Sodium Dodecyl Sulfate-polyacrylamide Slab Gel Research Articles

Identification of collagen types I, II, III, and V by two-dimensional fingerprinting of 125I-labeled peptides

Collagen types I, II, III, and V have been purified from chick tissues and separated into their component α chains by electrophoresis under reducing conditions in sodium dodecyl sulfate-polyacrylamide slab gels. The isolated collagen α chains contained within single Coomassie blue-staining bands from dried slab gels were then radiolabeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate (Bolton-Hunter reagent) and cleaved simultaneously with trypsin and chymotrypsin into peptides which were subsequently separated on thin-layer cellulose plates by electrophoresis (first dimension) and chromatography (second dimension). Two-dimensional peptide mapping of collagen α chains has been routinely performed on submicrogram amounts of collagens (less than 0.1 μg). Each type of collagen α chain reproducibly displays a peptide fingerprint distinct from those of the other collagen-type α chains. Thus, the identification of an individual collagen-type α chain can be readily accomplished even when it is present in a mixture with other α chains in the same gel slice.

The identification of αA and αB collagen chains in hypertrophic scar

Hypertrophic scarring develop as a result of full thickness dermal destruction. The connective tissue matrix of this lesion is mostly collagen. Collagen extracted by limited pepsin digestion was partially characterized, using bulk salt and heat-gelling techniques. The different collagen types isolated were Types I, III, and V or AB collagen composed of αA and αB chains. The identity of these collagen types was established by stained protein band patterns on sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The possible location of Type V or AB collagen in hypertrophic scar is discussed.

Investigation of a hemolysin produced by enteropathogenic Treponema hyodysenteriae.

A hemolysin produced by Treponema hyodysenteriae, the etiological agent of swine dysentery, was investigated. A virulent isolate (B204) was inoculated into a standard culture medium consisting of Trypticase soy broth without dextrose (BBL Microbiology Systems) supplemented with 10% fetal calf serum in an atmosphere of 70:30 deoxygenated H2-CO2. Sterile cell-free filtrates were prepared at 2-h intervals and assayed for hemolytic activity by using washed sheep erythrocytes. The maximum hemolytic titer was obtained during the early log phase of growth (4 h). A loss of hemolytic activity was observed when cell-free filtrates were stored at 23 and 4 degrees C. Storage at -20 or -80 degrees C after lyophilization resulted in retention of the hemolytic titer for periods of up to 30 days. Enzymatic inactivation of the hemolysin was accomplished with pronase, but not with deoxyribonuclease, ribonuclease, lipase, or trypsin. Addition of exogenous ribonucleic acid-core to the standard culture medium resulted in a dose-dependent increase in the amount of hemolysin produced. The hemolysin could be purified by acid and ammonium sulfate precipitation followed by ion exchange and molecular sieve chromatography. The molecular weight of the hemolysin was 68,000 when determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.

Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine.

Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, Mr = 60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50 mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker alpha-isopropyl-alpha-[(N-methyl-N-homoveratryl)-gamma-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 microM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+-dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 microM and 2.5 microM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.

Partial phosphorylation in vivo of the avian retrovirus pp32 DNA endonuclease.

Avian retrovirus pp32, a DNA endonuclease which is structurally related to the avian retrovirus DNA polymerase beta polypeptide, has been demonstrated to be partially phosphorylated in vivo. Unlabeled or [35S]methionine-labeled pp32 from avian sarcoma virus or avian myeloblastosis virus migrated as an electrophoretic doublet on discontinuous sodium dodecyl sulfate-polyacrylamide slab gels. However, pp32 immunoprecipitated from avian sarcoma virus labeled in vivo with [32P]orthophosphoric acid migrated as a single band, which co-electrophoresed with the slower-moving band of the doublet represented by unlabeled or 35S-labeled pp32. The presence of a slower-migrating phosphorylated band in pp32 suggests that the observed electrophoretic heterogeneity of purified pp32 is due to partial phosphorylation. Tryptic peptide analysis of 32P-labeled avian sarcoma virus beta and pp32 demonstrated that all the three labeled peptides in the beta polypeptide were also present in pp32. However, pp32 had one tryptic peptide which was preferentially labeled in comparison to the comigrating peptide found in beta digests, suggesting that phosphorylation may play a role in the processing of pp32 from beta or in the regulation of its associated DNA endonuclease activity.

