Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of proteins solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.     

Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis.

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of protein solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.     

Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents.

It was shown that in linear polyacrylamide gradient gels migration distance of a given protein increases as a function of the square root of the time of electrophoresis. The linearity between these two parameters is demonstrated by the statistical analysis of experimental data obtained with proteins of different shapes and a wide range of electrophoretic mobility. The slopes of the regression lines calculated by this method can be utilized to determine the molecular weight of a nondenatured protein. In fact, there is a linear relationship between the log of the molecular weights and the log of the slopes for proteins with M_rs between 20,000 and 950,000.     

Ribosomal proteins from rabbit reticulocytes: number and molecular weights of proteins from ribosomal subunits.

The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Biochemical, biophysical, and biological properties of densonucleosis virus. I. Structural proteins.

The polypeptide composition of highly purified densonucleosis virus was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viral proteins showed a different behavior in sodium dodecyl sulfate-gels in comparison with the marker proteins. Therefore, the molecular weights were estimated by analyzing the retardation of the electrophoretic mobility of these proteins in gels with increasing polyacrylamide concentrations. Four structural proteins with molecular weights of 49,000, 58,500, 69,000, and 98,000 were found, ant they were designated p49, p59, p69, and p98, respectively. There are several indications that p98 is a dimer of p49. The relative quantity of the structural proteins in a virion suggests that at least p49 (accounting for +/-70% of total protein mass) is a capsid protein and that there will be 12 capsomers per virion.     

Effect of ACTH on rabbit adrenal microsomal 17 alpha-hydroxylase activity, cytochrome P-450 and protein electrophoretic patterns

To determine whether a change in microsomal proteins can be correlated with adrenocorticotropic hormone (ACTH)-stimulation of rabbit adrenal 17 alpha-hydroxylase activity, rabbit adrenal microsomes were subjected to electrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate. Microsomes were obtained from rabbits stimulated with ACTH for 0, 2, 4, and 6 days. A protein band with a molecular weight of 53,000 was found to increase 31.1, 27.2 and 61.0 percent in 2-, 4-, and 6-day ACTH-stimulated microsomes as compared to controls; but 17 alpha-hydroxylase activity showed no apparent correlation, increasing 5-6 fold in all experiments. No new protein bands were found after ACTH stimulation, and no other changes in microsomal protein electrophoretic patterns after ACTH stimulation were found to correlate with the increases in 17 alpha-hydroxylase activity. The specific activity (nmol/mg protein) of cytochrome P-450 remained nearly the same throughout the stimulation periods. Tetramethylbenzidine staining for heme prosthetic groups on the electrophoretic gels displayed bands with molecular weights of 61,000, 58,000 and 53,000.     

An electrophoretic procedure for detecting proteins that bind actin monomers

The electrophoretic mobility of fluorescently labeled G-actin in polyacrylamide gels under nondenaturing conditions is altered by the formation of complexes with actin-binding proteins. This effect offers a convenient method for detecting and quantitating such proteins in tissue fractions and for monitoring their purification. When followed by second-dimension electrophoresis in the presence of sodium dodecyl sulfate, the method also gives the apparent molecular weights of the actin-binding components and the stoichiometry of the complexes. The method has also been used to identify actin-binding fragments in digests of actin-binding proteins, to investigate the formation of multicomponent complexes, and to determine the calcium-sensitivity of complexes.     

Electrophoretic and immunological charactorization of rat neurophysin

1. The electrophoretic properties of rat posterior pituitary proteins have been compared on starch gel with those of bovine and porcine neurophysins. 2. [(35)S]-Cysteine was injected into the supraoptic nucleus of male rats and 16-24h later the distribution of labelled neural-lobe protein in starch and polyacrylamide gels was determined. In both systems a single major protein component was found to contain more than 80% of the total recovered radioactivity. Between 5 and 10% of the radioactivity was found in a minor component in polyacrylamide gel. 3. In agar, microimmuno-diffusion and -electrophoresis of the rat neural-lobe proteins gave a single arc with neurophysin antiserum, and after starch-gel electrophoresis this arc was shown to be due to the major labelled component. 4. The molecular weights of the rat neural-lobe proteins were estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the major labelled component was found to be 12000. 5. It is concluded that the rat neurophysin consists of one major and possibly one minor component.     

