Changes in ribosomal proteins in developing Artemia salina embryos

Ribosomal proteins from cysts and nauplii of Artemia salina were analyzed by three kinds of two-dimensional polyacrylamide gel electrophoresis. The basic-acidic and basic-SDS gel systems were used to compare the basic ribosomal proteins, and some changes were observed between the cysts and nauplii in proteins S6, S14, and L24. The phosphorylation of protein S6 was increased in the nauplii. Basic proteins S14 and L24 in the cysts changed and none of the corresponding proteins in the nauplii were detected at the same positions on two-dimensional gels as in the cysts. The acidic-SDS gel system was used to compare the acidic proteins in ribosomes and it was revealed that an acidic protein, AX (Mr = 24,000), in the cysts was not present in the ribosomes from the nauplii. The ribosomal activities as to the formation of an 80S initiation complex with globin mRNA and poly(U)-directed polyphenylalanine synthesis were compared. There was no significant difference between the cyst and nauplius ribosomes.     

Ribosomal proteins of Tetrahymena thermophila. Correlation of one- and two-dimensional electrophoretic migration patterns and characterization of additional small and large subunit proteins

Further analysis of the protein complement of the cytoplasmic ribosome of the protozoon Tetrahymena thermophila has led to the identification and characterization of seven additional proteins, three in the small and four in the large subunit of this ribosome. Several of these proteins are poorly soluble or insoluble in the absence of high concentrations of urea and are not seen in the electrophoretic distribution patterns of ribosomal proteins in two-dimensional polyacrylamide gels unless 6 M urea is added to electrode buffers in contact with protein samples (first dimension) and first-dimension gels (second dimension). The migration patterns of the 40S and 60S subunits of the T. thermophila ribosome in one-dimensional polyacrylamide SDS gels and in two-dimensional gels prepared by means of the basic-acidic system of Kaltschmidt and Wittmann**, and the basic-SDS system of Zinker and Warner*** have been correlated.     

Ribosomal phosphoproteins in Microsporum canis

Ribosomal phosphoproteins of Microsporum canis labelled in vivo were characterised by two-dimensional and SDS polyacrylamide gel electrophoresis. A small subunit protein, S6, was the only phosphoprotein identified in 40S and 80S in basic-acidic two-dimensional gels. Three different forms of phosphorylated S6 were also observed in 40S subunit. On SDS gels five phosphoproteins were identified in 80S; of these three were present in 40S and two in 60S. S6 was the only basic phosphoprotein, while the other four were acidic.     

Yeast ribosomal proteins: V. Correlation of several nomenclatures and proposal of a standard nomenclature

Summary A tentative nomenclature (YP number) for yeast (Saccharomyces cerevisiae) cytoplasmic ribosomal proteins, which is used in our laboratory (Otaka and Kobata 1978; Higo and Otaka 1979), has been correlated with those of Warner and Gorenstein (1978) and several others. Our nomenclature is based on the two-dimensional gel electrophoretic pattern of proteins as analyzed by a modified method of Mets and Bogorad (1974), while others have used various modifications of Kaltschmidt and Wittmann's two-dimensional gel electrophoresis (1970). The method of correlation involved the examination in our twodimensional electrophoresis system of each protein spot excised from gel patterns prepared by Kaltschmidt and Wittmann's method or vice versa.The numbers of protein species recognized in this paper are 29 for small subunit, and 44 for large subunit. Based on these results, we propose a standard nomenclature for yeast ribosomal proteins, in which the designations YS1–YS29 and YL1–YL44 have been given to the small subunit proteins and the large subunit proteins respectively.     

Differences in electrophoretic behaviour of eight ribosomal proteins from rat and rabbit tissues and evidence for proteolytic action on liver proteins

Summary Divergence exists between eight ribosomal proteins of rat and rabbit. This is the first time such a divergence has been precisely demonstrated among mammals. In addition, a proteolytic activity, giving the appearance of modified proteins, is observed in the liver of both species but not in rabbit reticulocytes. These results were made possible by a recently developed method. Ribosomal proteins were compared by two-dimensional polyacrylamide gel electrophoresis in four different but related systems. The precise position of each individual protein was established in each of the four systems. Certain ribosomal proteins from different tissues, seemingly identical in one system were found to be different in other systems. Significant differences occurred between proteins of each ribosomal subunit from the two different species. Variation between reticulocyte and liver ribosomal proteins of the rabbit were minor. Several liver proteins of both species change position or disappear and apparently new proteins appear, if appropriate steps are not taken to prevent proteolysis. Differences in behavior of three small subunit and five large subunit proteins are attributable to an evolutionary divergence between the two species.     

