Sunflower Seed Proteins: Characterization and Subunit Composition of the Globulin Fraction

Salt soluble proteins from sunflower (Helianthus annuus) seedswere fractionated by isoelectric precipitation and analysedby electrophoresis. Three molecular species were detected bygradient polyacrylamide gel electrophoresis of the globulinfraction. Multi-dimensional gel electrophoresis analysis indicatesthat all these species contained similar intermediary subunitsof 60 000, 54 000, 48 000 and 40000 molecular weight, the twoformer being predominant. As shown by ion-exchange chromatographyunder dissociating and reducing conditions, the intermediarysubunits are composed of disulphide linked pairs of large ‘acidic’and small ‘basic’ subunits. Heterogeneity in molecularweight of these subunits was shown by electrophoretic studies.These results suggest that a major reserve protein in sunflowerseeds is similar to ‘legumin’ of plants of the familyLeguminosae. Key words: Sunflower, Seed globulin, Globulin subunits     

Purification and characterization of B-protein from human serum

B-Protein, present in the serum of individuals with cancer, has been purified to electrophoretic homogeneity. The purification procedure consisted of chromatography on Sephacryl S-200, Affi-Gel Blue, Con A--Sepharose 4B, wheat germ lectin--Sepharose and preparative polyacrylamide gel electrophoresis. The molecular weight of B-Protein is estimated to be 100 000 to 120 000. It is a glycoprotein which appears to be composed of two subunits, each with a molecular weight of approximately 52 000. Analytical polyacrylamide gel electrophoresis and analytical ultracentrifugation data indicate that purified B-Protein is homogeneous. Isoelectric focusing studies also show the purified B-Protein to be homogeneous in composition consisting of a single band of pI = 4.8. Amino acid analysis is consistent with this acidic isoelectric point. Other analyses indicate that B-Protein contains 7% carbohydrate and 7% lipid in the form of triglycerides.     

Multiple molecular forms of mouse liver arginase

Hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8-9.1) and acidic proteins (pI range 5.8-6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the same protein.     

Characterization of the protein moiety of messenger ribonucleoprotein complexes from duck reticulocytes by two-dimensional polyacrylamide gel electrophoresis.

The protein moiety of duck globin messenger ribonucleoprotein complexes isolated by oligo(dT)-cellulose chromatography or by sucrose gradient centrifugation was analysed by two-dimensional polyacrylamide gel electrophoresis under conditions where the separation in the first dimension occurs according to charge and in the second according to molecular weight. By comparing the pattern of protein from the mRNA - protein complex with that of ribosomal subunits we found that two acidic proteins with an identical molecular weight of about 49 000 and three basic proteins of about Mr 56 000, 64 000 and 73 000 were associated with the duck globin mRNA but were absent from either puromycin/high-salt-derived or 'run-off' ribosomal subunits. The comparison of the proteins from the complex with mRNA with those found in the 0.5 M KCl wash, commonly used as the source of initiation factors, showed also that only the 49 000-Mr protein from the complex could possibly be present in the 0.5 M KCl wash of polyribosomes; proteins with mobilities similar to the other three proteins complexed with mRNA were not detected in the salt wash of polyribosomes.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

Properties of urease from Ureaplasma urealyticum: kinetics, molecular weight, and demonstration of multiple enzyme isoelectric point forms

In the several strains of Ureaplasma urealyticum that we examined, all originally isolated from human sources, the ureases were found to have a pH optimum between 7.2 and 7.5, and the Km was approximately 2.5 mM urea. Using nonreducing, nondenaturing conditions for polyacrylamide gel electrophoresis, the molecular weight of the holoenzyme was determined to be approximately 380 000. Treatment with reducing agents did not affect the electrophoretic mobility and, therefore, the molecular weight of ureaplasma urease. Immunoblot analysis (using antiserum to U. urealyticum urease) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing, denaturing conditions revealed two antigenically reactive bands of molecular weight 174 000 and 179 000. Under reducing, denaturing conditions, a single band of molecular weight approximately 179 000 was detected. Multiple forms of urease were detected by isoelectrofocusing but not by zonal electrophoresis.     

Isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid

Canine prostatic fluid, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, is characterized by the presence of a single diffuse band (Mr approximately 30,000) which accounts for over 90% of the total protein. The biosynthesis of this protein is under androgen control. Castration results in the disappearance of this protein, whereas its presence in the prostate can be maintained in the castrated animal with exogenous androgen. Analysis of the native protein by isoelectric focusing revealed the presence of 10-13 charged variants with pI values in the range of 6.5 to 8.4. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed that each isoform is constructed of two dissimilar polypeptide subunits covalently linked through disulfide bonds. One subunit has a molecular weight of 15,000 (H chain); the second subunit (L chain) has a variable molecular weight in the 12,000-14,000 range. The H and L subunits have been purified by preparative isoelectric focusing and chemically characterized. Based on tryptic peptide mapping, NH2-terminal analysis, amino acid and carbohydrate composition, the H and L subunits are structurally unrelated and consequently appear to be unique gene products. Furthermore, the L subunit is glycosylated which potentially accounts for its size heterogeneity. Quantitative NH2-terminal analysis indicated that the H and L subunits are present in the native molecule in a ratio of 2:1, suggesting that the native molecule is a trimer with an apparent molecular weight of 43,000. Based on electrophoretic data, the glycoprotein also constitutes the major fraction of the soluble protein in canine prostatic tissue; its presence is organ specific. This glycoprotein should prove useful as a marker for prostatic function under varying hormonal and environmental conditions.     

Isoelectric-focusing properties and carbohydrate content of pea (Pisum sativum) legumin

Legumin from pea (Pisum sativum) is a molecule made up of six pairs of subunits, each pair consisting of an `acidic' subunit (mol.wt. about 40000) and a `basic' subunit (mol.wt. about 20000) linked by one or more disulphide bonds. The heterogeneity of legumin has been investigated by isoelectric focusing; undissociated legumin could not be focused satisfactorily, but legumin subunits could be analysed under dissociating conditions. 8m-Urea was not found to be a satisfactory medium for isoelectric focusing of legumin, as the `basic' subunits showed a shift in pI with time of incubation in urea. A new dissociating medium for isoelectric focusing, namely 50% (v/v) formamide, was used for analysis of legumin, which gave pI values of 5.0–5.3 for the `acidic' subunits, and 8.3–8.7 for the `basic' subunits. Both types of subunits were shown to be heterogeneous in charge and molecular weight by two-dimensional analysis employing isoelectric focusing in the first dimension and sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the second. The `basic' and `acidic' subunits of legumin were separated on the preparative scale by ion-exchange chromatography in 50% formamide. Carbohydrate attached to the protein was investigated as a possible cause of the heterogeneity of legumin subunits. However, both a fluorescent-labelling technique and a sensitive radioactive-labelling technique failed to show any carbohydrate bound to legumin subunits, and it was concluded that legumin is not a glycoprotein.     

The effect of reducing agents on the structure of mammalian beta-adrenergic receptors

Under reducing conditions (5% beta-mercaptoethanol) the mammalian beta-adrenergic receptor binding site from both beta 1 (porcine heart membranes) and beta 2 receptors (hamster lung and rat erythrocyte membranes) appears to reside on peptides of Mr 62,000-65,000 as determined by photoaffinity labeling with p-azido-m-[125I]iodobenzylcarazolol and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When similar experiments are performed in these same systems under a variety of non-reducing conditions, there are minimal changes in the apparent molecular weight of both the beta 1- and beta 2-adrenergic receptor binding subunits and no specifically labeled higher molecular weight proteins are observed suggesting that there are no disulfide linked subunits in mammalian beta-adrenergic receptors.     

Dissociation of human serum low density lipoproteins into several immunologically distinct subunits. Isolation and characterization of a B-III subunit

Low density lipoproteins (d = 1.030–1.055) were partially delipidated with ethyl-ether (LDLe). These LDLe exibit a spontaneous dissociation several days after dehpidanon. Four different immunoprecipitation complexes (B-I to B-IV) are observed when using two dimensional immunoelectrophoresis against anti LDLe immunoserum. The various subunits of LDLe have different behaviour upon electrophoresis. On disc gel electrophoresis containing urea three bonds can be seen; all are phospholipoproteins. The apolipoprotein moiety of LDL and LDLe have the same apparent molecular weight around 550 000. With time several subunits aappear in LDLe, the majority of them have a molecular weight around 370 000, 260 000 and 125 000.One of the components from dissociated LDLe containing the immunodeterminant B-III, has been separated by chromatography on heparin-agarose. This LDLe-B-III has the same phospholipid/protein ratio as total LDL protein moiety with an apparent molecular weight of 110 000. This part of apolipoprotein no affinity for heparin.     

