Correlation between the rate of exoprotein synthesis and the amount of the multiprotein complex on membrane-bound ribosomes (MBRP-complex) in Staphylococcus aureus

The membrane-bound ribosome protein (MBRP)-complex of Staphylococcus aureus was studied using antibodies to its individual components. The four polypeptides of the complex were firmly held together, and none were present in large excess. The membrane-bound fraction of the MBRP-complex was accessible to trypsin only after removal of the membrane-bound ribosomes; it also remained associated with the membrane-bound ribosomes even after solubilization of the membranes with Triton X-100. Furthermore, the amount of MBRP-complex in the membrane was proportional to the rate of exoprotein synthesis. These results strongly suggest a role for the MBRP-complex in protein secretion.     

the stability in various detergents of transferrin-transferrin receptor complexes from reticulocyte plasma membranes

Transferrin-membrane protein complexes were solubilized either with 0.4% sodium dodecyl sulfate (SDS), 1% Triton X-100 or 0.5% sulfobetaine 3-14 from the plasma membranes of rabbit reticulocytes previously labeled with 125I and then incubated with 131-labeled transferrin. When the solubilized membranes were analyzed by gel filtration fractionation, marked variation in the preservation of transferrin-transferrin receptor interaction was noted between the three detergents. After SDS solubilization, more than 80% of the 131I-labeled transferrin remained associated with membrane proteins with apparent molecular weight of the transferrin-receptor complexes of 1400 000 and 240 000. In contrast, after Triton X-100 solubilization only 40% of the transferrin was still complexed to membrane proteins with an apparent molecular weight of the complex of 450 000. Dissociation of transferrin from its receptor was most marked following sulfobetaine solubilization, with less than 30% of the transferrin still complexed. Following gel filtration 131I-labeled transferrin-125I-labeled membrane protein complexes were immunoprecipitated with goat specific anti-rabbit transferrin antibodies. The immunoprecipitates were analyzed under stringent dissociating conditions by two SDS-polyacrylamide gel electrophoretic techniques. In a linear 5-25% polyacrylamide gradient the 125I-labeled receptor obtained after membrane solubilization with all three detergents had an apparent molecular weight of 80 000. In contrast, in a different system using 10% polyacrylamide gel two 125I-labeled receptor components were detected wih apparent molecular weights of 90 000 and 80 000. These results demonstrate that estimates of the molecular weight of the transferrin receptor depended on the conditions of electrophoresis and suggest that the transferrin receptor is partially modified, perhaps by glycosylation.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [¹⁴C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Quantitative analysis of the protein composition of yeast ribosomes

The molecular weights of the individual yeast ribosomal proteins were determined. The ribosomal proteins from the 40-S subunit have molecular weights ranging from 11 800 to 31 000 (average molecular weight = 21 300). The molecular weights of the 60-S subunit proteins range from 10 000 to 48 400 (average molecular weight = 21 800). Stoichiometric measurements, performed by densitometric scanning on ribosomal proteins extracted from high-salt dissociated subunits revealed that isolated ribosomal subunits contain, besides some protein species occurring in submolar amounts, a number of protein species which are present in multiple copies: S13, S27, L22, L31, L33, L34 and L39. The mass fractions of the ribosomal proteins which were found to be present on isolated ribosomes in non-unimolar amounts, were re-examined by using an isotope dilution technique. Applying this method to proteins extracted from mildely isolated 80-S ribosomes, we found that some protein species such as S32, S34 and L43 still are present in submolar amounts. On the other hand, however, we conclude that some other ribosomal proteins, in particular the strongly acidic proteins L44 and L45 get partially lost during ribosome dissociation. Proteins L44/L45 appears to be present on 80-S ribosomes in three copies.     

Purification and characterization of amphiphilic trehalase from rabbit small intestine

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.     

Separation and reconstitution of sodium-dependent glucose transport activity from renal brush-border membranes using gel-filtration chromatography

Pig kidney brush-border membrane vesicles were solubilized using a final concentration of 1% Triton X-100, found optimal for quantitative reconstitution of d-glucose transport into liposomes. Using reconstituted proteoliposomes, selective permeability towards d-glucose compared to other sugars tested was shown as well as the main features of d-glucose transport in native membranes, namely sodium dependence and phlorizin inhibition of d-glucose accumulation. After removal of Triton X-100 from the detergent extract, some membrane proteins (about 40%), which are insoluble in the absence of detergent, were isolated. Among these proteins resolubilized by 1% Triton X-100, the component catalyzing the d-glucose transport was located by gel-filtration chromatography separation, using reconstitution of transport as the assay. The active fraction displayed a molecular size of 50 Å; when analyzed on SDS polyacrylamide gel electrophoresis, it contained one major protein subunit with an apparent molecular weight close to 65 000. We conclude that this protein fraction is involved in d-glucose transport by renal brush borders.     

