Changes in the protein composition of rat spermatozoa during maturation in the epididymis

Spermatozoa from the testis and cauda epididymidis were solubilized by detergent treatment and electrophoresis on SDS polyacrylamide gels revealed that the relative amounts of 13 detergent-extractable proteins decreased during passage of spermatozoa through the epididymis, 6 increased, whilst the remainder showed little or no change. Lactoperoxidase-catalysed iodination of plasma membrane proteins showed that the components carrying most of the label in testicular spermatozoa had Mr values of 110 000, 94 000, 84 000, 55 000 and 42 000 whereas on cauda epididymal spermatozoa the Mr values were 47 000, 24 000, 17 000, 14 500 and 13 500. Substantial differences were also noted in the protein composition of rete testis fluid and cauda epididymal plasma. The results support the concept that there is a considerable reorganization of the molecular architecture of the plasma membrane of spermatozoa during maturation in the epididymis.     

Molecular structure of the beta-adrenergic receptor

The beta-adrenergic receptor from several tissues has been purified to homogeneity or photoaffinity radiolabeled and its subunit molecular weight determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In this study we have examined the oligomeric structure of nondenatured beta 1- and beta 2-adrenergic receptor proteins, as solubilized with the detergent digitonin. Model systems used were frog and turkey red blood cell as well as rat, rabbit, and bovine lung plasma membrane preparations. To correct for the effects of detergent binding, sedimentation equilibrium analysis in various solvents, as adapted for the air-driven ultracentrifuge, was used. With this approach an estimate of 6 g of digitonin/g of protein binding was determined, corresponding to a ratio of 180 mol of digitonin/mol of protein. Protein molecular weights estimated by this method were 43 500 for the turkey red blood cell beta 1 receptor and 54 000 for the frog red blood cell beta 2 receptor. Molecular weights of 60 000-65 000 were estimated for beta 1 and beta 2 receptors present in mammalian lungs. These values agree with estimates of subunit molecular weight obtained by SDS gel electrophoresis of purified or photoradiolabeled preparations and suggest beta-adrenergic receptors to be digitonin solubilized from the membrane as single polypeptide chains.     

Light induced changes in the polypeptide composition of Sorghum bicolor

Two dimensional gel electrophoresis resolved total protein extracted from Sorgum bicolor (L.) Moench shoots into 430 polypeptides detectable by silver staining. The relative amunts of 82 polypeptides increased and 192 decreased upon exposure of etiolated shoots to light. Total protein extracted from etiolated mesocotyls was resolved into 360 polypeptides. The relative amounts of 23 mesocotyl polypeptides increased and 5 decreased after light exposure. Of 274 polypeptides whose relative amounts was altered in shoots exposed to light, 189 were found in mesocotyls. The relative amounts of only 6 polypeptides showed the same response to light in mesocotyls as in shoots. Immunoblotting identified 2 subunits of phosphoenolpyruvate carboxylase (PEPCase) differing in isoelectric point and molecular weight. The 81 000 dalton subunit was the major subunit found in mesocotyls while the 95 000 dalton subunit was the major subunit found in green shoots suggesting that these subunits are derived from a non-photosynthetic and photosynthetic isozyme of PEPCase. In etiolated shoots but not in mesocotyls, light induced the 81 000 and 95 000 apparent molecular weight (MW) subunits of PEPCase, the subunit of pyruvate orthophosphate dikinase (PPDK) as well as the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase). Specific activity measurements indicated that light induced the accumulation of the peroxisomal enzyme hydroxypyruvate reductase and inhibited the accumulation of the mitochondrial enzyme serine hydroxymethyltransferase in both mesocotyls andshoots. NADP-glyceraldehyde-3-phosphate dehydrogenase activity was light induced in shoots but undetectable in mesocotyls. In shoots and meocotyls, light had little effect on the mitochondrial enzyme fumarase or the cytoplasmic enzyme NAD-glyceraldehyde-3-P-dehydrogenase.     

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.     