Solubilization and characterization of adrenal and uterine angiotensin II receptors after photoaffinity labeling.

The physical characteristic of the receptors for angiotensin II in dog adrenal cortex and uterus were determined after affinity labeling. 125I-nitro-5-azido-benzoyl-angiotensin II, a photosensitive angiotensin II analogue which retained aldosterone-stimulating activity, was used to couple the octapeptide specifically and irreversibly to its membrane receptors. After solubilization with Triton X-100, the covalent hormone . receptor complex was analyzed by gel filtration and sucrose density gradient centrifugation. Two radioactive species were consistently observed, with calculated Mr values of 126,000 +/- 10,000 and 64,500 +/- 11,000. the elution profiles of solubilized adrenal and uterine particles were almost identical. When the solubilized complexes were subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, a single radioactive band was detected upon autoradiography, with Mr - 65,000 +/- 6,000 for adrenal cortex and 68,000 +/- 7,000 for myometrium. These results indicate that the receptors for angiotensin II in adrenal cortex and uterus are composed of two subunits of similar molecular weight, and that the common functional properties of the receptors from both tissues are probably related to their similar physicochemical characteristics.

Evidence that arylhydroxamic acid N,O-acyltransferase and the genetically polymorphic N-acetyltransferase are properties of the same enzyme in rabbit liver.

Arylhydroxamic acid N,O-acyltransferase and Co-ASAc-dependent N-acetyltransferase activities were measured simultaneously in liver cytosols from rabbits of known acetylator phenotype. Both activities were high in rapid acetylator rabbits and low in slow acetylator rabbits indicating that these two acetyl transfer steps in the metabolic activation of certain arylamines are under common genetic control in this species. The two enzyme activities could not be resolved by sequential centrifugation, fractional precipitation with ammonium sulfate, ion exchange chromatograhy on DEAE-cellulose, gel filtration on Sephacryl, and electrophoresis on polyacrylamide gels. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis of extracts of disc gel electrophoretic slices showed that a single symmetrical protein band with a molecular weight of 33,000 was associated with both activities. The results obtained strongly suggest that the CoASAc-dependent N-acetyltransferase reaction and the intramolecular N,O-acetyl transfer by arylhydroxamic acid N,O-acyltransferase are properties of the same enzyme.

Virion envelope glycoproteins as epidermiological markers of Venezuelan encephalitis virus isolates.

Virion polypeptide compositions of 26 isolates of Venezuelan encephalitis virus were analyzed by a reproducible and comparative technique of discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Although the molecular weight of the core polypeptide for each isolate was 36,000, numbers and molecular weights of envelope glycoproteins were heterogeneous. Isolates associated with human, but not equine, disease usually had two glycoproteins of 50,000 to 51,000 and 51,000 to 55,000 molecular weight, whereas isolates associated with both human and equine disease usually had an additional, third polypeptide band of either 45,000 to 46,000 or 56,000 to 58,000 molecular weight. The former isolates were in hemagglutination inhibition subtypes I-D, I-E, III, or IV, and the latter were in subtypes I-A, I-B, I-C, or II. Thus virion envelope glycoproteins should be useful markers of Venezuelan encephalitis virus isolates in epidemiological investigations.

De novo synthesis and secretion of heterogeneous forms of human chorionic gonadotropin and its free alpha-subunit in the human choriocarcinoma clonal cell line JEG-3.

