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.     

Characterization of cytoplasmic arginine-rich basic protein of Guerin epithelioma

Summary Arginine-rich basic protein from cytoplasma of Guerin epitheliomas has been isolated and characterized. It contains five amino acids: arginine, lysine, glycine, alanine and glutamic acid which make together 74 per cent of all amino acid residues. The protein has a cationic character with an isoelectric point of 8.2. No carbohydrate component was found in this protein. The significance of arginine-rich basic protein in the cytoplasma of Guerin epithelioma is discussed briefly.     

Amino terminal amino acid sequences and carbohydrate of the two major forms of rabbit plasminogen

Two major forms of plasminogen exist in the plasma of many animal species and are distinguished by their affinities for certain antifibrinolytic amino acids. Quantitative end group analysis demonstrated that each isolated form of rabbit plasminogen possessed a single amino terminal residue of glutamic acid. Amino acid sequence analysis indicated that at least the first twelve amino terminal amino acids were identical in the two forms. The unique amino terminal sequence obtained for each form was NH₂-glu-pro-leu-asp-asp-tyr-val-asn-thr-gln-gly-ala-. Analysis of the carbohydrate content of each major plasminogen form revealed some striking differences. The first major form of rabbit plasminogen isolated from affinity chromatography columns contained 1.5–1.7 percent neutral carbohydrate and 3.0–3.3 moles of sialic acid per mole of protein. The second major form of rabbit plasminogen isolated from affinity chromatography columns contained 0.6–0.8 percent neutral carbohydrate and 1.8–2.2 moles of sialic acid per mole of protein.     

Avian thrombocyte thrombospondin

A high molecular weight glycoprotein (450,000) was obtained from thrombin-treated duck thrombocytes by barium citrate adsorption technique followed by heparin-agarose affinity chromatography. Amino acid composition (high number of acidic amino acids and cystine) as well as carbohydrate contents (1.3 per cent hexosamine, 0.9 per cent sialic acid and 1.5 per cent hexose) showed similarity to mammalian platelet thrombospondin.     

Study of the structure of human serum albumin, liberated from fatty acids, by a tritium marker method]

To elucidate the natural fatty acids effect on the human serum albumin (HSA) structure a new method of tritium labelling was used. The main peculiarity of the method consists in the possibility to get information on the qualitative and quantitative amino acid composition of the surface layer of the protein globule at different conformational states of the globule. Defatted HSA was shown to be characterized a higher accessibility of Asx, Glx, Thr, Ser, Gly, Pro, Ile, Tyr residues while the other residues remain unchanged. Asx residues are characterized by the largest changes (about 8 folds). Full accessible protein surface during defatting increases from 39,000 to 48,000 A2. Fatty acids connected with albumin in the relation 1-3 moles/mol of protein are noted to be the factor increasing the globule compactness and stipulating for the conformational protein stability to warmth, urine and guanidine salts effect.     

ISOLATION AND CHARACTERIZATION OF A SOLUBLE GLUCOSE-CONTAINING SIALOGLYCOPROTEIN FROM THE CORTICAL GREY MATTER OF CALF BRAIN

A sialoglycoprotein has been isolated from the cortical grey matter of calf brain after homogenization in 0.32 M-sucrose or in 0.15 M-NaCl. The sialoglycoprotein is present in the supernatant obtained after centrifugation at 100,000 g for 60 min. It is designated GP-350 on account of its elution with 350 mM-NaCl on a DEAE-cellulose column. From DEAE-cellulose chromatography it is evident that compounds comparable to GP-350 occur in the brain of calf and sheep, whereas they seem to be absent in calf liver and kidney. After purification, with polyacrylamide gel electrophoresis only one band can be shown both at pH 8.9 and 7.5. GP-350 consists of about 83 percent of protein and about 17 per cent of carbohydrate. The polypeptide core has an acidic character: amino acid analysis gives 26 per cent for glutamic acid plus aspartic acid and their amides, with a ratio of acidic to basic amino acids of 3.3. The carbohydrate moiety contains 2.4% sialic acid, 5.5 % hexosamine and 9.4% hexose. It is remarkable that this brain sialoglycoprotein comprises 4% glucose. Care was taken to prevent contamination with glucose-containing materials during the purification procedure of GP-350. The complete absence of other glucose-containing compounds which occur in brain, Le. glycogen and gangliosides, was demonstrated. GP-350 accounts for at least 3 per cent of the total saline-extractable protein and about 20 per cent of the total saline-extractable protein-bound sialic acid of the cortical grey matter of calf brain. These percentages correspond to 390 pg of protein and to 14 μg of sialic acid per g wet weight. GP-350 remains soluble when the pH is brought to 3.9 or when ethanol is added to 70 % (v/v).     