Detection of a membrane-associated protein on detached membrane ribosomes in Staphylococcus aureus

Membrane ribosomes from Staphylococcus aureus which were detached from the membrane by extraction with the nonionic detergent Triton X-100 retained a protein (MBRP) with a molecular weight of 60 000, which was absent from cytoplasmic ribosomes. MBRP was detected and quantified by immunological methods. When membrane ribosomes were dissociated into 50S and 30S subunits, MBRP remained associated with the 50S particle. MBRP was found both on membrane ribosomes and in the cytoplasm in roughly equal amounts. When added to Triton X-100-solubilized protoplasts, antibodies to MBRP produced immunoprecipitates which contained a complex of MBRP and three other proteins with molecular weights of 71 000, 46 000 and 41 000. Four proteins with the same molecular weights as those of the MBRP complex were found associated with membrane ribosomes. The proteins of molecular weight 71 000, 60 000, 46 000 and 41 000 seemed to be present in stoichiometrically equivalent amounts in the complex.     

Adenosine-3':5'-monophosphate-binding proteins from bovine kidney. Isolation by affinity chromatography and limited proteolysis of the regulatory subunit of protein kinase II.

Affinity chromatography on cyclic AMP columns allowed a two-step isolation of the cyclic-AMP-binding proteins from bovine kidney cytosol. An AMP-binding protein (apparent molecular weight approximately 60 000) and large amounts of a low affinity binding protein ('P35'; apparent subunit size approximately 35 000) were obtained in practically pure form besides the high affinity binding proteins of the R type. Among the R proteins the dimer R2 of the regulatory subunit of protein kinase II (apparent subunit size approximately 54 000) represented the bulk material. Small amounts of monomer, of higher aggregates, and of a protein 'P49' (subunit size approximately 49 000) presumably identical with the regulatory subunit of protein kinase I were also detected. The R protein fraction of kidney also contained a high affinity binding protein of smaller size (designated as R'; molecular weight approximately 37 000) which appeared to be derived from protein R2 of protein kinase II by limited proteolysis. At all stages of purification, R protein and its aggregates could be quantitatively transformed into R' protein (or a closely related polypeptide) by several proteases including the relatively unspecific proteinase K. The degradation product exhibited unchanged cyclic-AMP-binding capacities but had largely lost the ability to inhibit the catalytic subunit C of protein kinase, to be phosphorylated by C, and to form a dimer. Preliminary experiments indicate that protein R' may be a natural component of kidney tissue.     

Further studies on bilitranslocase,a plasma membrane protein involved in hepatic organic anion uptake

Bilitranslocase, a plasma membrane protein involved in bilirubin and other organic anion uptake by the liver, exhibits a high molecular weight (170 000) when isolated in the presence of deoxycholate. This value is decreased to approx. 100 000 if deoxycholate is not included in the isolation medium. Both preparations can be resolved into two kinds of subunit, α and β, of 37 000 and 35 500, respectively, by reduction with 2-mercaptoethanol and addition of sodium dodecyl sulfate. Under these conditions the two subunits are still capable of high-affinity sulfobromophthalein binding and, despite the presence of the detergent, may be isolated by preparative polyacrylamide gel electrophoresis still associated with the dye. It may be suggested that the physiological subunit composition of bilitranslocase is α₂-β.     

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.     

Localisation of the subunits of the photosynthetic reaction centers in the chromatophore membrane of Rhodospirillum rubrum.

Reaction centers were isolated with the detergent lauryl dimethyl amine oxide from chromatophore membranes of Rhodospirillum rubrum. The subunit composition of these reaction centers is similar to the one obtained from Rhodopseudomonas spheroides: three subunits with the molecular weights of 21 000, 24 000 and 29 000. Reaction centers prepared from chromatophores labeled with 131I were heavely labeled in their large subunit (H). The smaller subunits (L and M) contained only little label. Sonication during labeling yielded a slightly higher incorporation of 131I in subunit H compared to the smaller ones. It is concluded that the H protein is largely exposed at the cytoplasmic side of the membrane but might also be accessible for iodination on the inside of the membrane while the L and M proteins are almost completely embedded in the membrane. Iodination of spheroplasts results in only a slight binding of 131I to chromatophores and reaction centers.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.     

Nuclear protein synthesis and phosphorylation in Friend erythroleukemia cells stimulated with DMSO

Patterns of nuclear protein synthesis and phosphorylation have been investigated in Friend erythroleukemia cells. The rate of incorporation of [³H]leucine and [³²P]phosphate remains relatively constant during the first 48 h of dimethylsulfoxide (DMSO) stimulation, when more than 90% of the cells commit to erythroid differentiation, but falls to 20% by 120 h. Histone H2A phosphorylation is greatly increased during DMSO treatment, but no significant changes were found in the non-histone phosphoprotein patterns as determined by gel electrophoresis. There is also a small, but reproducible, change in the relative amounts of the two sub-fractions of histone H2A. There are no striking changes in the electrophoretic patterns of [¹⁴C]leucine-labelled nuclear proteins during the first 48 h, but the amount and the synthesis of two proteins of 46 000 and 280 000 D are increased somewhat during this period. Another protein, of molecular weight 65 000, appears to be induced in low amounts.     