Protein constituents of the mouse spermatozoon: I. An electrophoretic characterization

Total protein constituents of the mouse spermatozoon have been fractionated and characterized by polyacrylamide gel electrophoresis. Three spermatozoan fractions were obtained following homogenization with 1% sodium dodecylsulfate (SDS) and sucrose gradient centrifugation: SDS-soluble proteins, SDS-insoluble tail components, and SDS-insoluble head components. Purities of these fractions were assessed at greater than 95% using Nomarksi differential interference microscopy. Subsequently, the SDS-insoluble sperm heads were further fractionated into five protein subclasses by ultracentrifugation and ion-exchange chromatography. SDS-Polyacrylamide gel electrophoresis indicates that each of these spermatozoan fractions contains distinct protein species. Furthermore, the electrophoretic profiles are highly reproducible and show no evidence of cross-contamination or proteolysis. The SDS-soluble fraction, which includes proteins from the plasma membrane, acrosome, axoneme, matrix and cristae of the mitochondria, contains one major 39,000-molecular weight band and numerous minor bands with molecular weights ranging from ～30,000 to greater than 100,000. In contrast, electrophoresis of the SDS-insoluble tail proteins reveals the presence of at least nine prominent bands with apparent molecular weights between 21,000 and 89,000. Ultrastructural analysis suggests that this fraction contains proteins from the outer dense fibers, fibrous sheath, outer mitochondrial membranes, and structural elements of the neck region of the sperm tail. Two subfractions of the SDS-insoluble sperm heads each contain one of the two mouse protamines. In addition, the acidic and moderately basic head fractions each contain a limited number of distinct protein bands with molecular weights ranging from 14,000 to 76,000. These proteins are apparently derived from either the spermatozoan nucleus or the associated perinuclear material, since all other sperm head structures are solubilized during SDS treatment. One- and two-dimensional electrophoresis on acetic acid-urea polyacrylamide gels indicates that the moderately basic fraction may contain minor components that resemble certain histones and/or spermatidal basic nuclear proteins.     

An electroblotting apparatus for multiple replica technique and identification of human serum proteins on micro two-dimensional gels

A new apparatus for electrophoretic transfer of proteins from micro polyacrylamide slab gels has been developed. The apparatus enabled the easy changing of nitrocellulose sheets and was suited for obtaining multiple blots from a gel. Electrophoretic conditions were determined so that all of the blots obtained sequentially from one slab gel were successfully used to visualize specific proteins irrespective of their molecular weight. Combining the transfer technique with the technique of parallel micro two-dimensional electrophoresis, 20 blots could be obtained within 1 h of electroblotting time. The locations of 28 human serum proteins were determined simultaneously on these blots using commercial specific antisera.     

A simplified gel electrophoretic system and its validity for molecular weight determinations of protein-cetyltrimethylammonium complexes

Proteins ranging in molecular weight from 90,000 to 15,000 daltons were studied by polyacrylamide gel electrophoresis in the presence of cetyltrimethylammonium bromide (CTAB) at pH 4.6. The results showed a linear relationship between log molecular weight and relative electrophoretic mobility. The simplicity and reliability of the method offer an alternative means of determination of molecular weights of a wide variety of proteins by gel electrophoresis.     

Tissue-specific secretory proteins of the salivary glands of Chironomus thummi An electrophoretic and immunochemical analysis

The salivary proteins of Chironomus thummi larvae were separated by SDS gel electrophoresis and characterized by immunological techniques. As a result, five protein fractions (sp-220, sp-180, sp-35, sp-18, and sp-16) with molecular weights Mr=220000, 180000, 35000, 18000 and 16000 were identified in 6%–20% polyacrylamide gradient gels. In addition, three giant proteins fractions (molecular weights exceeding Mr=800000) were detected in composite polyacrylamide-agarose gels. Crossed immunoelectrophoresis allowed us to identify five immunochemically dissimilar organ-specific antigen fractions in the salivary gland secretion. Data were obtained indicating that the protein fractions, sp-220, sp-180, sp-35, sp-18, and sp-16, are immunochemically and structurally similar. The giant secretory proteins and the secretory fractions with low molecular weights were found to be immunochemically unrelated.     

Characterization and Comparison of Neurofilament Proteins from Rat and Mouse CNS

Rat and mouse CNS neurofilament proteins (NFPs) were characterized and compared, in terms of electrophoretic properties on polyacrylamide gels and by peptide mapping, with one another and with other co-purifying lower-molecular-weight CNS proteins, including α and β tubulin. NFPs were partially purified by modification of the axon flotation procedure of Norton and co-workers and were demyelinated with Triton X-100. On one-dimensional SDS polyacrylamide gels the molecular weights of the triad of NFPs from both rat and mouse were approximately 200,000, 140,000, and 70,000. Prominent lower-molecular-weight proteins (63,000-16,000) as well as minor amounts of tubulin and actin were observed after gel electrophoresis. On two-dimensional gels (isoelectric focusing followed by SDS gel electrophoresis) each of the NFPs appeared to be composed of more than one component and the corresponding NFPs from rat and mouse had similar isoelectric points. Gel electrophoresis peptide mapping using Staphylococcus aureus V8 protease indicated the following: (1) the triad of NFPs of different sizes have different peptide maps; (2) α and β tubulin have nonidentical digestion products, which are dissimilar to those of the NFPs; (3) other proteins that co-purify by the axon flotation procedure also have nonidentical peptide maps; and (4) the corresponding NFPs from rat and mouse have similar peptide maps. The co-purifying proteins examined in detail (63,000–49,000) do not appear to be derived by proteolytic cleavage of NFPs and may represent other cytoskeletal constituents.     

