Identification of sperm forward motility-related proteins in human seminal plasma

Seminal plasma, an amorphous material that exists in semen, contains proteins related to sperm forward motility. Employing affinity chromatography with ConA beads and protein ultrafiltration, we isolated and concentrated proteins from heated human seminal plasma. Results of computer-assisted semen analyses (CASA) demonstrated that the forward motility index of bovine spermatozoa from the epididymal caput, incubated with proteins and theophylline, was significantly different from that of spermatozoa incubated with theophylline alone (P < 0.01). The electrophoreses revealed that the protein bands with high molecular weights in the gel of PAGE changed into low molecular weights in the gel of SDS-PAGE. Furthermore, proteins from a separated portion of the PAGE gel were still able to stimulate spermatozoa from the epididymal caput to gain forward motility. Two-dimensional (2D)-gel electrophoresis and mass spectrometry indicated that spots focused on the portion seemed, according to their amino acid sequences, to be like human alpha-1-antitrypsin and zinc-alpha-2-glycoprotein (ZAG) precursors. Western blot analysis showed the presence of these two proteins in seminal plasma. These proteins, related to the forward motility of spermatozoa in human seminal plasma, may play important roles during maturation of spermatozoa, from the epididymis through fertilization in the female reproductive tract.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

Glucosamine-labelled envelope proteins of Escherichia coli K-12 I. Electrophoretic studies and partial fractionation of the phenol-soluble proteins

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-^{1 4}C]glucosamine and L-[4,5-³H]leucine were obtained by electrophoretic separation. Envelope were isolated from cells labeleed with D-[1-^{1 4}C]glucosamine—HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. Te glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-^{1 4}C₂]pimelic acid, while ortho[^{3 2}P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAS-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.     

Glucosamine-labelled envelope proteins of Escherichia coli K-12 II. Location in inner and outer membranes

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-^{1 4}C]glucosamine and L-[4,5-³H]leucine were obtained by electrophoretic separation. Envelope were isolated from cells labeleed with D-[1-^{1 4}C]glucosamine—HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. Te glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-^{1 4}C₂]pimelic acid, while ortho[^{3 2}P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAS-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.     

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.     

Products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: methanol

Rat liver mitochondria were incubated in vitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electrophoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000.Mitochondria were incubated for 5 min with [³H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M_r, range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the M_r 27,000–40,000 range.Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform: methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform: methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel.Yeast cells were pulse labeled in vivo with [³H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform:methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a 6-fold stimulation of labeling into the band of lowest molecular weight of the chloroform: methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform: methanol.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Biogenesis of erythrocyte membrane proteins in vitro studies with rabbit reticulocytes

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [³H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polycrylamide gel electrophoresis of labeled membranes on large (19 mm) gel which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [³H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removal of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

EVIDENCE THAT THE MAJOR PROTEIN IN RAT SCIATIC NERVE MYELIN IS A GLYCOPROTEIN

Evidence is presented that the major protein of rat sciatic nerve myelin is a glycoprotein. When myelin proteins were separated by polyacrylamide gel electrophoresis, the major band which was stained with amido black–Coomassie blue was also stained with periodic acid-Schiff reagents for carbohydrate. Radioactive labelling of myelin in vivo with [³H]leucine and [¹⁴C]fucose, followed by electrophoresis of the proteins, indicated that with both isotopes the major labelled peak corresponded to the major stained band. In addition, a second smaller peak of [¹⁴C]fucose migrated ahead of the major peak. Delipidated myelin contained galactose, mannose, fucose and sialic acid.     

Cell cycle-related transfer of proteins between the nucleus and cytoplasm of Physarum polycephalum

The proteins of wild-type and polyploid plasmodia of P. polycephalum were prelabelled with [³H]leucine and [¹⁴C]leucine. The two types of plasmodia were then fused for 2 h. Following fusion the nuclei were isolated and the smaller wild-type cell nuclei separated from the larger polyploid cell nuclei. The proteins were isolated from the recipient cell nuclei and the recipient nuclear proteins extracted. Ratios of ³H/¹⁴C in the various nuclear protein fractions show that during fusion differential transfer of labelled preformed proteins from the donor cell into the recipient cell nucleus occurs. The quantity of proteins transferred varies among the different fractions and with the phase of the cell cycle. Isotopic dilution experiments indicate that these differences in protein transfer are, in part, due to a high rate of synthesis and turnover of the nuclear proteins.     

