Allelic variants of acidic proline-rich proteins observed in Japanese,Chinese, and Malays

Three new variants of acidic proline-rich proteins (At, Au, Aw) were found in human parotid saliva by isoelectric focusing and basic gel electrophoresis. Electrophoretic comparison of the purified proteins and their tryptic peptides suggested minor charge and size differences from other acidic PRPs. Genetic and biochemical studies indicate that the At and Aw proteins are allelic products of thePRH1 locus. Gene frequencies of the At productive allele (PRH1 ⁶) in Japanese, Chinese, and Malays were 0.008, 0.012, and 0.004, respectively. The Au protein was also found in Japanese (2 in 746 samples), Chinese (1 in 215 samples), and Malays (1 in 220 samples), however, the Aw protein was found only in one Japanese (n=746). These three proteins were not found in 106 Indian subjects.This study was supported in part by a grant from International Scientific Research Program of the Ministry of Education, Science and Culture of Japan, No. 62043071 (representative: K.S.).     

Charge-dependent staining of proteins after electrophoretic separation in polyacrylamide gels

A staining procedure is described which allows for the identification of basic and acidic proteins after gel electrophoresis. This includes electrophoresis of sodium dodecylsulfate (SDS) membrane proteins not accessible to isoelectric focusing as a means of charge-dependent separation. Nonfixative charge-dependent staining can be used for detection of proteins after separation in gels, when further investigation of the intact protein is desired.     

Isoelectric analysis of 3H-pargyline-labelled monoamine oxidase in rat and carp

1. After selective binding of [3H]pargyline to either monoamine oxidase (MAO) A or MAO B in the rat liver, MAO B alone in the rat brain and MAO in carp brain and liver, molecular weight and isoelectric points (pI) of these MAO were determined by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing and results obtained were compared. 2. For all tissues tested, SDS-polyacrylamide gel electrophoresis of [3H]pargyline-bound samples revealed a labelled protein band of an apparent mol. wt of 60,000 da. 3. Estimation of radioactivity of [3H]pargyline bound after isoelectric focusing revealed a single protein band with acidic pI values of about 5.5 for rat brain and liver MAO B. 4. Moreover, the pI values of about 7.5 were obtained for carp brain and liver MAO. This basic value was also found for MAO A in the rat liver MAO A.     

Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary

Isoelectric focusing has allowed us to fractionate pituitary extracts into basic (pI 8-9) and acidic (pI 4-5) fibroblast growth factor. The acidic fibroblast growth factor (a) is stable upon refocusing, (b) migrates as an acidic protein in urea-containing gel electrophoresis; (c) is not cell-specific, being active with fibroblasts, adrenal, and glial cells, and (d) is a heterogeneous protein fraction with active components of different pI values. The component of pI 4.7, purified to or near homogeneity by isoelectric focusing shows a single peak of activity (Mr = 12,000) in gel chromatography and a single protein band of apparent Mr = 15,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal restimulation of DNA synthesis initiation on serum-deprived 3T3 fibroblasts is achieved at 1-2 ng/ml; activity with rat glial cells (C6-3D) is less pronounced than with 3T3 fibroblasts.     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

Isoelectric points and molecular weights of salt-extractable ribosomal proteins.

Rat liver ribosomes prepared in low salt buffer contain basic and acidic proteins not found on ribosomes washed in high salt buffer. Proteins extracted from liver ribosomes by 500 mM KCL were characterized by acid urea-polyacrylamide gel electrophoresis, sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel isoelectric focusing. The salt-solubilized proteins contain 12 polypeptides with a molecular weight over 67000, several polypeptides with molecular weights less than 67 000, and three polypeptides whose molecular weight exceeded 130 000. Ten to 12 of the proteins were basic, and about 24 acidic proteins were partially or wholly extracted from the ribosomes. Four of the acidic proteins have isoelectric points less than 4.5.     

DNA-dependent RNA polymerases from Acanthamoeba castellanii. Comparative subunit structures of the homogeneous enzymes.