Some molecular properties of human seminal transferrin (HSmT) in comparison with human serum transferrin (HSrT)

In this paper we report some structural features of human seminal transferrin (HSmT) in comparison with the homologous protein purified from human serum (HSrT). In particular, the sequence of the first 13 N-terminal amino acids of HSmT shows 12/13 of identity with the first 13 N-terminal amino acids of HSrT, the ninth residue of the former protein being not definitely determined. Moreover, HSrT and HSmT analysed under the same conditions, by means of reversed phase HPLC, thiol groups determination and second derivative spectroscopy, show a different content of amino acids. In particular, HSmT exhibits mainly: i) a lower Asx/Glx ratio; ii) a reduction of about 50% in Cys residues; iii) a decrease of Tyr and Trp residues. Eventually oligosaccharide parallel analyses of HSmT and HSrT show the same glycosidic bond and almost the same sugar content (around 5.5% w/w); conversely, HSmT lacks of sialic acid residues and probably it contains fucose. These results, taken all together, could be sound of interest to a better understanding of the possible physiological roles of HSmT.     

Pig plasma protease inhibitor gene complex: isolation and partial characterization of three inhibitors

1. Pig plasma alpha-protease inhibitors (protease inhibitor-1, PI1; protease inhibitor-2, PI2; postalbumin-1A, PO1A; postalbumin-1B, PO1B), all encoded by one gene complex (gene cluster), were isolated by rivanol-ammonium sulphate fractionation and double-one dimensional IPG-PAGE. The proteins were recovered from the polyacrylamide gel by a combination of electrophoresis and isoelectric focusing. 2. Molecular wt estimated by SDS-PAGE under reducing conditions was 63,000 each for PI1 and PI2 and 60,000 each for PO1A and PO1B. The two main components of a genetic variant of PI2 differed in mol. wt by approx. 1000. 3. PO1A, PO1B and PI2 were shown to be glycoproteins. The major component of both PO1A and PO1B contained about 15% carbohydrate and the two components of PI2 had about 24 per cent and 21 per cent carbohydrate, respectively. 4. Neuraminidase treatment showed that the main component of PO1A had 8 sialic acid residues and fast and slow components of PI2 had respectively 11 and 10 residues. 5. Amino acid compositions of PO1A, PO1B and PI2 were very similar to one another, indicating that the genes for these proteins have evolved by regional duplications of a common ancestral gene. 6. The results (mol. wt, amino acid and carbohydrate compositions) confirm that pig PI2 is homologous to human plasma alpha 1-antichymotrypsin.     

Purification and characterization of a sulfated glycoprotein secreted by Sertoli cells

Sulfated glycoprotein 2 (SGP-2), the major secretion product of Sertoli cells, was purified from cell culture medium by reverse-phase high-performance liquid chromatography. The native protein consists of disulfide-linked monomers of 41,000 and 29,000 daltons which have a strong tendency to associate into multimers. The purified SGP-2 was subjected to amino acid analysis and contained high levels of Asx (11.1%), Glx (15.1%), and leucine (11.5%). The oligosaccharides on the purified SGP-2 were analyzed to determine the monosaccharide compositions and the molecular weights of the intact carbohydrate moieties. SGP-2 was shown to be 23.7% carbohydrate and consisted of 1% fucose, 3.5% mannose, 4.1% galactose, 7.1% N-acetylglucosamine, and 8.0% N-acetylneuraminic acid. No N-acetylgalactosamine was detected. When the SGP-2 was digested with proteases, the intact oligosaccharides were chromatographed over a Bio-Gel P-6 column and found to elute in a single symmetrical peak of approximately 3,300 g/mol. On the basis of these results, the oligosaccharides on SGP-2 were proposed to consist of triantennary chains similar to those found on fetuin. When the 35SO4(2-)-labeled SGP-2 was digested with Pronase, the free amino acids could be separated by chromatography from the oligosaccharide. The 35SO4(2-) was shown to be associated with the oligosaccharide portion of SGP-2.     

Purification and properties of the gamma-butyrobetaine-binding protein from an Agrobacterium sp.

A binding protein for gamma-butyrobetaine was purified from osmotic shock fluid of an Agrobacterium sp. It was a monomeric protein with an apparent molecular weight of 52,000 or 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The isoelectric point was 4.3, as determined by isoelectric focusing. Amino acid analysis of the protein showed that Asx and Glx were predominant components and that the protein contained no cysteine. The dissociation constant of this protein for gamma-butyrobetaine was found to be 0.7 microM by equilibrium dialysis. Attempts to sequence the amino-terminal end with the Edman method failed, suggesting that this region of the protein is blocked.     