A simplified electrophoretic system for determining molecular weights of proteins.

Electrophoresis of 31 different proteins in commercially prepared polyacrylamide gradient gels, Gradipore, yields a linear relationship between a hypothetical limiting pore size (the reciprocal of a limiting gel concentration, GL) and the cube root of the mol.wt., over the range 13 500-9000 000. A regression analysis of these data reveals that 98.6% of all variability in 1/GL is explained by the molecular weight, and this degree of accuracy compares favourably with existing methods for the determination of molecular weight by retardation of mobility in polyacrylamide. This new procedure has the additional advantages that molecular-weight standards can be obtained from readily available body fluids or tissue extracts by localizing enzymes and other proteins by standard histochemical methods, and that the same electrophoretic system can be used in determining molecular weights as is used in routine surveys of populations for individual and species variation in protein heterogeneity.     

Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes

The proteins of Sarcoplasmic reticulum membranes were resolved by polyacrylamide gel electrophoresis into several fractions ranging in mol wt from 300,000 to about 30,000. The ATPase enzyme involved in Ca²⁺ transport is associated with a major protein fraction and its molecular weight based on its electrophoretic mobility on polyacrylamide gels in the presence of sodium dodecylsulfate is about 106,000. Reducing agents (β-mercaptoethanol or dithiothreitol) cause the dissociation of membrane proteins into subunits of 20,000–60,000 mol wt, which can be separated by electrophoresis or Sephadex G-150 chromatography.     

Cellulolytic enzyme system of Acetivibrio cellulolyticus

Polyacrylamide gel electrophoresis of the cellulolytic system from culture supernates of Acetivibrio cellulolyticus showed the presence of four major enzymes: a beta-glucosidase, an exoglucanase, and two endoglucanases. The relative proportions of these enzymes in the culture supernate were affected by the nature of the cellulosic substrate and by the length of the incubation period. The molecular weights of the cellulolytic enzymes were beta-glucosidase, 81 000; exoglucanase, 38 000; endoglucanase C2, 33 000; and endoglucanase C3, 10 400, as estimated by their electrophoretic mobilities relative to proteins of known molecular weight. Treatment of the high molecular weight endoglucanase with SDS--mercaptoethanol led to reversible dissociation of the enzyme into polypeptide subunits similar to the low molecular weight endoglucanase. Endoglucanase activity could be assayed for directly using a novel method of incorporating carboxymethyl cellulose in the polyacrylamide gels. The molecular weights and functions of these enzymes are compared with those detected in culture filtrates of various fungi.     

Studies of basic proteins of 60S- and 40S-subunits of pea seed ribosomes by two-dimensional polyacrylamide gel electrophoresis]

Basic proteins of 60S- and 40S-subunits of pea seed ribosomes were studied by two-dimensional electrophoresis in polyacrylamide gel (PAAG) with subsequent electrophoresis of separated proteins in the gels containing sodium dodecyl sulfate. The proteins under study were found to be electrophoretically heterogenous and showed considerable variations in the staining by amido black and a specific distribution between the two subunits. 47 protein components were detected in the protein preparations of the 60S subunit: 18--as intensively stained, 12--as moderately stained and 17--as weakly stained spots. Presumably, the 60S subunit does not contain proteins whose molecular weights are over 60.000 or below 14.000. Two proteins have mol. weight over 50.000; other proteins have mol. weights varying between 15.000 and 30.000. 32 proteins components were revealed in the protein preparations of the 40S subunit: 15--as intensively coloured, 8--as moderately coloured and 9--as weakly coloured spots. The 40S subunit does not contain proteins whose molecular weights are over 33.000 and below 10.000. Three proteins have mol. weights over 30.000, the other proteins have mol. weights within the interval of 15.000--30.000. The amount of basic proteins in the 80S plant ribosomes is, in all probability, higher as compared to that in animal ribosomes, and this is due to the 60S subunit.     

Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride

Several proteins, which are used as molecular weight markers in polyacrylamide gel electrophoresis, were reacted with dabsyl chloride. This labeled them deep orange and the chromophore attachment was stable throughout the electrophoretic procedure and fixation. Small amounts (10-50 micrograms) of the labeled proteins could be loaded onto gels and seen with the unaided eye so that the separation during electrophoresis could be followed. Dabsylation did not affect the mobility of the proteins. The location of the orange band gave a good indication of the position of the protein in the gel so that molecular weight estimations could be made during and immediately following electrophoresis.