The constituent polypeptides of the three classes of DNA-dependent RNA polymerase from Acanthamoeba castellanii were compared by several electrophoretic methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) reveals that a number of polypeptide components of the isozymes have identical molecular weights. Two-dimensional electrophoresis (isoelectric focusing in 8 M urea:SDS-polyacrylamide gel electrophoresis) demonstrates that the polypeptides of identical molecular weights also have identical isoelectric pH values. These polypeptides were also coincident after electrophoresis in 8 M urea at acidic or basic pH values followed by a second electrophoretic separation in the presence of SDS. By these criteria, subunits of molecular weight 13,300, 15,500, 17,500, 22,500, 37,000, and 39,000 are indistinguishable in polymerase I and III. The 13,300, 15,500, and 22,500 subunits are also shared by the class II polymerase. In addition, electrophoresis in 8 M urea under basic conditions reveals microheterogeneity in the 17,500 molecular weight subunit. The strikingly similar pattern of common subunits between yeast and Acanthamoeba suggests that a universal arrangement of functional units may be an essential feature of the eukaryotic polymerases.     

RAPID CONCURRENT AUTOMATED FLUORIMETRIC ASSAY OF NORADRENALINE, DOPAMINE, 3, 4-DIHYDROXYPHENYLACETIC ACID, HOMOVANILLIC ACID AND 3-METHOXYTYRAMINE IN MILLIGRAM AMOUNTS OF NERVOUS TISSUE AFTER ISOLATION ON SEPHADEX G10

Abstract— Brain tubulin subunits were separated by a combination of isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a two-dimensional polyacrylamide slab gel technique. Isoelectric focusing separated tubulin subunits into two major groups of bands, such that the more acidic group corresponded to the α subunit and the less acidic group corresponded to the β subunit. In addition, isoelectric focusing resolved the β subunit into two subspecies which differed slightly in isoelectric properties but were the same apparent molecular weight. The a subunit was resolved into many subspecies that appear to differ from each other by both apparent molecular weight and isoelectric properties.     

RAT BRAIN TUBULIN: SUBUNIT HETEROGENEITY AND PHOSPHORYLATION

Abstract— Evidence for multiple forms of the α and β subunits of tubulin isolated from rat brain has been obtained by means of SDS polyacrylamide gel electrophoresis, isoelectric focusing, and SDS hydroxylapatite column chromatography. Fourteen distinct bands, localized near pH 5.4, were formed when tubulin was subjected to isoelectric focusing in a gradient established with a very narrow range ampholyte mixture. Three tubulin subunits, a₁., α₂, and β, were separated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in a second dimension. The β subunit was more acidic than the α subunits. Brain sections were incubated in tissue culture medium containing ³²P₁ and radiolabeled tubulin was subsequently isolated and subjected to electrophoresis. Only the β subunit was labeled. All radioactivity was associated with two or three adjacent bands on isoelectric focusing gels.     

Scanning isoelectric focusing of cholera enterotoxin in polyacrylamide gels

Scanning isoelectric focusing has been employed for continuous monitoring of the isoelectric spectrum of highly purified cholera enterotoxin in 4% polyacrylamide gels containing 2% ampholytes pH 3–10. The resolution obtained by this technique is of high order because at no instance during focusing interruption of current occurs and thus diffusion of the isolated protein moieties is suppressed. An added aspect of scanning isoelectric focusing was that it allowed estimation of the minimal focusing time of cholera enterotoxin. Thus under the standard assay procedure, the main basic component of cholera enterotoxin was focused in 5800 sec, while the other at least 3 minor acidic and anodic components were focused in approximately 19000 sec. Focusing of cholera enterotoxin in the presence of 6m urea allowed the visualization of 5 well defined and about equal components. The proteinaceous nature of the observed peaks was verified by scanning at wavelengths other than 280 nm, staining of gels for protein, and varying the concentration of the enterotoxin. The design of scanning isoelectric focusing equipment is presented. Reproducibility, economy of sample, and ampholytes and simplicity of experimental technique were some of the features of this apparatus. The resolution of scanning isoelectric focusing was found to be superior to that of ordinary disc and SDS gel electrophoresis.     

A two-dimensional polyacrylamide gel electrophoresis (PAGE) system using sodium dodecyl sulfate-PAGE in the first dimension.

A two-dimensional gel electrophoretic system for the separation of cellular proteins is described. The system utilizes sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in the first dimension and polyacrylamide gel isoelectric focusing in the second dimension. The system offers a good starting point for many difficult protein separations requiring SDS.     

High resolution two-dimensional electrophoresis of basic as well as acidic proteins.