Ribosomal proteins ofThermomyces lanuginosus — characterisation by two-dimensional gel electrophoresis and differential disassembly

One- and two-dimensional gel electrophoresis were employed to characterise the proteins derived from the ribosomes of the thermophilic fungusThermomyces lanuginosus. Approximately 32 (29 basic and 3 acidic) and 45 (43 basic and 2 acidic) protein spots were resolved fromTh. lanuginosus small and large ribosomal subunits, respectively. The molecular weight of the small subunit proteins ranged from 9,800–36,000 Da with a number average molecular weight of 20,300 Da. The molecular weight range for the large subunit proteins was 12,000–48,500 Da with a number average molecular weight of 25,900 Da. Most proteins appeared to be present in unimolar amounts. These data are comparable with but not identical to those from other eukaryotic ribosomes. The sensitivities of the ribosomal proteins to increasing concentrations of NH₄Cl were also evaluated by two-dimensional gel electrophoresis. Most ribosomal proteins were gradually released over a wide range of salt concentrations but some were preferentially enriched in one or two salt conditions.     

Identification of a basic hybrid glutathione S-transferase from human liver. Glutathione S-transferase delta is composed of two distinct subunits (B1 and B2).

The purification of a hybrid glutathione S-transferase (B1 B2) from human liver is described. This enzyme has an isoelectric point of 8.75 and the B1 and B2 subunits are distinguishable immunologically and are ionically distinct. Hybridization experiments demonstrated that B1 B1 and B2 B2 could be resolved by CM-cellulose chromatography and have pI values of 8.9 and 8.4 respectively. Transferase B1 B2, and the two homodimers from which it is formed, are electrophoretically and immunochemically distinct from the neutral enzyme (transferase mu) and two acidic enzymes (transferases rho and lambda). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that B1 and B2 both have an Mr of 26 000, whereas, in contrast, transferase mu comprises subunits of Mr 27 000 and transferases rho and lambda both comprise subunits of Mr 24 500. Antisera raised against B1 or B2 monomers did not cross-react with the neutral or acidic glutathione S-transferases. The identity of transferase B1 B2 with glutathione S-transferase delta prepared by the method of Kamisaka, Habig, Ketley, Arias & Jakoby [(1975) Eur. J. Biochem. 60, 153-161] has been demonstrated, as well as its relationship to other previously described transferases.     

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37, 000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 μ;mol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100.

The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29, 000 D and 26, 000 D to give a native molecular weight of 55, 000 D.

The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. l-chloro-2, 4-dinltrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse.

Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.     

Characterization of the Antigenic Subunits of the Envelope Protein of Yersinia pestis

Chemical, physical, and immunological properties of the envelope antigen of Yersinia pestis strains have been investigated. The antigen consists of two components with isoelectric points (pI) of 4.6 and 4.8. One component (pI 4.6) is a protein bound to a small carbohydrate moiety identified as an oligomeric galactan; the other component (pI 4.8) is a simple protein. These two components are antigenically identical. In buffered solution, the antigen exists as aggregates of molecular weights larger than 300,000. The aggregates dissociate into a variety of smaller molecular weight forms depending on the nature of the treatment for dissociation. Each aggregate can be further dissociated into a single antigenic subunit fraction containing protein and glycoprotein species with molecular weights in the range from 15,000 to 17,000. The subunits can be obtained by a dissociation treatment with 0.1% mercaptoethanol in 0.25% sodium dodecyl sulfate at 95 C for 5 min. The subunits will readily reaggregate into a variety of larger molecular weight forms on the removal of dodecyl sulfate.     

