 20%. Lower quantifiable limits range from 2.5 to 5 ng ml-1. This approach considerably reduces the number of animals needed to screen drug candidates and its power is illustrated by determination of the pharmacokinetics of 10 substances after their simultaneous administration to dogs.
Assuntos
Autoanálise/instrumentação , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Preparações Farmacêuticas/análise , Animais , Pressão Atmosférica , Cães , Masculino , Sistemas On-Line , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
MK-434 is a new 5 alpha-reductase inhibitor. A sensitive and specific assay based on combined liquid chromatography-mass spectrometry (LC-MS) has been developed for the determination of this compound in plasma. The analyte was isolated from plasma by solid-phase extraction on a C18 cartridge. A related substance, L-654,066, was used as the internal standard. Extracts were separated on a 5-cm C18-reversed-phase high performance liquid chromatography column interfaced via the heated nebulizer probe to a corona discharge chemical ionization source. The mass spectrometer was operated in the positive ion MS-MS mode. The method had sufficient sensitivity, precision, accuracy, and selectivity for the analysis of clinical samples containing MK-434 and its two principal metabolites at concentrations in the range 0.5-50 ng ml-1. The chromatographic run time was < 5 min.
Assuntos
Inibidores de 5-alfa Redutase , Azasteroides/sangue , Finasterida/análogos & derivados , Calibragem , Cromatografia Líquida de Alta Pressão , Finasterida/sangue , Espectrometria de Massas , Radioimunoensaio , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
A sensitive and specific method based on radioimmunoassay (RIA) has been developed for the analysis of L-691,121, a new antiarrhythmic agent, and its major metabolite, L-692,199, in plasma. Two RIAs using immunogens and radioligands prepared from different derivatives of L-691,121 were used in conjunction to determine both parent compound and metabolite concentrations by solving simultaneous equations, since neither assay alone was adequately specific. Variable cross-reactivity factors were incorporated into the calculations to correct for non-parallel drug and metabolite displacement curves. The direct assay using 30 microliters of plasma is sensitive to 0.1 ng ml-1 and has sufficient precision, accuracy and specificity for the analysis of clinical samples.
Assuntos
Antiarrítmicos/sangue , Piperidonas/sangue , Compostos de Espiro/sangue , Animais , Especificidade de Anticorpos , Reações Cruzadas , Humanos , Indicadores e Reagentes , Ligantes , Coelhos/imunologia , RadioimunoensaioRESUMO
MK-383 is a novel, non-peptide fibrinogen receptor antagonist. A sensitive and specific radioimmunoassay has been developed for the determination of this drug candidate in plasma and urine. The immunogen was prepared by coupling to albumin via the N-hydroxysuccinimide ester from which the radioligand was also prepared by reaction with [I125]iodotyrosine. The method was specific and no immunoreactive material other than the parent drug was detectable in plasma and urine from dosed volunteers. This direct assay, using 5 microliters of plasma or 0.5 microliter of urine, is sensitive to 1 and 10 ng ml-1, respectively, without matrix interference and has sufficient sensitivity, specificity, accuracy, and precision for the analysis of clinical samples.
Assuntos
Fibrinolíticos/sangue , Fibrinolíticos/urina , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Tirosina/análogos & derivados , Animais , Especificidade de Anticorpos , Feminino , Fibrinolíticos/imunologia , Heparina/química , Humanos , Radioisótopos do Iodo , Marcação por Isótopo , Coelhos/imunologia , Radioimunoensaio , Tirofibana , Tirosina/sangue , Tirosina/imunologia , Tirosina/urinaRESUMO
MK-852 is a novel fibrinogen receptor antagonist. A sensitive and specific radioimmunoassay has been developed for the determination of this drug candidate in plasma and urine. The immunogen was prepared by coupling to albumin via a dinitrophenylene bridge and the radioligand by reaction of the drug with the 125I-labelled Bolton-Hunter reagent. The method was specific and no immunoreactive material other than parent drug was detectable in plasma from dosed volunteers. The direct assay using 0.05 ml of plasma is sensitive to 0.2 ng ml-1 without matrix interference and has sufficient sensitivity, precision, accuracy, and selectivity for the analysis of clinical samples. The lower quantifiable limit in (diluted) urine is 50 ng ml-1.
Assuntos
Oligopeptídeos/urina , Peptídeos Cíclicos/urina , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Radioimunoensaio , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas , Reações Cruzadas , Feminino , Heparina/farmacologia , Humanos , Dados de Sequência Molecular , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Soroalbumina Bovina , TiazolidinasRESUMO
An analytical method based on radioimmunoassay (RIA) has been developed for the determination of the antiarrhythmic agent, MK-0499, in plasma and urine. Owing to the potency of the drug, the specificity of this assay in human plasma could not be adequately determined using conventional RIA procedures. A highly specific procedure, based on LC/MS-MS, was developed to cross-validate the RIA. The lower quantifiable limits of the RIA and LC/MS-MS-based methods were 0.05 and 0.013 ng ml-1, respectively. Cross-validation data, compared using paired student's t-test regression analysis, showed excellent correlation between methods. The mass spectrometric assay was also used to simultaneously measure plasma concentrations of unlabeled and 14C-labeled MK-0499 following administration of the drug at high specific activity to volunteers.