A previously described two-dimensional electrophoresis procedure (O'Farrell, 1975) combined isoelectric focusing and sodium dodecylsulfate slab gel electrophoresis to give high resolution of proteins with isoelectric points in the range of pH 4–7. This paper describes an alternate procedure for the first dimension which, unlike isoelectric focusing, resolves basic as well as acidic proteins. This method, referred to as nonequilibrium pH gradient electrophoresis (NEPHGE), involves a short time of electrophoresis toward the cathode and separates most proteins according to their isoelectric points. Ampholines of different pH ranges are used to optimize separation of proteins with different isoelectric points. The method is applied to the resolution of basic proteins with pH 7–10 Ampholines, and to the resolution of total cellular proteins with pH 3.5–10 Ampholines. Histones and ribosomal proteins can be readily resolved even though most have isoelectric points beyond the maximum pH attained in these gels. The separation obtained by NEPHGE with pH 3.5–10 Ampholines was compared to that obtained when isoelectric focusing was used in the first dimension. The protein spot size and resolution are similar (each method resolving more than 1000 proteins), but there is less resolution of acidic proteins in this NEPHGE gel due to compression of the pattern. On the other hand, NEPHGE gels extend the range of analysis to include the 15–30% of the proteins which are excluded from isoelectric focusing gels. The distribution of cell proteins according to isoelectric point and molecular weight for a procaryote (E. coli) was compared to that of a eucaryote (African green monkey kidney); the eucaryotic cell proteins are, on the average, larger and more basic.     

Isolation and partial characterization of a alpha 2-beta 1-glycoprotein from horse plasma

A alpha 2-beta 1-glycoprotein was isolated from horse plasma by classical methods. The final product appeared homogeneous by agarose gel and pore limit SDS polyacrylamide gel electrophoresis, immunoelectrophoresis and crossed immunoelectrophoresis. The protein moved in agarose gel electrophoresis just above the beta 1 region and seemed composed of a single polypeptide chain. A highly heterogenic banding pattern, focused between pH 5.1 and 6.5 was revealed by isoelectric focusing. The molecular weights determined by gel filtration on Sephadex G100 and by a pore limit polyacrylamide gel electrophoresis in presence of SDS were 65,000 and 82,300 dalton, respectively. No serological relation was found between the horse alpha 2-beta 1-glycoprotein and human and bovine plasma proteins.     

Partial purification and characterization of a polymorphic protein (Pa) in human parotid saliva.

A polymorphic acidic protein (Pa) has been isolated from human parotid saliva by the use of ion-exchange and gel filtration chromatography. Following these purification procedures, analytical anionic polyacrylamide disc gel electrophoresis revealed a single stainable band. Amino acid analysis demonstrated a protein particularly rich in proline, glutamic acid, and glycine, but with reduced amounts of threonine and no tyrosine. Only a very small percentage of carbohydrate was detected. Isoelectric focusing at pH 3-10 verified the acidic character of this protein with an isoelectric point in the range pH 3.9-4.5. Other salivary proteins called Pa-II, possibly related physiologically and genetically to the Pr system, were also partially purified and studied. Differences were noted between Pa and Pa-II proteins in molecular size and amino acid composition.     

Biochemical characterization of six trisomics of grain sorghum,Sorghum bicolor (L.) Moench

To determine protein differences of grain sorghum disomics and trisomics, we analyzed leaf extracts from six trisomics and a disomic control by disc gel, gel isoelectric focusing, and SDS gel electrophoresis. Based on the number and position of protein bands revealed by Commassie blue staining, the disomic control could be differentiated from the trisomics, and trisomics could be shown to differ among themselves in most cases. SDS gels revealed the most protein bands, followed by isoelectric focusing and disc gel. However, disc gel electrophoresis was the simplest technique of the three and was just as effective in identifying trisomics and differentiating trisomics from the disomic control.Contribution 1596-j, Department of Agronomy, and 182-j, Department of Biochemistry, Kansas State University, Manhattan, Kansas.     

Characteristics of membrane protein phosphorylation in Plasmodium berghei-infected mouse erythrocytes

Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (32P)orthophosphate and incubating isolated membrane with (gamma-32P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with Mr 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Purification and Characterization of the Acidic Lectin from Winged Bean Seeds

Winged bean acidic lectin was purified by DEAE-Sephadex A-50 and affinity chromatography on N-acetylgalactosamine-agarose gel. The purified lectin was a glycoprotein homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and gel filtration. The molecular weight of the lectin was 52,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single component of molecular weight of 27,000. Its isoelectric point was 5.5. The acidic lectin was rich in acidic amino acids, and contained 2mol of methionine but no cystine. It also agglutinated both trypsinized and untreated human erythrocytes (types A, B, AB and O), but not rabbit erythrocytes. The hemagglutination was inhibited by d-galactose and related sugars. Modification of the acidic lectin with N-bromosuccinimide caused a concomitant loss of the hemagglutinating activity with oxidation of tryptophan residue. The acidic lectin was immunologically different from the purified winged bean basic lectin by double immunodiffusion using antiserum raised against the basic lectin.     