Purification, physicochemical characterization, and immunohistochemical localization of a major 11.7 S glycoprotein from the jelly coats of the anuran Lepidobatrachus laevis

Embryos of the frog Lepidobatrachus laevis are encased by a fertilization envelope and two jelly layers, termed J1 (innermost) and J2 (outermost). From preparations of total jelly solubilized from cleavage-stage embryos by a solution of alkaline beta-mercaptoethanol we have purified one jelly coat glycoprotein to homogeneity via FPLC gel permeation chromatography on Superose 6H. The purified glycoprotein was 94% protein and 6% carbohydrate, had an s0(20),w of 11.7 S, with a molecular weight of 245,000 measured by sedimentation equilibrium and 263,000 by gel permeation chromatography. SDS-PAGE revealed that the glycoprotein is composed of a single subunit near 29,700 molecular weight; thus we propose that eight of these subunits comprise the native molecule. Amino acid analysis of the glycoprotein indicated a high content of Glx + Asx (32.4 mole%), a low content of basic amino acids (Arg + Lys = 12.2 mole%), and a single cysteine residue per subunit. The N-terminal amino acid was threonine and the sequence of the first twenty amino acids was determined. Monospecific antisera to the glycoprotein were prepared in rabbits and were used to immunohistochemically localize the glycoprotein throughout the matrix of both jelly layers. Antiserum against the glycoprotein had virtually no effect on the fertilizability of jellied eggs in vitro; thus we hypothesize that the glycoprotein fulfills a structural role in both jelly layers.     

ISOLATION OF AN ACID-SOLUBLE PROTEIN WITH LOW MOLECULAR WEIGHT FROM MONKEY BRAIN

Abstract— The isolation of a perchloric acid-soluble low molecular weight protein from brain of Macaca irus is reported. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing indicate that the protein is free of impurities. The molecular weight, as determined by gel filtration and sodium dodecyl sulphate gel electrophoresis, is shown to be 10,400 and 9900, respectively. This is in agreement with the value of 10,700 obtained from amino acid analysis. The protein contains 27 per cent acid amino acids and 15 per cent basic amino acids. However, the relatively high amide content gives the protein a neutral nature as shown by isoelectric point determination using gel isoelectric focusing.     

The presence of fatty acids in human alpha-fetoprotein.

alpha-Fetoprotein has been prepared from human fetal tissue by procedures utilizing DEAE-Sephadex, concanavalin A-Sepharose, and isoelectric focusing. A major and a minor component with isoelectric points of 4.7 and 5.3, respectively, have been isolated and are similar to those prepared under various conditions by other investigators. The 4.7 material contains 2.4 mol of fatty acids/mol of protein, whereas the minor component is fat-free. The relative amounts of fatty acid vary somewhat with different preparations. The ranges found in three isolates were as follows: palmitic acid (8 to 11%), stearic acid (2 to 5%), oleic acid (10 to 28%), linoleic acid (7 to 15%), arachidonic acid (12 to 39%), and 4,7,10,13,16,19-docosahexaenoic acid (16 to 42%). Human fetal serum albumin contained 0.7 mol of fatty acid/mol of protein, with arachidonic acid and the docosahexaenoic acid comprising only 11.4% of the total. Removal of fatty acids by treatment with charcoal converted alpha-fetoprotein into material with an isoelectric point of pH 5.3. Addition of arachidonic acid to the lipid-free protein restored it to protein with a pH 4.7 isoelectric point, typical of the major native component. The possible role of the fatty acids in alpha-fetoprotein on the inhibition of various lymphocyte functions is projected.     

Isolation and partial characterization of the heterophile antigen of infectious mononucleosis from bovine erythrocytes

The heterophile antigen (Paul-Bunnell antigen, PBA) of infectious mononucleosis was isolated by extraction of an aqueous suspension of bovine erythrocyte stromata with chloroform-methanol (2:1). The upper aqueous layer contained gangliosides, PBA, and a high-molecular-weight glycoprotein. PBA and gangliosides were separated from the high-molecular-weight glycoprotein by extraction of lyophilized upper layer with chloroform-methanol solvents. Separation of PBA from gangliosides was carried out by chromatography on DEAE-cellulose with chloroform-methanol solvents. PBA appeared to be a minor glycoprotein component of the erythrocyte membrane and had both hydrophobic and hydrophilic properties. It was soluble in either organic or aqueous solvents. On SDS-polyacrylamide gel electrophoresis, it migrated as a single component that stained for protein with Coomassie blue, for carbohydrate with periodic acid-Schiff reagent, and for lipid with oil red 0; it had an apparent molecular weight of 26,000. It was composed of 62% protein with major amino acids: glutamic acid, proline, glycine, isoleucine, leucine, and threonine (158, 116, 98, 90, 85, and 82 residues per 1,000 residues, respectively). Carbohydrate content was 9.2% with major sugar constituents: sialic acid, galactosamine, and galactose. Serologic activity of PBA was destroyed by pronase but not by trypsin.     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.     