Assuntos
Antiarrítmicos/análise , Benzopiranos/análise , Piperidinas/análise , Animais , Antiarrítmicos/sangue , Antiarrítmicos/urina , Especificidade de Anticorpos , Benzopiranos/sangue , Benzopiranos/urina , Calibragem , Cromatografia Líquida , Feminino , Congelamento , Haptenos/química , Humanos , Indicadores e Reagentes , Marcação por Isótopo , Espectrometria de Massas , Piperidinas/sangue , Piperidinas/urina , Controle de Qualidade , Coelhos , Radioimunoensaio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Assays have been established for the quantitation of timolol and its 13C3- and 2H9-stable-isotope-labeled analogs in plasma and urine using liquid chromatography with atmospheric-pressure chemical-ionization tandem mass spectrometry. For the analysis of urine, underivatized timolol and its labeled analogs are monitored while timolol in plasma is assayed down to concentrations of 0.2 ng/mL after derivatization with phosgene. The great power of this technique is illustrated by simultaneously assaying three different species of timolol in plasma and urine obtained from dogs receiving simultaneous ophthalmic, oral, and intravenous doses of unlabeled and [2H9]- and [13C3]-labeled timolol. Thus, the ophthalmic and oral bioavailabilities of timolol are measured in a single experiment rather than as a three-phase crossover experiment. This approach yields accurate and precise analytical data, obviates intrasubject variability, and saves both analytical and animal resources.
Assuntos
Antagonistas Adrenérgicos beta/análise , Antagonistas Adrenérgicos beta/farmacocinética , Timolol/análise , Timolol/farmacocinética , Administração Oral , Animais , Área Sob a Curva , Disponibilidade Biológica , Calibragem , Cromatografia Líquida , Cães , Masculino , Espectrometria de Massas , Soluções OftálmicasRESUMO
Liquid chromatography, combined with tandem mass spectrometry (LC/MS/MS) has been rapidly embraced by the pharmaceutical industry as the definitive method for the determination of drug levels in biological fluids obtained from pharmacokinetic and toxicological studies. This technique has proved to be reliable, accurate and precise for the determination of drugs and related substances (e.g. metabolites and isotope-labeled compounds) in support of preclinical and clinical studies. Our group has recently expanded the use of quantitative LC/MS/MS into the area of discovering new substances as potential drug candidates. When used as an accurate mass detector, triple quadrupole instruments have the ability to simultaneously and specifically detect minute quantities of closely-related drug substances in the extracts of biological fluids. Analytical procedures have been developed and validated that simultaneously determine plasma concentrations of up to 12 drug candidates over a concentration range of 1-1000 ng mL-1 in single analytical occasions. This approach is used to support drug discovery by rapidly providing pharmacokinetic data to a wide range of compounds following either the administration of multiple compounds to single animals, or by increasing the speed and efficiency of analyzing samples following the administration of single compounds to multiple animals. Currently, we have screened over 400 compounds in two different target classes in a period of 24 weeks. A standard operating procedure defining the acceptability of quality control data obtained during such screening experiments is described.
Assuntos
Preparações Farmacêuticas/análise , Animais , Área Sob a Curva , Cromatografia Líquida , Cães , Espectrometria de Massas , Farmacocinética , Controle de Qualidade , RatosRESUMO
L-654,066 is a new 5 alpha-reductase inhibitor. A sensitive and specific assay based on combined liquid chromatography/mass spectrometry has been developed for the determination of this drug candidate in plasma. The analyte was isolated from plasma by solid-phase extraction on a C-18 cartridge. A related substance, L-683,838, was used as the internal standard. Extracts were chromatographed on a 5 cm C18 reverse-phase high-performance liquid chromatography column interfaced via the heated nebulizer probe to a corona discharge chemical ionization source. The mass spectrometer was operated in the positive ion tandem mode. The method has sufficient sensitivity, precision, accuracy and selectivity for the analysis of clinical samples containing L-654,066 at concentrations in the range 0.5-20 ng ml-1. The chromatographic run time is less than 2 min.