The clonal human choriocarcinoma cell line JEG-3 secretes hCG and heterogeneous forms of its free alpha-subunit. We have studied the relationship of these forms in de novo biosynthesis experiments. Cells at near confluence were labeled with [35S]methionine by continuous (10-min to 24-h exposure) and by pulse-chase (5-min exposure, 10-min to 4-h chase) techniques. Media and cell lysates, chromatographed in Sephadex G-100 or, after reduction, electrophoresed on 12-20% gradient sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAG:), were analyzed by immunoprecipitation with antisera to hCT-alpha and hCG-beta. The intracellular labeled free alpha-subunit observed in lysates at all time points of either continuous or pulse-chase experiments was the same size or was slightly smaller on G-100 (apparent mol wt, 21,600 +/- 3,900) than urinary standard alpha-subunit (CR119 alpha; apparent mol wt, 22,700 +/- 1,500) and also somewhat smaller on SDS-PAGE (apparent mol wt, 19,400 +/- 600) than urinary standard alpha-subunit (apparent mol wt, 20,200 +/- 1,100). However, the predominant form of secreted free alpha-subunit, at chase times as early as 30 min, migrated with a higher apparent molecular weight in both systems (SDS-PAGE, 22,100 +/- 400; G-100, 29,300 +/- 2,700). We have not observed this secreted large free alpha-subunit in the cell lysate, and our data suggest that the small intracellular alpha-subunit is a precursor of the larger secreted alpha-subunit and not vice versa. The beta-subunit of secreted hCG was somewhat larger (apparent mol wt, 31,500 +/- 1,000) on SDS-PAGE than standard beta-subunit (CR119-2 beta and CR115 beta; apparent mol wt, 29,900 +/- 1,900). Secreted intact hCG migrated with urinary standard hCG (CR119) on G-100, but analysis of 35S-labeled intracellular hCG was complicated by co-precipitating large mol wt proteins. De novo synthesis and secretion of a large form of free alpha-subunit as well as a large beta-subunit in hCG may be due to posttranslational oligosaccharide addition during the secretory process.

Biosynthesis of polymyxin by Bacillus polymyxa: II. On the nature and interaction of the multienzyme complex with the end product polymyxin

The interaction of polymyxin with the producer organism Bacillus polymyxa has been shown to be at the level of membranes, resulting in an enhancement of the activities of its own biosynthetic enzymes. This enhancement has been shown to be due to the solubilization of the membrane-associated multienzyme complex by polymyxin in a specific manner. The relevance of this physiological feature was also indicated by the appearance of the soluble multienzyme complex activity only in cells, which synthesize maximal amounts of polymyxin. Purification of the polymyxin released multienzyme complex from the membranes and the soluble form of the complex from the stationary phase cells has revealed several similarities between them. Both contain two major fractions of the molecular weights of 300,000 and 170,000, harboring all the polymyxin component amino acid-activating enzymes. Their multisubunit nature was established by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Using mutants blocked in sporulation and/or antibiotic synthesis, it was shown that the interaction of polymyxin with the producer organism was inoperative when antibiotic production was curtailed. This interaction has been suggested as one of the early sporulation-specific phenemenon.

Assembly of the sarcoplasmic reticulum. Biosynthesis of the high affinity calcium binding protein in rat skeletal muscle cell cultures.

Temporal patterns of biosynthesis of the high affinity calcium binding protein from the sarcoplasmic reticulum were determined and compared with rates of ATPase ane cells. Cells at various stages of differentiation were incubated for 2 h with [35S]methionine. Specific proteins were isolated from detergent extracts of cells by incubation with antibodies specific against the various proteins and immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Radioactivity incorporated into specific bands was analyzed by counting gel slices and incorporation data were used to obtain relative rates of individual protein sys found to be indistinguishable from that of calsequestrin when cells were grown in standard medium, in medium containing 60 microM Ca2+ which prevented fusion of cells, or in enriched medium which delayed cell fusion. The high affinity calcium binding protein had a relatively high turnover rate with a half-life of about 10 h. These studies suggest that synthesis of calsequestrin and the high affinity calcium binding protein are coordinated even though calsequestrin is a glycoprotein, whereas the high affinity calcium binding protein is not glycosylated.

Germination-induced Changes in Chromosomal Proteins of Spring and Winter Wheat Embryos

The template activity of chromatin from winter wheat embryos gradually increased during germination and was regulated with some nonhistone proteins different from the two major ones, molecular weight 39k and 59k polypeptides, previously reported.To clarify chromosomal proteins which are involved in regulation of template activity of chromatin, we studied the quantitative and qualitative changes in chromosomal proteins. Differences in acid-soluble and acid-insoluble proteins between chromatins from wheat germ and embryos germinated for various times were visualized by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.Nonhistone proteins of 39k, 41k, and 50k molecular weights were specifically present in wheat germ and in 24- or 48-hour germinated wheat embryos, thereafter greatly reduced or finally disappeared. In contrast, nonhistone protein of 37k was absent in germ and in embryos germinated for 24 hours and appeared after 48 hours of germination. Thereafter it was present in abundant amounts in 96-hour germinated winter wheat embryos and in 72-hour germinated spring embryos, corresponding to 7 and 10% of total nonhistone proteins, respectively. Histone H1, especially H1d, was slightly reduced after 48-hour germination, as much as basic nonhistone proteins having electrophoretic mobilities between H1d and H2B. Further-more, similarity and diversity of chromosomal proteins between spring and winter wheat embryos are shown in this study. A subspecies of histone H1c of spring wheat had faster electrophoretic mobility than that of winter wheat.