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Determination of Triton X-100 binding to membrane proteins by polyacrylamide gel electrophoresis

The molecular weight of proteins in protein-detergent complexes can be determined from ultracentrifugation experiments if the amount of bound detergent is known. A new sensitive method to measure the binding of the nonionic detergent Triton X-100 to proteins has been developed. For the membrane proteins studied, less than 50 μg of protein was required to achieve an accuracy of 10% in the determination of the detergent-protein weight ratio.The proteins were equilibrated with the detergent by electrophoresis into polyacrylamide gels containing radioactively labelled Triton X-100. The gels were then sliced and the amount of bound detergent calculated from the increase in radioactivity in the slices containing the protein zone. The amounts of protein were determined by amino acid analysis of identical protein zones cut from gels running parallel .     

Disruption of the quaternary structure of (Na+ + K+)-dependent adenosine triphosphatase by Triton X-100.

(Na⁺ + K⁺)-dependent adenosine triphosphatase (NaK ATPase) consists of two polypeptide chains, a large polypeptide with a molecular weight of about 100,000, and a sialoglycoprotein with a molecular weight of about 40,000. In the presence of Triton X-100 both polypeptides react to form high molecular weight aggregates with apparent molecular weights of 168,000, 200,000 and 260,000. These aggregates arise as a result of disulfide bond formation which results from the autooxidation of sulfhydryl groups on the two polypeptides of NaK ATPase. These data are discussed in light of studies aimed at determining the size and subunit structure of membrane proteins with Triton X-100.     

Two-dimensional gel electrophoresis of ribosomal proteins from streptomycin-sensitive and streptomycin-resistant mutants of Chlamydomonas reinhardi.

Ribosomal proteins from three mutant strains of Chlamydomonas reinhardi were analysed and compared by one-dimensional and two-dimensional gel electrophoresis. One mutant was streptomycin-sensitive the other two were streptomycin-resistant, one with a Mendelian the other with a non-Mendelian pattern of inheritance. In the 30-S subunits of chloroplast ribosomes approximately 25 proteins are found and in the 50-S subunits 34 proteins. The 40-S subunits of cytoplasmic ribosomes contain about 31 proteins and the 60-S subunits 44 proteins. The molecular weights of most proteins in all subunits are in the range of 10 000 to 35 000. However, the 60-S subunits contain in addition a protein of molecular weight 50 000 and the 30-S subunits show 6-7 bands of molecular weights from 50 000 to 83 000. The proteins of the cytoplasmic 80-S ribosomes or of their subunits from all three mutants are electrophoretically identical. The proteins of the 70-S organellar ribosomes and both of their subunits show distinct differences between the three strains. Our results indicate that organellar ribosomal proteins are in part controlled by nuclear DNA and in part by organellar DNA.     

Interactions of cytoskeletal elements with the plasma membrane of Sarcoma 180 ascites tumor cells

The association of cytoskeletal proteins with cell surface envelopes from Sarcoma 180 ascites cells has been studied by several techniques previously used successfully in studying the interaction of spectrin with erythrocyte membranes. By electron microscopy the envelopes exhibit irregular exterior surfaces and the presence of substantial amounts of “fuzz” at the interior surface. Extraction of the envelopes at low ionic strength and alkaline pH fragments the membranes and depletes them of the “fuzz” with concomitant elution of four major polypeptides of mol. wt >300 000 (Band E), 250 000, 100 000 and 43 000 D. The last three of these have been tentatively identified as actin-binding protein (ABP), α-actinin and actin. Membrane-associated myosin is not eluted under these conditions. Neither actin nor myosin is eluted under conditions commonly used to depolymerize them. However, myosin can be eluted at high salt concentrations if the envelopes have been previously extracted and fragmented with alkaline buffer as above. Extraction of the envelopes with Triton X-100 removes 60% of the membrane lipid and 70–80% of lactoperoxidase-iodinated cell surface proteins without removal of significant amounts of the cytoskeletal proteins. The Triton residues maintain the shape of the original envelopes but have lost the trilaminar membrane structure. Proteolysis of intact envelopes with trypsin or papain cleaves the high molecular weight polypeptides in the order E > ABP > myosin. Fragmentation occurs with cleavage of E or ABP, but does not appear to require cleavage of myosin. Actin and α-actinin are not appreciably cleaved when associated with the membrane. The results, combined with previous observations, suggest an extensive complex of cytoskeletal proteins attached to the membrane interior surface.     