Molecular weight hetergeneity of proteins of the ribosomes and ribosomal subunits from dry pea seeds]

The molecular weight distribution of the total protein of ribosomes and ribosomal subunits isolated from dry pea seeds was studied by electrophoresis in polyacrylamide gel, containing sodium dodecyl sulfate. It was demonstrated that overall protein of 80 S ribosomes is separated into a number of fractions with molecular weights of 10000-64000. Treatment of ribosomes with 0.5 per cent tritone, 0.5 per cent and 1 per cent deoxycholate does not change the general pattern of the molecular weight distribution of ribosomal proteins. The large subunit reveals 19 protein zones (14 major and 5 minor zones), their molecular weights are varying from 10000 to 54000. The majority of proteins of the large subunit have molecular weights of 14000--32000. The molecular weights of 17 protein zones of the small subunit (7 major and 10 minor zones) vary from 10000 to 64000. The majority of proteins of both large and small subunits have molecular weights of 14000--32000. Electrophoretic separation of proteins in the split gel confirmed the fact that the proteins of large subunit differ in molecular weights from those of the small subunit. Thus, ribosomal proteins of pea seeds are shown to produce a typical (for 80S ribosomes) pattern of molecular weight distribution under polyacrylamide gel electrophoresis in the presence of sodium dodecul sulphate.     

Ribosomal proteins of HeLa cells

Ribosomal proteins from HeLa cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt-Wittmann) and dodecylsulfate polyacrylamide gel electrophoresis (Laemmli). 35 proteins are associated with the small ribosomal subunit and 47 proteins with the large ribosomal subunit. The HeLa ribosomal proteins S6, S32, L40b,c, L41 and L42 are phosphorylated in vivo and in vitro. Minor differences between HeLa and rat liver ribosomal proteins were revealed by their direct coelectrophoresis.     

Ribosomal proteins ofThermomyces lanuginosus — characterisation by two-dimensional gel electrophoresis and differential disassembly

One- and two-dimensional gel electrophoresis were employed to characterise the proteins derived from the ribosomes of the thermophilic fungusThermomyces lanuginosus. Approximately 32 (29 basic and 3 acidic) and 45 (43 basic and 2 acidic) protein spots were resolved fromTh. lanuginosus small and large ribosomal subunits, respectively. The molecular weight of the small subunit proteins ranged from 9,800–36,000 Da with a number average molecular weight of 20,300 Da. The molecular weight range for the large subunit proteins was 12,000–48,500 Da with a number average molecular weight of 25,900 Da. Most proteins appeared to be present in unimolar amounts. These data are comparable with but not identical to those from other eukaryotic ribosomes. The sensitivities of the ribosomal proteins to increasing concentrations of NH₄Cl were also evaluated by two-dimensional gel electrophoresis. Most ribosomal proteins were gradually released over a wide range of salt concentrations but some were preferentially enriched in one or two salt conditions.     

Ribosomal proteins in the fungus Podospora anserina: evidence for an electrophoretically altered 60S protein in a cycloheximide resistant mutant

Summary Proteins of cytoplasmic ribosomes of the Podospora anserina were analyzed by two dimentional gel electrophoresis. The numbers of proteins were estimated to be 28 in the small subunit and 41 in the large subunit. The L21 protein of the large subunit was found to migrate differently in a cycloheximide resistant mutant.     

The protein composition of HeLa ribosomal subunits and nucleolar precursor particles

Using two-dimensional polyacrylamide gel electrophoresis, the protein patterns from HeLa 80S and 55S nucleolar precursor particles have been compared with those of cytoplasmic 40S and 60S ribosomal subunits. The 55S particle was found to have 21 anionic and 52 cationic proteins, including 18 large subunit ribosomal proteins. The 80S precursor pattern was identical to the 55S pattern except three anionic and four cationic proteins were absent. Of those missing cations, three were large subunit proteins. However, no small subunit ribosomal proteins were detected on either precursor. Numerous high molecular weight non-ribosomal proteins were found in both precursor particles and may correspond to a class of stable nucleolar proteins.     

Characterization of cytoplasmic and chloroplast ribosomal proteins of Euglena gracilis.

Active cytoplasmic ribosone subunits 41 and 62S were prepared by treatment with 0.1 mM puromycin in the presence of 265 mM KCl. Active chloroplast subunits 32 and 49S were obtained after dialysis of chloroplast ribosomal preparations against 1 mM Mg(2+)-containing buffer. Proteins from these different ribosomal particles were mapped by two-dimensional gel electrophoresis in the presence of urea. The 41S small cytoplasmic ribosomal subunit contains 33-36 proteins, the 62S large cytoplasmic ribosomal subunit contains 37-43, the 32S small chloroplast ribosomal subunit contains 22-24, and the 49ts large chloroplast ribosomal subunit contains 30-34 proteins. Since some proteins are lost during dissociation of monosomes into subunits, the 89S cytoplasmic monosome would have 73-83 proteins and the 68S chloroplast monosome, 56-60. The amino acid composition of ribosomal proteins shows differences between chloroplast and cytoplasmic ribosomes.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

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.     

The polysomal proteins of L cells. Discrimination between the structural ribosomal proteins, the exchangeable ribosomal proteins and the non-ribosomal proteins by two-dimensional dodecylsulfate electrophoresis and autoradiography.