Specific non-histone protein fractions associated with ‘active’ chromatin in immunized rats

Summary Non-histone proteins of normal, non-immunized rats and rats immunized with mouse spleen cells were labelled with three different amino acids: [³H]tryptophan, [³H]methionine and [³H]leucine. Chromatin was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and residual. Non-histone protein fractions F (M_r 12 000) and H (M_r 3 000) highly labelled with [³H]tryptophan, lower with [³H]methionine but not with [³H]leucine, were present mainly in the residual fraction. After DNAse II treatment non-histone protein fractions F and H disappeared in chromatin fractions and were present in Mg^2– soluble fractions which suggests that, similar to the fractions I (M_r below 3 000) and B (M_r 120 000) described previously (5), these fractions may be associated with active transcribed genes.     

Localization of epididymal secretory proteins on rat spermatozoa

Spermatozoa from the testis and cauda epididymidis of the rat were surface labelled with radioactive iodide. Detergent extracts of radioiodinated spermatozoa immunoprecipitated with antisera against specific epididymal proteins, followed by polyacrylamide gel electrophoresis, revealed two proteins (D and E of Mr 27 000 and 28 000, respectively) which became associated with spermatozoa during epididymal transit. These proteins were observed by immunofluorescence microscopy to be located over a restricted area of the head surface. Proteins with similar molecular weight were labelled on spermatozoa from the cauda epididymidis, but not from the testis, by reaction with sodium boro[3H]hydride in the presence of galactose oxidase. However, failure to immunoprecipitate with antibodies to Proteins D and E and non-coincident migration on two-dimensional gel electrophoresis established the non-identity of these proteins. Compared with Proteins D and E, two other major epididymal secretory proteins (Proteins B and C of Mr 16 000) associated with spermatozoa to a relatively minor extent during epididymal transit.     

Inhibitor-“resistant” labelling of rat ventral prostate nuclear proteins : Further evidence consistent with protein synthesis associated with cell nuclei

When minced rat ventral prostate was incubated with labelled amino acids and cycloheximide or puromycin, the specific radioactivity of proteins associated with Triton X 100-washed nuclei exceeded that of the 105 000 g cytosol. The distribution of radioactive proteins from incubated mince, examined by SDS polyacrylamide gel electrophoresis was also consistent with labelling of some nuclear proteins that was resistant to inhibitors. Highly purified prostate nuclei, washed with detergent, labelled proteins of from 1–6 × 10⁴ D with radioactive amino acids. When these proteins were fractionated according to solubility, NaOH-soluble ‘acidic’ proteins, examined by SDS polyacrylamide gel electrophoresis, were highly labelled, with a distribution of radioactivity that differed from the patterns of 0.4 N H₂SO₄-soluble basic proteins (including histones), and proteins soluble in Krebs-Ringer-phosphate buffer. Although these results cannot be interpreted unambiguously, they are consistent with the synthesis of certain nuclear proteins at a site(s) sequestered from cycloheximide and puromycin. Nuclei may represent one such site.     