Subunits of the extracellular hemoglobin of Tubifex tubifex.

The molecular weight of the extracellular hemoglobin of Tubifex tubifex determined by equilibrium sedimentation is 3.0 +/- 0.2 . 10(6). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the hemoglobin dissociated into four subunits: 13 000 (subunit 1), 21 000 (subunit 2), 23 000 (subunit 3) and 47 000 (subunit 4); in the presence of mercaptoethanol two subunits were observed, 13 000 +/- 1000 (subunit I) accounting for 70--80% of the whole molecule, and 26 000 (subunit II). Electrophoresis of the subunits obtained in the absence of mercaptoethanol showed that subunit I originated from subunits 1 and 4, while subunit II originated from subunits 2 and 3. These relationships were supported by N-terminal group determinations. Gel filtration in 6 M guanidine hydrochloride showed that the molecular weight of subunit I is 17 500 and that of subunit II, 36 000. Tubifex hemoglobin appears to consist of at least seven polypeptide chains.     

Studies of a large transformation-increased membrane protein in the BHK21 cell system

We have recently described in BHK cells a plasma membrane protein of molecular weight 177 000, which is significantly increased in Hamster Sarcoma Virus-transformed cells (Lage-Davila, A. and Montagnier, L. (1977) Biochem. Biophys. Res. Commun. 79, 577–584). We present now a study of proteins from purified plasma membrane fractions in the same pair of clones. Solubilization conditions, cross-linking experiments, metabolic labelling and enzymatic radioiodination allow to characterize this 177 000 transformation-increased protein as an integral membrane glycoprotein partially exposed at the outer cell surface. Additional information on other membrane proteins in this system is also given.     

Changes in the amount and distribution of prosomal subunits during the differentiation of U937 myeloid cells: high expression of p23K

Abstract. Prosomes (Proteasomes/Multicatalytic proteinase (MCP)-complexes) are protein particles built of 28 subunits in variable composition, having proteinase activity. We have studied the changes in prosomal subunits p29K, p31K and the highly expressed p23K during the differentiation of U937 cells. Control cells had little prosomal subunit p31K in the cytoplasm, while p29K antigen was detected in both the nucleus and cytoplasm; more p23K antigen was found in the cytoplasm than in the nucleus. Flow cytometry demonstrated a biphasic intracellular decrease in prosomes during differentiation induced by phorbol-myristic-acetate (PMA) and retinoic acid plus 1,25-dihydroxycholecalciferol (RA + VD). p23K and p29K decreased both in the cytoplasm and the nucleus of differentiated cells, though the p23K antigen was concentrated near vesicles and the plasma membrane in PMA-induced cells. The p31K antigens disappeared from RA + VD-induced cells, while in PMA-induced cells, cytoplasmic labelling was unchanged and nuclear labelling was increased. Small amounts of prosomal proteins p23K and p29K were found on the outer membrane of un-induced cells. While there was no labelling on the outer membrane of RA + VD-induced cells, p23K protein increased on the plasma membrane of PMA-induced cells. The prosome-like particle protein p21K was not present to any significant extent in the intracellular compartment of control or induced cells; however, p21K was detected on the outer surface of control cells and was increased only in PMA-induced cells. The culture medium of control and induced cells contained no p21K, p23K, p29K or p31K. RA + VD seemed to induce a general decrease of prosomal subunits within the cells and at the outer surface, whereas PMA caused a migration toward the plasma membrane and an increase at the outer surface. These changes in the distribution and type of prosomes in RA + VD- and PMA-induced cells indicate that prosomes may play a part in differentiation, especially p23K which is the most highly expressed protein among those studied and presents the more important changes.     

Analysis of electrophoretic behavior of cytochrome c oxidase subunits. Retardation coefficient versus molecular weight in dodecyl sulfate urea gels.