Three groups of proteins can be clearly discriminated in the total protein of L cell polysomes by selective labelling in the presence of low doses of actinomycin D and two-dimensional polyacrylamide/dodecylsulfate gel electrophoresis followed by autoradiography: (a) structural ribosomal proteins which are not labelled in the presence of actinomycin D and form stained non-radioactive spot in gels; (b) exchangeable ribosomal proteins which are labelled in the presence of actinomycin D and stained radioactive spots; (c) non-ribosomal proteins which are detectable only by autoradiography of gels. The large and small subunits of L cell ribosomes contain respectively 45 and 34 ribosomal proteins with molecular weights less than or equal to 50 000; seven of the large subunit proteins and nine of the small subunit proteins are exchangeable. Most of the non-ribosomal proteins migrate in the region of the related to the separation of the ribosomal proteins of mammalian cells and the possible significance of the presence of non-ribosomal proteins in polysomes are discussed.     

Study on proteins from yeast cytoplasmic ribosomes by two-dimensional gel electrophoresis

Summary Proteins of yeast cytoplasmic ribosomes were analyzed by two different methods of two-dimensional gel electrophoresis: run at pH 8.6 in 1-D¹ and at pH 4.6 in 2-D (Method A); run at pH 5.0 in 1-D and in the presence of sodium dodecyl sulfate in 2-D (Method B). The numbers of proteins estimated were 28 (Method A) and 29 or 30 (Method B) in the 40S small subunit, and 40 (Method A) and 41 (Method B) in the 60S large subunit, respectively. Molecular weights of proteins in the small and the large subunits were found to be less than 40,000 and 60,000 respectively.     

Characterization of the ribosomal proteins from mosquito (Aedes albopictus) cells

Proteins from the large and small subunits of Aedes albopictus (mosquito) cytoplasmic ribosomes were characterized by two-dimensional polyacrylamide gel electrophoresis. The small subunit contained 28-31 proteins ranging in molecular mass from 10 to 49 kDa. The large subunit contained 36-39 proteins that ranged in molecular mass from 11 to 53 kDa. The largest protein on the small subunit, S1, was the predominant phosphorylated ribosomal protein. Under long-term labelling conditions, L4 and L33 were also phosphorylated. Peptide mapping by partial proteolysis indicated that Ae. albopictus S1 may share partial amino acid homology with the phosphorylated ribosomal protein S6 from Drosophila melanogaster. Unlike Drosophila S6, however, Aedes S1 was not dephosphorylated during heat shock. Treatment of mosquito cells with the insect molting hormone 20-hydroxyecdysone did not affect phosphorylation of ribosomal proteins.     

Spatial arrangement of proteins within the small subunit of rat liver ribosomes studied by cross-linking

Cross-linking of proteins within the small subunit of rat liver ribosomes by the bifunctional reagent dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate produced numerous covalently linked protein dimers which could be separated by a combination of ion-exchange chromatography on carboxymethyl cellulose and polyacrylamide gel electrophoresis. The protein components of the dimers were identified electrophoretically after periodate cleavage of the cross-link(s). The analysis revealed 42 cross-linked dimers involving 25 different proteins. Among these, proteins S3, S4 and S20 occurred in combinations with six, eight and seven different proteins, respectively. For proteins S13, S14 and S17 five protein neighbours could be identified, while 13 of the remaining proteins were linked to three or four different protein partners. The involvement of the majority of proteins in the formation of multiple cross-linked dimers implies that a large number of protein-protein interaction sites exist within the ribosomal subunit. A preliminary model illustrating the arrangement of 16 proteins in the small ribosomal subunit is presented and discussed with respect to possible functions, especially in the event of translation initiation.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

E. coli ribosomal proteins are cross reactive with antibody prepared against Chlamydomonas reinhardi chloroplast ribosomal subunit

Summary Antisera prepared against purified Chlamydomonas reinhardi small chloroplast ribosomal subunit, judged homogenous by sucrose gradient velocity sedimentation and RNA gel electrophoresis was immunologically cross reactive with E. coli ribosomal proteins. The results of three different experimental approaches, namely Ouchterlony double diffusion, sucrose gradient velocity sedimentation and two dimensional crossed immunoelectrophoresis indicate that both E. coli ribosomal subunits and the chloroplast large ribosomal subunit contain proteins which show antigenic similarity to the chloroplast small ribosomal subunit proteins. However, cytoplasmic ribosomal subunits did not contain proteins which were cross reactive with immune antisera.     

Ribosomal proteins in the cyanobacterium Anabaena variabilis strain M3: presence of L25 protein

Ribosomal proteins from a cyanobacterium Anabaena variabilis were analyzed by two-dimensional gel electrophoresis. We detected 21 protein spots of the small subunit and 29 protein spots of the large subunit. One of the spots was identified as L25 protein, which suggests that the reading frame sll1824 of Synechocystis is the L25 protein.