Biogenesis of erythrocyte membrane proteins in vivo studies in anemic rabbits

To study the process of red cell membrane protein synthesis we have followed the time course of [³H]leucine appearance in total protein and individual peptides of the erythrocyte membrane following injection of the amino acid into phenylhydrazine-anemic rabbits. Multiple peripheral blood samples were taken from single animals over a 5-week period. Erythrocyte membrane proteins were separated by polycrylamide gel electrophoresis in sodium dodecylsulfate and dithiothreitol; incorporation of radioactivity was determined by gel slicing and liquid scintillation spectrometry. Appearance of [³H]leucine in circulating erythrocytes reached a peak at 1–3 days, with a steady decline thereafter. The radioactive amino acid appeared first in the lowest molecular weight peptides and last in the largest peptides; at the earliest time point (8 h), little radioactivity was observed in any of the four largest peptides present in the membranes (bands A, 1, 2 and 3). Certain smaller peptides (bands 4, 5 and 9) were the predominant species labeled at this time. By 24 h all peptides showed significant incorporation. With maturation of the red cells, label largely disappeared from bands A, 9 and several smaller peptides; this was confirmed by finding that the peptides are virtually absent from mature circulating erythrocytes. These data are interpreted as showing that red cell membrane proteins are synthesized asynchronously during the life cycle of the erythrocyte; the largest peptides are made predominantly in the earlier marrow stages of development, while certain of the smaller peptides are still being synthesized in the reticulocyte stage. Several membrane proteins appear to be specific to the reticulocyte and are lost during the process of cell maturation in the circulation.     

Addition of proteins to the cylindrical gel embedding medium for transverse molecular-weight markers in two-dimensional gel electrophoresis

As an aid in the comparison of different complex mixtures of proteins resolved by two-dimensional electrophoresis, a simple method which results in the electrophoresis of molecular-weight standards as appropriately migrating, highly resolved bands extending across the entire second-dimension slab gel is described. The proteins to be used as markers are included in the molten agarose mixture used to affix the first-dimension cylindrical gel atop the second-dimension slab gel. As the proteins which are resolved in the first dimension migrate through the second-dimension slab gel, the marker proteins also migrate, experiencing the same electrophoretic conditions as the sample proteins in the immediate vicinity. If the same protein is resolved in the first dimension and also used as a marker, it electrophoreses in the second dimension as a spot intersected by a band traversing the entire gel. This sensitive method is applied to a comparison of soluble seed proteins of two cotton species, Gossypium hirsutum and G. arboreum, using G. hirsutum seed protein as the molecular-weight marker. Other applications are described.     

Rabbit epididymal secretory proteins. I. Characterization and hormonal regulation

Analyses of samples of luminal fluid from the rete testis, distal efferent ducts, and epididymal regions 2-5 and 8 revealed that 91% of the fluid leaving the testis is reabsorbed by the efferent ducts, 79% of the remainder is reabsorbed proximal to epididymal regions 4 and 5, and there is a net secretion of fluid into the duct caudally. There is a net reabsorption by the efferent ducts of 73% of the protein leaving the testis and then a net secretion along the epididymis. SDS-PAGE of the luminal fluids indicated that four new protein bands that were not present in blood appeared in the efferent ducts, 5 in epididymal regions 1-5, 6 in regions 6 and 7, and one in region 8. Two bands in samples from the efferent ducts were absent caudally, and one band present in region 7 was absent in region 8. The rates of incorporation of (35)S-methionine into minced duct in vitro varied among regions when expressed per milligram of wet weight of tissue (region 2-5 > region 7 > region 6 > region 1 > region 8 > ductuli efferentes), and orchidectomy had little effect on the rates. Incorporation into four proteins that were secreted in vitro (M(r) 38 000, 20 000, 15 000, and 13 000) was reduced or abolished by orchidectomy and restored by testosterone therapy. The secretion of three proteins (M(r) 52 000, 23 000, and 22 000) was reduced or abolished by orchidectomy and not restored by testosterone therapy. SDS-PAGE of detergent extracts of sperm indicated that five proteins were lost and nine were gained during epididymal transit. Seven of the proteins gained were about the same molecular weight as proteins secreted by the epididymis (M(r) 94 000, 52 000, 38 000, 36 000, 22 000, 20 000, and 13 000) and were analyzed using N-terminal amino acid microsequencing.     