The proteins of nonoccluded Autographa californica nuclear polyhedrosis virus produced in an established cell line of Spodoptera frugiperda

Nonoccluded virions have been isolated from the extracellular fluid of cultured Spodoptera frugiperda cells that have been infected with Autographa californica nuclear polyhedrosis virus. By sucrose density gradient centrifugation, infectious virus particles have been obtained that exhibit densities of about 1.19 g/cm 3. Using staining and autoradiographic techniques, about 35 polypeptides, including some high molecular weight proteins, have been detected by analysis of the virion proteins on sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAGE). Two major proteins of MW 65,000 and 42,000 have been found.

Fat cell protein phosphorylation. Identification of phosphoprotein-2 as ATP-citrate lyase.

We have purified to apparent homogeneity a phosphoprotein from rat adipose tissue which is rapidly phosphorylated in vitro by ATP. The native phosphoprotein has an approximate sedimentation coefficient of 14.8 S. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the protein dissociated into identical subunits of Mr = 128,000. A phosphoprotein with similar properties was also isolated from liver. Purified phosphoproteins from fat cells and liver had ATP-citrate lyase activity and co-migrated on sodium dodecyl sulfate gels with fat cell phosphoprotein-2, the phosphorylation of which is increased by incubating fat cells with insulin. The phosphoamino acid residue of the cells with insulin. The phosphoamino acid residue of the phosphoprotein was identified as tau-phosphohistidine. These evidences suggest that fat cell phosphoprotein-2 is ATP-citrate lyase.

Turnover of prolyl hydroxylase and an immunologically related protein in rabbit tissue

The turnover rates of prolyl hydroxylase and immunologically related (cross reacting) protein were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and immunologically cross-reacting protein back to basal levels. Prolyl hydroxylase and immunologically cross-reacting protein were purified from neonatal rabbit skin at various times following the administration of [ 3H]leucine. Prolyl hydroxylase was purified by affinity chromatography. Immunologically cross-reacting protein was purified by antibody precipitation from the dialyzed 70% (NH 4)SO 4 supernatants and subsequent electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide slab gels. The radioactivity of the species isolated, which corresponded to the two major subunits of prolyl hydroxylase, was used in the turnover studies of immunologically cross-reacting protein. The peak incorporation of label into prolyl hydroxylase was found to be 12 h while for immunologically cross-reacting protein this occured within 2 h. The loss of radioactivity from these protein pools denotes an apparent t 1 2 for prolyl hydroxylase of 73 h and a 1 2 for immunologically cross-reacting protein of 53 h. From the specific activity of free skin leucine pools, the effect of reutilization could be corrected and a true t 1 2 for prolyl hydroxylase of 45 h was determined. The t 1 2 values of these proteins were determined by a second method in which prolyl hydroxylase and immunologically cross-reacting protein in the aorta and liver of adult male rabbits were elevated by daily epinephrine-thyroxine treatment for 12 days. The decline of prolyl hydroxylase and immunologically cross-reacting protein with termination of treatment in the aorta denotes values of 42 h for enzyme and 53 h for immunologically cross-reacting protein. Calculated enzyme κ d values, by both methods, indicate that breakdown of enzyme does not account for tissue immunologically cross-reacting protein.