Characterization of hepatic lactogen receptor. Subunit composition and hydrodynamic properties

The structure of the membrane-bound and Triton X-100-solubilized female rat liver prolactin receptor has been studied by affinity cross-linking/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and sucrose/H2O and sucrose/D2O density gradient centrifugation. Hydrodynamic characterization revealed that the 125I-human growth hormone receptor-detergent complex represents a molecular species with a Stokes radius of 61 A, a sedimentation coefficient of 5.0 s, and a calculated molecular weight of 158,000. The molecular weight of the receptor was calculated to be 92,000. Three lactogenic hormone-binding species with Mr values of 87,000, 40,000, and 35,000, respectively, were repeatedly found when detergent-solubilized preparations were analyzed using an affinity cross-linking technique. Estrogen treatment of female rats increased the intensity of these bands. Occasionally, an Mr 165,000 hormone-binding species was also found. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies (first dimension, nonreducing; second dimension, reducing) demonstrated that disulfide- and nondisulfide-linked hormone-binding species with Mr values of 40,000 and 35,000 are contained within the Mr 87,000 species. It is concluded that the Triton X-100-solubilized female rat liver prolactin receptor has a molecular weight of about 90,000. This molecular species contains Mr 40,000 and Mr 35,000 hormone-binding subunits. It cannot be determined whether these subunits are combined with each other or with as yet undetected subunit(s) to make up the Mr 90,000 species, or whether each one of these subunits is a proteolytic fragment of the Mr 90,000 species.     

Immunologically related multimeric forms of 30-40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000-40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high-molecular-weight proteins (greater than 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30-40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Purification and properties of α-1,4-glucan phosphorylase from the red seaweed Gracilaria sordida (Gracilariales)

α-1,4-Glucan phosphorylase (EC 2.4.1.1) from the red seaweed Gracilaria sordida (Harv.) W. Nelson was adsorbed onto starch-Sepharose 6B and Sephacryl S-300 under specified conditions. The algal enzyme was purified to homogeneity by these two steps. A molecular weight of 97.4 kDa was observed on SDS-polyacrylamide gel electrophoresis under reducing conditions, while the native molecular weight was 240 kDa asrevealed by 8-25% native gradient gel electrophoresis or 245 kDa by gel filtration. The pI of the enzyme was 5.4. It had a Km of 227, 264, 285, and 453 μg ml-1, respectively, towards glycogen, amylopectin, amylose, and maltodextrin. The enzyme activity was inhibited by cyclohexaamylose, ADP-glucose, and UDP-glucose. In contrast to other plant sources, cell-free extracts of G. sordida contained only one form of phosphorylase.     

A homologue of alpha 2-macroglobulin purified from the hemolymph of the horseshoe crab Limulus polyphemus

A high molecular weight protease inhibitor has been purified from the cell-free plasma of the horseshoe crab Limulus polyphemus using high speed centrifugation, polyethylene glycol precipitation, and gel filtration. The inhibitor is sensitive to mild acidification, methylamine treatment, and inhibits the proteolytic activity of a variety of endopeptidases. The molecule does not inhibit trypsin-mediated hydrolysis of low molecular weight substrates and protects the active site of trypsin from inactivation by soybean trypsin inhibitor. These properties are diagnostic of the alpha 2-macroglobulin (alpha 2M) class of protease inhibitors found in vertebrates. Like vertebrate alpha 2M the Limulus alpha 2M molecule is composed of subunits of molecular weight 180,000-185,000 as determined by polyacrylamide gel electrophoresis under reducing conditions. The apparent native molecular weight for the Limulus molecule as determined by both gel filtration and gel electrophoresis under nonreducing conditions is 500,000-550,000, compared to a native molecular weight of 700,000-750,000 for human alpha 2M, determined in parallel under identical conditions. These results suggest that alpha 2M appeared in evolution at least 550 million years ago before the divergence of the lineages that gave rise to present-day arthropods and mammals.     

Isolation and reconstitution of the membrane-bound form of dopamine beta-hydroxylase

Dopamine beta-hydroxylase is present in the bovine adrenal medulla in two forms, soluble and membrane bound. Previous isolation procedures for the membranous hydroxylase have resulted in a form of enzyme identical in subunit structure with the soluble type. We report here the isolation of a membrane-bound form of dopamine beta-hydroxylase which is structurally different from the soluble form. The isolated membranous enzyme has a large apparent molecular weight on gel filtration, is amphiphilic, and contains bound phospholipid which is predominantly phosphatidylserine. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows that the membranous hydroxylase contains two nonidentical subunits under both reducing and nonreducing conditions. Under reducing conditions the apparent molecular weights of the two subunits are 70,000 and 75,000 and both contain carbohydrate. The purified membranous hydroxylase binds to phospholipid vesicles and chymotryptic digestion of the bound enzyme suggests that two forms of the membranous hydroxylase exist.