Polypeptide synthesis by mitoplasts isolated from mouse liver

Polypeptide synthesis by mouse liver mitochondria was studied by incubating purified mitoplasts (mitochondria treated with digitonin) with [³⁵S]methionine. The products were separated either by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, or by isoelectric focusing, followed by SDS polyacrylamide gel electrophoresis. At least 14 distinct bands with molecular weights (mol. wt) ranging from about 8 000 to about 70 000 were found upon radioautography of the gels. When the samples were incubated in the presence of chloramphenicol, only a single weak band was found, whereas the protein pattern was unaffected by the presence of cycloheximide in the medium. The newly synthesized proteins were all acidic and evidence was obtained that they were hydrophobic in nature. Virtually all the labelled polypeptides were present in the membrane fraction, whereas the matrix showed little radioactivity. The data indicate that the proteins synthesized by mammalian mitochondria, like those in yeast, are components of the inner mitochondrial membrane. One protein of mol. wt 22 000 D was detected in the incubation medium. Since more of this component was present in the medium than in the pelleted mitoplasts and since this protein was not found in the matrix fraction of sonicated mitoplasts, it is believed that it had been excreted from the inner mitochondrial membrane. The finding that the number of proteins synthesized in mitoplasts isolated from mouse liver is considerably higher than that synthesized in yeast mitochondria reflects a most efficient utilization of the mammalian mitochondrial genome.     

Microsomal target proteins of metabolically activated aromatic hydrocarbons.

The specificity of binding to microsomal proteins of metabolically activated hydrocarbons has been studied. Radioactively labelled benzene, phenol, chlorobenzene, BP and MC were incubated with liver microsomes from control, phenobarbital- and MC-treated rats in the presence of an NADPH-generating system. The patterns of metabolite binding to microsomal proteins were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. Benzene, phenol and chlorobenzene metabolites showed one type of binding pattern dominated by a band at 72 000 Mr. This band was strong both in control and induced microsomes. Additional radioactive bands were seen in the 50 000--60 000 Mr region particularly in MC-induced microsomes. BP and MC metabolites showed a different type of binding pattern with incorporation of radioactivity into several fractions in the 50 000--60 000 Mr region of MC-induced microsomes. Two other strongly labelled fractions occurred at 68 000 and 72 000 Mr. The incorporation was low into control and phenobarbital-induced microsomes. Two labelled bands (Mr 56 000 and 72 000) were common for all hydrocarbons in MC-induced microsomes. The 56 000 Mr band had the same mobility in the gel as an MC-induced protein likely to be cytochrome P-448. The NADPH-generating system was essential for metabolite binding and GSH and UDPGA greatly reduced binding. We suggest that differences in metabolite binding patterns reflect differences in the routes of metabolite formation and that activated hydrocarbons are likely to bind to proteins close to their site of formation.     

Blood protein synthesis in pupae of the silkmoth, Hyalophora cecropia

The blood proteins of pupae of Hyalophora cecropia were studied by acrylamide gel electrophoresis. Analyses of fractions separated on Sephadex G-75 columns revealed that a series of low molecular weight proteins previously reported to be absent in diapausing pupae are a normal component of diapausing pupal blood. Incorporation of isotopically labelled leucine into blood protein bands of injured pupae, perfused pupae, and perfused isolated pupal abdomens (lacking a midgut) revealed that each of these three preparations could synthesize all of the low molecular weight protein bands, with maximal incorporation in the perfused whole pupae.     

HeLa cell plasma membranes : IV. Iodination of membrane proteins in synchronized cells

Intact HeLa cells and isolated HeLa cell plasma membranes were subjected to lactoperoxidase-catalysed iodination. The ¹²⁵I-labelled proteins were separated by SDS-polyacrylamide gel electrophoresis. Six protein species with apparent molecular weights from 32 000 to 200 000 were accessible to labelling from the outer cell surface, while most of the proteins present in the plasma membrane were labelled when isolated plasma membranes were iodinated. Iodination of synchronized intact cells revealed that the labelling obtained was cell cycle dependent with maximal labelling at mitosis. No changes in the distribution of radioactivity among the labelled proteins were observed when cells from different phases were iodinated.