Protein composition of human submandibular secretions

The protein composition of human submandibular saliva obtained from a single donor has been investigated, and 21 proteins have been resolved. On Sephadex G-100, submandibular secretions (unstimulated) separated into four fractions, I, II, III, and IV. Each fraction was analyzed further by isoelectric focusing and disc gel electrophoresis. The major components detected in each fraction along with their isoelectric point (pI) are as follows: I, blood group specific substance (2.3), immunoglobulin A (5.0–6.0), and immunoglobulin G (4.5–6.5); II, albumin (4.9), two glycoproteins (5.0), and acid phosphatase (5.2); III, three phosphoproteins (4.3–4.4), isoamylase 1A (5.9), isoamylase 1B (6.4), unidentified protein (7.1), lysozyme (>10), and a basic protein (>10); and IV, isoamylase 2A (5.9) and isoamylase 2B (6.4). Isoamylase 1A and IB are glycoproteins. Stimulated submandibular secretions were also resolved into four protein fractions by gel filtration. Fraction III, compared with unstimulated secretions, showed the greatest percent increase in protein. Analysis of this fraction by disc gel electrophoresis demonstrated the presence of four protein bands which were not detected in the unstimulated secretion. One of these proteins is tentatively identified as a phosphoprotein and two as basic proteins (pI > 10). The protein composition of submandibular, parotid, and sublingual secretions is compared.     

Production and characterization of antisera against three individual NHC proteins; a case of a generally distributed NHC protein

In order to assess the selectivity of the distribution patterns of individual nonhistone chromosomal proteins (NHC proteins), immunofluorescent staining experiments were performed on Drosophila polytene chromosomes. Antisera have been prepared against three individual NHC proteins which were isolated by sequential preparative slab gel isoelectric focusing and SDS polyacrylamide gel electrophoresis. In two cases, immunofluorescent staining of the chromosomes indicated a specific limited distribution pattern; apparently the antigen in each case is present at a reproducible and distinct subset of chromomeres. This type of pattern has also been obtained with antisera prepared against molecular weight subfractions of NHC proteins (Silver and Elgin, 1977). Each selective fluorescence distribution pattern obtained so far is reproducible and unique to the antiserum under study. In a third case, an antiserum caused prominant staining at dense chromomeres and the chromocenter in a pattern mimicking DNA (and presumably histone) distribution. Indirect radioimmunostaining of SDS and isoelectric focusing gels on which total NHC proteins had been separated confirmed that this antiserum reacted specifically with a protein(s) of molecular weight 21,000 D and pI 5.2. The data in conjunction with absorption experiments indicates that the chromosomal staining is due to an interaction of antibodies with NHC protein(s) and not with histones. This finding suggests that at least one major acidic NHC protein plays a very general role (comparable to that of the histones) in maintaining chromatin structure.     

Electrophoretic isolation and partial characterization of a glycoprotein of the submandibular glands of the mouse

A protein has been isolated from the water-extract of the submandibular glands of the mouse, after Biogel P-300 column passage, followed by preparative polyacrylamide gel electrophoresis at pH 4.3 and subsequently at pH 8.9, designated the AM1 protein. The isolated protein was electrophoretically pure in 7.5, 10 and 15% polyacrylamide gels both at pH 4.3 and at pH 8.9. Likewise, by electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel only one protein band could be detected. Of the total amount of the water-extractable submandibular proteins the recovery of this protein component comprised 3 to 5 per cent. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 28 000, both in 7.5 and 15% gel. The isoelectric point was determined by isoelectric focusing in 4.8% polyacrylamide slabgel to be 4.85. The amino acid analysis showed that the ratio of acidic amino acids (Glx plus Asx) to basic amino acids (Lys plus Arg) is 2.3. The glycoprotein consists of protein for 77.4 per cent and of carbohydrate for 22.6 per cent. The molar ratio of the carbohydrates was GlcNH2:GalNH2:Man:Gal:Glc:Fuc:sialic acid = 22.0:1.3:3.0:1.7:10.0:2.6:0.3. The glycoprotein was not secreted from the submandibular glands by stimulation with cholinergic (acetylcholin) or adrenergic (noradrenalin) drugs both in vitro and in vivo. So, it appeared that this glycoprotein could be characterized as a cellular, non-secretory component of these salivary glands.