Evidence for a proteolytic modification of liver pyruvate kinase in fasted rats

Liver pyruvate kinase was purified to homogeneity from rats fed a high carbohydrate, low protein diet (LPK-C) and from rats fasted for 84 h (LPK-F). Although the enzymes have similar electrophoretic mobilities in 7% polyacrylamide disc gels, the specific activity of LPK-C was two to three times the value of the specific activity of LPK-F. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPK-C yields a single protein band of 56,000 daltons. In contrast, LPK-F yields two bands of protein. Approximately one-third of the LPK-F has an electrophoretic mobility similar to the 56,000-dalton LPK-C peptide. The remaining two-thirds of the LPK-F protein migrates as a 51,000-dalton peptide. Cyanogen bromide was used to cleave LPK-C and LPK-F. Similar peptide patterns were obtained from LPK-C and LPK-F when the cyanogen bromide fragments were resolved by 12% polyacrylamide gel electrophoresis in 7.5 m urea containing 6 m m Triton X-100 and 5% acetic acid. Separation of the two peptides from LPK-F was accomplished by selective immunologie absorption of the 56,000-dalton peptide with anti-LPK-C gammaglobulin immobilized on Sepharose 4B. Tryptic digests of LPK-C, LPK-F and the 51,000-dalton peptide yield similar peptide patterns when analyzed via sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. These results suggest that the 51,000-dalton peptide could be derived by a proteolytic cleavage or limited digestion of the 56,000-dalton subunit. Phosphorylation of LPK-C and LPK-F by [γ- 32P]ATP in vitro with cyclic AMP-activated protein kinase results in covalent incorporation of 32 P into only the 56,000-dalton subunit. These results suggest that an in vivo proteolytic modification that yields the 51,000-dalton subunit.

The tritlum labeling of small amounts of protein for analysis by electrophoresis on sodium dodecyl sulfate-polyacrylamide slab gels

A simple and reproducible method for the tritium labeling of small amounts of proteins prior to analysis under denaturing conditions on polyacrylamide slab gels is described. The method involves the in vitro labeling of proteins by reductive methylation using formaldehyde and high specific activity [ 3H]potassium borohydride. Labeled proteins were detected by fluorography after fractionation on polyacrylamide slab gels in the presence of sodium dodecylsulfate. The overall procedure allows the analysis and molecular weight estimation of submicrogram quantities of protein.

Peptide mapping of heterogeneous protein samples.

A simple two-dimensional electrophoretic method for peptide mapping of heterogeneous protein samples is presented. The reduced and denatured proteins of the mixture are separated in a first dimension by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. After completion of the electrophoresis, the whole gel lane is equilibrated in stacking gel buffer and is transferred at right angles onto a second slab gel. A protease solution is overlayed on the gel lane and a partial proteolysis of the proteins to be analyzed is performed during the stacking phase of the second electrophoresis. The second electrophoresis resolves the characteristic pattern of peptides of each individual protein as a series of spots located below the original position of the undigested protein. The peptide maps of the following samples are presented as examples: protein P23 and P23* of bacteriophage T4, membranes of Dictyostelium discoideum, membranes of human erythrocytes, and 35S-labeled proteins of D. discoideum synthesized in vivo or in a cell-free wheat germ extract. In complex samples, up to 20 individual proteins can be analyzed at once and a protein comprising only 1% of the total sample generates a clearly identifiable peptide pattern. Good reproducibility of the patterns obtained allows the comparison of samples of different origins.

Penicillin-binding proteins in Proteus species

Penicillin-binding proteins in three species of Proteus, Proteus mirabilis, P. morganii, and P. rettgeri, were investigated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Penicillin-binding proteins in these Proteus species were compared with those in Escherichia coli K-12. An approximate correlation between penicillin-binding proteins in E. coli and those in Proteus species was shown by several criteria: electrophoretic mobilities; affinities of several beta-lactam antibiotics which show characteristic patterns of binding to penicillin-binding proteins in E. coli; relation between affinities of antibiotics to the proteins and effects on morphological changes in Proteus species; location of beta-lactamase activity among penicillin-binding proteins; and thermostability. The electrophoretic mobilities and several other characteristics of penicillin-binding proteins among the Proteus species examined were found to be similar from species to species and differed only slightly from those of E. coli.

Cell surface changes in the proteins of rabbit spermatozoa during epididymal passage

AbstractDifferences in the exposure of spermatozoa surface components during epididymal passage have been examined using lactoperoxidase‐catalyzed 125I‐iodination or labeling with 125I‐diazodiiodosulfanilic acid. Labeled surface proteins obtained from caput and cauda epididymides were solubilized in detergent, separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis, and identified by radiography. Densitometer scans of autoradiograms revealed increased amounts or exposures of surface proteins of ∼35,000, ∼39,000, ∼50,000, and ∼78,000 molecular weight on the cauda epididymal spermatozoa.