Assuntos
Azasteroides/sangue , Espectrometria de Massas/métodos , Oxirredutases/antagonistas & inibidores , Calibragem , Colestenona 5 alfa-Redutase , Humanos , RadioimunoensaioRESUMO
In this first of two consecutive papers, the main objective of which is to present a new approach to the systematic localization of individual proteins located in the Escherichia coli ribosome by immunoelectron microscopy, we describe the derivatization of several purified 30 S proteins (S12, S21, S14, S19, S18, S17) with 2,4-[3,5-3H]dinitrofluorobenzene at pH 7.4 and 8.4 and the uptake of each dinitrophenylated protein in place of the corresponding unmodified protein into totally reconstituted 30 S subunits. Reverse-phase high performance liquid chromatography is used to purify the proteins, to separate and characterize the products of 2,4-[3,5-3H]dinitrofluorobenzene modification, and to analyze the protein composition of the reconstituted subunits. The extent of dinitrophenyl (DNP) modification is estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214 and 360 nm. DNP derivatives of each of the six proteins are efficiently incorporated into reconstituting 30 S subunits. Incorporation of any of the six DNP-modified proteins does not interfere with binding of Phe-tRNA(Phe) in a poly(U)-dependent manner. This result, as well as data showing that unmodified protein competes with DNP-protein for uptake during reconstitution, provide evidence that each DNP-protein occupies the same position in 30 S subunit as does unmodified protein. In general, for a given protein, unmodified and/or less modified forms are incorporated in preference to more modified forms. Modification of protein S19 at pH 7.4 proceeds with selective formation of one derivative in high yield. Reverse-phase high performance liquid chromatography analysis of acid hydrolysates of a purified sample of this derivative, as well as of peptides derived from it by digestion with Staphylococcus aureus protease, show the N-terminal proline to be the predominant site of DNP-derivatization.
Assuntos
Dinitrofluorbenzeno/farmacologia , Escherichia coli/metabolismo , Nitrobenzenos/farmacologia , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão , Dinitrofluorbenzeno/metabolismo , Escherichia coli/ultraestrutura , Ligação Proteica , Proteínas Ribossômicas/isolamento & purificação , Ribossomos/ultraestrutura , EspectrofotometriaRESUMO
Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.
Assuntos
Dinitrofluorbenzeno , Escherichia coli/ultraestrutura , Nitrobenzenos , Proteínas Ribossômicas/análise , Ribossomos/ultraestrutura , Anticorpos , Imunoensaio , Microscopia Eletrônica , Modelos Estruturais , Proteínas Ribossômicas/imunologiaRESUMO
PURPOSE: The objective of this study was to compare plasma concentrations of timolol following multiple dosing of the therapeutic regimens of timolol maleate ophthalmic gel-forming solution (Timolol GS; TIMOPTIC-XE) and timolol maleate ophthalmic solution. Timolol maleate ophthalmic gel-forming solution is also referred to as Timolol GS, i.e. gel-forming solution. METHODS: This was a masked observer, two-period crossover study in six normal male subjects randomized to receive either Timolol GS, 0.5% (TIMOPTIC-XE,) once daily (0530 hours) or timolol maleate ophthalmic solution (0.5% TIMOPTIC) twice daily (0530 and 1730 hours) for 8 days, in both eyes. On Day 8, a blood sample was obtained prior to treatment, as well as 1, 2, 4, 8, 10, 12, 13, 14, 16, and 24 hours following the morning instillation. After a 7-day inter-period washout interval, subjects received the opposite treatment. RESULTS: Timolol GS (TIMOPTIC-XE): Plasma concentrations of timolol rarely exceeded 0.375 ng/ml (the lower limit of assay quantification). For all subjects, peak plasma concentrations of timolol averaged <0.3 ng/ml within 4 hours after the last dose. The highest single observation was 0.49 ng/ml in one subject (at hour 2). Timolol solution: For all subjects, peak plasma concentrations of timolol averaged about 0.5 ng/ml and 0.3 ng/ml within 4 hours following the first and second dose, respectively, on Day 8. The highest single observation was 0.95 ng/ml in one subject (at hour 2). CONCLUSIONS: The data suggest that there is less systemic exposure to timolol following once-daily therapy with Timolol GS 0.5% compared with twice daily therapy with timolol maleate ophthalmic solution 0.5%.
Assuntos
Antagonistas Adrenérgicos beta/farmacocinética , Timolol/farmacocinética , Absorção , Antagonistas Adrenérgicos beta/administração & dosagem , Adulto , Disponibilidade Biológica , Estudos Cross-Over , Método Duplo-Cego , Sistemas de Liberação de Medicamentos , Géis , Humanos , Masculino , Soluções Oftálmicas/administração & dosagem , Soluções Oftálmicas/farmacocinética , Timolol/administração & dosagemRESUMO
Purified Escherichia coli ribosomal proteins S6, S13, S16, and S18 were dinitrophenylated at their amino termini and/or at one or more internal lysine residues. Each dinitrophenyl protein was then separately incorporated into reconstituted small ribosomal subunits. Modified proteins were localized on the 30 S subunit surface by electron microscopy of reconstituted subunits complexed with antibodies to dinitrophenol (DNP). DNP protein S13 was placed on the subunit head above the platform and on the surface that faces the large subunit. DNP-S18 was localized to the subunit platform below the tip and in a region associated with binding to 50 S subunits. DNP proteins S6 and S16 were both localized near the junction of the subunit body and platform; DNP-S6 was available to antibody in 70 S ribosomes and was placed on the cytoplasm-facing side of the subunit in an area that overlaps the platform and body of the particle. DNP-S16 in 70 S ribosomes was not bound by antibody. It was localized to the 30 S body near its junction with the platform and on the surface facing the 50 S particle. The results complement and clarify data obtained using other approaches.