Amino acid analysis by high-performance liquid chromatography of a single stained protein band from a polyacrylamide gel

A single stained band containing approximately 5 micrograms of protein was cut out of a polyacrylamide gel and subjected to hydrolysis together with the gel. The hydrolysate was subsequently analyzed for its amino acid content by high-performance liquid chromatography and postlabeling with o-phthalaldehyde. Bovine serum albumin, ribonuclease B, ovalbumin, pepsin, and chymotrypsinogen A were analyzed by this method, and their amino acid compositions were found to be in good agreement with the reported values. By this method, it is possible to quantitate 16 amino acids: Asx, Thr, Ser, Glx, Pro, Cys, Gly, Ala, Val, Ile, Leu, Tyr, Phe, His, Lys, and Arg. Thioglycolic acid is effective protection against the decomposition of Tyr, Cys, and Met; however, the recovery of Met is inconsistent. This method might be very helpful for the amino acid analysis of proteins of multicomponent systems, especially, those which can be resolved only by polyacrylamide gel electrophoresis.     

GANGLIOSIDES OF HUMAN MYELIN: SIALOSYLGALACTOSYLCERAMIDE (G7) AS A MAJOR COMPONENT

Abstract— Gangliosides were isolated from purified human myelin in a yield of 62 μg of lipid-bound sialic acid per 100 mg of dry myelin. Sialosylgalactosyl ceramide (G₇) was found to be a major component of the ganglioside fraction, amounting to 15 per cent of the total sialic acid. It accounted for 10 per cent of lipid-bound sialic acid in adult human white matter, making it the third most abundant ganglioside on a molar basis. These results were obtained with an improved method for isolating total gangliosides in high yield, by employing DEAE-Sephadex column chromatography. Myelin from other mammalian species had considerably less G₇, and there were also indications of maturational changes. Both 2-hydroxy and unsubstituted fatty acids were components of the ceramide unit, in a ratio of 3:2, respectively. The overall fatty acid pattern was very similar to that for myelin cerebroside and sulphatide. Long-chain bases included only C₁₈ species, with sphingosine predominating (>90 per cent). These observations suggest a metabolic relationship between G₇ and either cerebroside or sulphatide.     

Isolation and characterization of pregnancy associated alpha2 glycoprotein]

A method using immunoadsorbents for the isolation of pregnancy-associated alpha2-glycoprotein (alpha2-PAG) from the extract of human placentae is described. The physical properties and the chemical composition of the purified protein are determined: alpha2PAG sediments with 11,5 S, has a molecular weight of 360 000 daltons and is composed of subunits having a molecular weight of 180000, which are held together by disulfide bonds. The isoelectric point was found to be pH 4,7 and the extinction coefficient (E1%1cm) was determined to be 9,7 at 277 nm. The carbohydrate content of the molecule amounts to 12,1% (hexose 6,0%, hexosamine 3,7%, fucose 0,06%, sialic acid 2,4%). An analysis of the amino acids is reported, too. The purified alpha2PAG was used to determine the absolute concentrations of this protein in a reference standard and in sera.     

Purification, composition, and structure of macrophage adhesion molecule

Macrophage adhesion molecule (MAM) is a surface heterodimer consisting of the trypsin- and plasmin-sensitive glycopeptide gp160 (MAM-alpha) and the glycopeptide gp93 (MAM-beta). MAM, which is the guinea pig analogue of Mo1 and Mac-1, was purified from detergent lysates of peritoneal neutrophils by lentil lectin chromatography and M2-antibody chromatography. The pure heterodimer molecule was dissociated by acidic conditions (pH 3.5), and MAM-alpha and MAM-beta were separated by M7-antibody chromatography. MAM-beta is an approximately 640 amino acid residue polypeptide with exceptionally high cysteine content. At 7.2 residues per 100 amino acids, Cys/2 of MAM-beta is more than 3 times the mean for 200 purified proteins. Reactivity with six beta-subunit-specific monoclonal antibodies recognizing at least four epitopes demonstrated that intrapeptide disulfide bonds are required to maintain the structure of MAM-beta. All six antibodies failed to react when MAM-beta was treated with reducing agents. MAM-beta is 18% carbohydrate; the major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid. MAM-beta is estimated to contain five to six N-linked carbohydrate units. MAM-alpha is an approximately 1100-residue polypeptide with lower Cys/2 content (2.0 residues per 100 amino acid residues). MAM-alpha is 21% carbohydrate. The major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid; the mannose content is higher in MAM-alpha than MAM-beta. MAM-alpha is estimated to contain 12 N-linked carbohydrate units.     

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.