Physical properties of the purified cardiac muscarinic acetylcholine receptor

The physical properties of the cardiac muscarinic acetylcholine receptor (mAcChR) purified from porcine atria as recently described [Peterson, G.L., Herron, G.S., Yamaki, M., Fullerton, D.S., & Schimerlik, M.I. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4993-4997] have been examined by D2O/H2O sucrose gradient sedimentation and Sephacryl S-300 gel filtration in Triton X-405 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the sedimentation experiments the partial specific volume and sedimentation constant for the mAcChR-Triton X-405 complex were determined to be 0.813 cm3/g and 5.30 S, respectively, which lead to an estimate of the molecular weight of the complex of 143 000. Gel filtration in Triton X-405 gave an estimate of the Stokes radius (4.29 nm) and an apparent molecular weight of 116 000. Combination of sedimentation and gel filtration gave an apparent molecular weight of 137 000 and a frictional ratio (f/f0) of 1.21 for the complex. The partial specific volume of the receptor calculated from composition was 0.717 cm3/g assuming 26.5% by weight carbohydrate. The amount of bound Triton X-405 was estimated at 1.011 g/g of mAcChR, which gave an apparent molecular weight of 70 900 (sedimentation) or 68 200 (sedimentation plus gel filtration) for the uncomplexed receptor. SDS-PAGE experiments at acrylamide concentrations ranging from 6% T [monomer plus bis(acrylamide)] to 17% T gave a linear range of apparent molecular weight from 67 600 (6% T) to 98 600 (17% T), and calibration against the retardation coefficient, Kr, determined from Ferguson plots gave an apparent molecular weight of 89 100 +/- 6700. From a newly developed, novel evaluation scheme the anomalous migration of the mAcChR in SDS-PAGE was found to be due to both an excess charge density and an abnormally large shape parameter (Kr), and the true molecular weight of the protein portion of the mAcChR ligand binding polypeptide was estimated to be between 50 000 and 60 000.     

A study of mitochondrial ribosomes from the higher plant Solanum tuberosum L.

Ribosomal subunits are isolated from potato tuber mitochondria devoid of contaminating organelles. The sedimentation constants of the two mitochondrial ribosomal subunits are 33S and 50S respectively. The apparent sizes of the high molecular weight RNAs are 19S and 25S.The proteins of these ribosomes have been analyzed by two-dimensional electrophoresis in SDS polyacrylamide gels to determine their number and molecular weights. The small subunit contains 35 protein species ranging from 8 to 60 kDa. The 50S large subunit contains 33 protein species ranging from 12 to 46 kDa. These preliminary results are the first analysis made on mitochondrial ribosomes from a higher plant.     

Molecular characteristics of the transferrin-receptor complex of the rabbit reticulocyte

A macromolecular complex of transferrin and a membrane component was isolated by gel filtration chromatography from Triton X-100-solubilized ghosts of reticulocytes previously incubated with ¹²⁵I-labeled transferrin. This complex is believed to be transferrin specifically associated with its primary receptor. Following the procedures of Clark [14], the complex in Triton X-100 was found to behave as an asymmetric molecule with a molecular weight of approximately 250,000 and an axial ratio of 9:1. On SDS-polyacrylamide gel electrophoresis the complex displays, in addition to transferrin, components of molecular weights 176,000 and 95,000, respectively. The larger component may be a dimer of the smaller. Each appears to crosslink, with dimethyl suberimidate, to transferrin. These results are compatible with the hypothesis that the transferrin receptor itself has a molecular weight near 175,000 and may be a dimer of two smaller components each of molecular weight near 95,000.     

Purification and characterization of the two molecular forms of membrane acid protease from Aspergillus oryzae.