1. Standard proteins were examined by electrophoresis in highly cross-linked polyacrylamide gels containing sodium dodecyl sulfate and urea. Their behavior was analyzed at a single gel concentration (by molecular weight vs. relative mobility) and at several gel concentrations (molecular weight vs. retardation coefficient). The validity of the latter method of analysis was established for this gel system. 2. Cytochrome c oxidase was subjected to analysis by this method. Compared to standards, subunits I and III showed increased free electrophoretic mobility, while that of subunit V was slightly decreased. The molecular weight values derived were: I, 44 600; II, 22 700; III, 23 500; IV, 16 900; V, 9400; VI, 7600; VII, 4300. The standard errors were all less than +/- 7%. 3. Isolated V and VI were analyzed by two dimensional dodecyl sulfate electrophoresis, in which the second dimension also contained urea. In contrast to their behavior when the holo-enzyme was examined, these isolated subunits V and VI no longer exchange migrating positions relative to each other during the two dimensional analysis. The molecular weight values of the isolated subunits agree with those of the holo-enzyme.     

Two microsomal-associated iron-binding proteins observed in rat small intestinal cells during iron absorption

Acrylamide gel electrophoresis of microsomal protein obtained from rat small intestinal mucosal cells, after an injection of [³H]leucine, demonstrated increased quantities of two soluble iron-binding proteins during iron absorption, one with a high molecular weight (about 400 000) and the other of intermediate molecular weight (80 000). Both proteins were present in a ribosomal-enriched sub-fraction obtained during purification of the microsomal membrame but were not identified among the purified membrane proteins.     

Determinants in the β and δ Subunit Cytoplasmic Loop Regulate Golgi Trafficking and Surface Expression of the Muscle Acetylcholine Receptor

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25–28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.     

Polypeptides of the synaptic membrane antigens D1, D2, and D3

The rat brain synaptic membrane antigens D1, D2, and D3 were labelled by 125I and precipitated by antibodies in a crossed immunoelectrophoresis. The precipitates were stained, scraped off, reduced, and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The D1 antigen was composed of two polypeptide chains, apparent molecular weights 50 300 and 116 000 D2 of only one polypeptide chain, apparent molecular weight 139 000, and D3 of three polypeptides, apparent molecular weights 14 100, 23 500, and 34 400. Higher apparent molecular weight polypeptides were present in variable amounts in the D3 precipitate, except when the synaptic membrane extracts had been pre-treated with phospholipase D.     

Internalization of insulin receptors in the isolated rat adipose cell. Demonstration of the vectorial disposition of receptor subunits

The internalization of the insulin receptor in the isolated rat adipose cell and the spatial orientation of the alpha (Mr = 135,000) and beta (Mr = 95,000) subunits of the receptor in the plasma membrane have been examined. The receptor subunits were labeled by lactoperoxidase/Na125I iodination, a technique which side-specifically labels membrane proteins in intact cells and impermeable membrane vesicles. Internalization was induced by incubating cells for 30 min at 37 degrees C in the presence of saturating insulin. Plasma, high density microsomal (endoplasmic reticulum-enriched), and low density microsomal (Golgi-enriched) membrane fractions were prepared by differential ultracentrifugation. Receptor subunit iodination was analyzed by immunoprecipitation with anti-receptor antibodies, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and autoradiography. When intact cells were surface-labeled and incubated in the absence of insulin, the alpha and beta receptor subunits were clearly observed in the plasma membrane fraction and their quantities in the microsomal membrane fractions paralleled plasma membrane contamination. Following receptor internalization, however, both subunits were decreased in the plasma membrane fraction by 20-30% and concomitantly and stoichiometrically increased in the high and low density microsomal membrane fractions, without alterations in either their apparent molecular size or proportion. In contrast, when the isolated particulate membrane fractions were directly iodinated, both subunits were labeled in the plasma membrane fraction whereas only the beta subunit was prominently labeled in the two microsomal membrane fractions. Iodination of the subcellular fractions following their solubilization in Triton X-100 again clearly labeled both subunits in all three membrane fractions in identical proportions. These results suggest that 1) insulin receptor internalization comprises the translocation of both major receptor subunits from the plasma membrane into at least two different intracellular membrane compartments associated, respectively, with the endoplasmic reticulum and Golgi-enriched membrane fractions, 2) this translocation occurs without receptor loss or alterations in receptor subunit structure, and 3) the alpha receptor subunit is primarily, if not exclusively, exposed on the extracellular surface of the plasma membrane while the beta receptor subunit traverses the membrane, and this vectorial disposition is inverted during internalization.