Two forms (M1 and M2) of the membrane-bound acid protease of Aspergillus oryzae have been purified by extraction with Triton X-100, washing with cold acetone, and repeated gel filtration on Bio-Gel A-15 m in the presence and absence of Triton X-100. The purified membrane enzymes, M1 and M2, moved as a single band in acrylamide gel electrophoresis and had apparent molecular weights of 150 000 and 60 000, respectively, as estimated by sodium dodecyl sulfate/acrylamide gel electrophoresis. These two membrane enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They immunologically cross-reacted with each other and with an extracellular acid protease from A. oryzae, and contained carbohydrate, ranging from 52.5 to 80.5% and comprising three hexoses, glucose, galactose, and mannose. While these catalytic, chemical and immunological properties are similar to those of the extracellular acid protease from A. oryzae, both membrane enzyme differed in their hydrophobic properties from external enzymes. Thus they are activated by the detergent Triton X-100 and some polar lipids.     

Comparison of the size and physical properties of gamma-glutamyltranspeptidase purified from rat kidney following solubilization with papain or with Triton X-100.

gamma-Glutamyltranspeptidase is associated with the brush border membrane of kidney proximal straight tubule cells. It can be solubilized qualitatively by treatment with papain or Triton X-100. Neither procedure affects its catalytic activity but the two resulting forms of the enzyme differ considerably in their physical properties. The papain-solubilized transpeptidase is soluble in aqueous buffers and was purified 430-fold. It has an s20,w of 4.9 S, a Stokes radius of 36 A, and a calculated molecular weight of 69,000. It appears homogeneous by sedimentation equilibrium centrifugation (Mr=66,700). In contrast, the Triton-solubilized transpeptidase is soluble only in the presence of detergents and was purifed 300-fold. This form of the enzyme has a Stokes radius of 70 A but an s20,w of only 4.15 S. Aggregation of the enzyme just below the critical micelle concentration of Triton X-100 and its ability to bind 1.16 mg of Triton X-100-protein complex was calculated to be 169,000, but the glycoprotein portion of the complex is 52% of the total mass (87,000). The mass of Triton X-100 (82,000) is consistent with its reported micelle molecular weight. Treatment of the Triton-purified transpeptidase with papain or bromelain results in a form of the enzyme identical in all respects with the papain-purified enzyme. Both the Triton- and papain-purified transpeptidase exhibit two protein bands on sodium lauryl sulfate-polyacrylamide gel electrophoresis. The smaller subunits of the two forms appear identical (Mr=27,000), while the larger subunits of the Triton- and papain-purified enzyme have apparent molecular weights of 54,000 and 51,000, respectively. These data suggest that a peptide (3,000 to 19,000) in the larger subunit of gamma-glutamyltranspeptidase is responsible for its binding to Triton micelles and probably for holding the enzyme in the brush border membrane.     

Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes

Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis. The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000. The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000. The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively. Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively. On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions.     

The major polypeptides of the nuclear pore complex

Nuclear envelopes of maturing oocytes of various amphibia contain an unusually high number of pore complexes in very close packing. Consequently, nuclear envelopes, which can be manually isolated in great purity, provide a remarkable enrichment of nuclear pore complex material, relative to membranous and other interporous structures. When the polypeptides of nuclear envelopes isolated from oocytes of Xenopus laevis and Triturus alpestris are examined by gel electrophoresis, visualized either by staining with Coomassie blue or by radiofluorography after in vitro reaction with [³H]dansyl chloride, a characteristic pattern is obtained (10 major and 15 minor bands). This polypeptide pattern is radically different from that of the nuclear contents isolated from the same cell. Extraction of the nuclear envelope with high salt concentrations and moderately active detergents such as Triton X-100 results in the removal of membrane material but leaves most of the non-membranous structure of the pore complexes. The dry weight of the pore complex (about 0.2 femtograms) remains essentially unchanged during such extractions as measured by quantitative electron microscopy. The extracted preparations which are highly enriched in nuclear pore complex material contain only two major polypeptide components with apparent molecular weights of 150 000 and 73 000. Components of such an electrophoretic mobility are not present as major bands, if at all, in nuclear contents extracted in the same way. It is concluded that these two polypeptides are the major constituent protein(s) of the oocyte nuclear pore complex and are specific for this structure. When nuclear envelopes are isolated from rat liver and extracted with high salt buffers and Triton X-100 similar bands are predominant, but two additional major components of molecular weights of 78 000 and 66 000 are also recognized. When the rat liver nuclear membranes are further subfractionated material enriched in the 66 000 molecular weight component can be separated from the membrane material, indicating that this is relatively loosely associated material, probably a part of the nuclear matrix. The results suggest that the nuclear pore complex is not only a characteristic ubiquitous structure but also contains similar, if not identical, skeletal proteins that are remarkably resistant to drastic changes of ionic strength as well as to treatments with detergents and thiol reagents.