Alkaline Urea Polyacrylamide Gel Research Articles

The effect of dephosphorylation on the properties of αS1-casein enriched protein

αS1-Casein was dephosphorylated using alkaline phosphatase without the interference from proteolysis. The progressive dephosphorylation was monitored by alkaline urea polyacrylamide gel electrophoresis and mass spectrometry techniques. Little change was observed in the SDS-polyacrylamide gel patterns for the native and dephosphorylated αS1-casein. Dephosphorylation modified the zeta-potential of the αS1-casein, shifting the pH versus zeta potential curves to higher pH as dephosphorylation increased. The isoelectric pH of the dephosphorylated αS1-casein increased by about 0.55 pH units when compared with the native protein. Dephosphorylation increased the surface hydrophobicity of αS1-casein and a negative linear correlation was observed between the surface hydrophobicity and the isoelectric pH of αS1-casein. Dephosphorylation did not affect the secondary structure of the αS1-casein.

Characterization of Silter Cheeses Produced in Valley and Alpine Pastures by a Proteomic Approach

Silter is an Italian hard cheese manufactured with milk produced by cows fed at different altitude, valley or alpine pasture. The chemical, rheological and sensory properties of cheeses can be affected by the modification in milk composition due to the breed which at different altitude causes the modification of protein content, κ-CN glycosylation, plasmin activity, and coagulation properties. The influence of milk plasmin activity on dairy production was investigated in seven Silter cheeses, four produced in the valley and three from alpine mountain through alkaline urea-polyacrylamide gel electrophoresis; two-dimensional gel electrophoresis coupled to mass spectrometry and image analysis. Results demonstrated that Silter cheese obtained from cows reared in alpine pasture is characterized by a more evident proteolysis, determining high levels of β-CN and αs1-CN fragments. Therefore, the most relevant fragmentation was attributed to a more intense activity of plasmin and to a different dosage of rennet to make up for the reduced coagulation properties of alpine milk.

Polymorphic AP-1 Binding Site in Bovine CSN1S1 Shows Quantitative Differences in Protein Binding Associated with Milk Protein Expression

Polymorphisms in 5′-flanking regions of milk protein encoding genes can influence the binding activity of the affected response elements and thus have an impact on the expression of the gene products. However, precise quantitative data concerning the binding properties of such variable response elements have so far not been described. In this study we present the results of a quantitative fluorescent electromobility shift assay comparing the allelic variants of a polymorphic activator protein-1 binding site in the promoter region of the bovine αs1-casein encoding gene (CSN1S1), which is affected by an A→G exchange at −175 bp (CSN1S1−175bp). A supershift assay using a commercial c-jun antibody was carried out to verify the specificity of protein binding. The gel shift analysis revealed specific and significantly reduced protein binding of oligonucleotides containing the G variant of the CSN1S1−175bp binding site. Further investigations comprised genotyping of the variable CSN1S1−175bp activator protein-1 element by an NmuCl restriction fragment length polymorphism in 62 cows of the breed Simmental and 80 cows of the breed German Holstein. Single milk proteins from at least 4 milk samples per cow were quantified by alkaline urea polyacrylamide gel electrophoresis. Homozygotes for CSN1S1−175bp*G were not observed, and the allele frequencies were 0.19 in Simmental and 0.05 in German Holstein. Carriers of CSN1S1−175bp*G showed higher content (%) as well as quantity (g/d) of αs1-casein than CSN1S1−175bp*A homozygotes, independent of breed. We assume that the positive association of the CSN1S1−175bp*G variant with CSN1S1 expression is likely to be caused by a reduced affinity of the affected response element to a c-jun-containing CSN1S1 dimer with repressor properties.

Casein fraction of bulk milks from different caprine breeds

The relative amounts of the major caseins in the total casein fraction from bulk milks of four different caprine breeds (two indigenous Greek and the highly selected Alpine and Saanen) were determined by alkaline urea polyacrylamide gel electrophoresis and by reversed-phase HPLC. Both methods revealed differences with regard to the α s1-casein fraction of the different breeds. The estimated values, expressed as percentage of total caseins, ranged as follows: for α s1-casein from 9.9% to 25.6%, for α s2-casein from 12.5% to 18.9%, for β-casein from 51.1% to 58.4% and for κ-casein from 9.9% to 13.5%. The lowest α s1-casein and the highest β-casein and κ-casein percentages were found in the casein fraction of the Saanen breed.

Heat-induced hydrolysis of sodium caseinate

Abstract The formation of pH 4.6- and 2% trichloroacetic acid-soluble peptides from sodium caseinate solutions (2.5%, w v ) on heating at 140 °C in the pH range 1–11 was studied using alkaline urea-polyacrylamide gel electrophoresis (urea-PAGE) and reversed phase (RP)-HPLC. The pH of the caseinate solution had marked qualitative and quantitative effects on the soluble peptides produced on heating. Soluble peptides were formed linearly on heating at 140 °C and pH 7.0 for 100 min. Fifteen of the pH 4.6-soluble peptides produced at pH 7.0 were isolated and identified: 9 peptides originated from αs1-casein (5 from the C-terminal region), 1 from αs2-casein, 3 from β-casein and 2 from κ-casein.

Biochemical characteristics of the Mr 52,000 component of Akazara scallop troponin.

Some biochemical properties of the Mr 52,000 component of Akazara scallop striated adductor troponin, which had been tentatively identified as troponin-I, were compared with those of rabbit troponin-I. Both the Mr 52,000 component and rabbit troponin-I together with rabbit tropomyosin inhibited the Mg-ATPase activity of rabbit reconstituted actomyosin to 1/10 of the original activity. The inhibition was neutralized by the addition of Akazara scallop and rabbit troponin-C or Patinopecten scallop calmodulin. The Mr 52,000 component and rabbit troponin-I were insoluble below 0.15 M KCl, but were solubilized by complexing with an equimolar amount of troponin-C or calmodulin. On alkaline urea-polyacrylamide gel electrophoresis, the Mr 52,000 component as well as rabbit troponin-I was found to form a stable complex with troponin-C or calmodulin in the presence of Ca2+.

Differential Proteolysis of Glycinin and β-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and beta-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and beta-conglycinin specific antibodies. The three subunits of beta-conglycinin were preferentially metabolized. Of the three subunits of beta-conglycinin, the larger alpha and alpha' subunits are rapidly degraded, generating new beta-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the beta-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of beta-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.

Purification and characterization of a senile amyloid-related antigenic substance (apoSASSAM) from mouse serum. apoSASSAM is an apoA-II apolipoprotein of mouse high density lipoproteins.

Two putative serum precursors which cross-react with antiserum against murine senile amyloid protein (ASSAM) were isolated from the high density lipoprotein (HDL) of normal mouse serum. Apolipoproteins designated "apoSASSAM-1" and "apoSASSAM-2" have the same molecular weight as tissue amyloid fibril protein. ApoSASSAM-1 and apoSASSAM-2 migrate to an intermediate position between apoA-I and apoC on alkaline-urea polyacrylamide gel electrophoresis and are present mainly in HDL apoproteins and to a slight extent in very low density lipoprotein apoproteins when compared to apoC. ApoSASSAM-1 and apoSASSAM-2 are polymorphic; there are two apparent isoproteins of apoSASSAM-1 with isoelectric points of 4.72 and 4.79 and two major isoproteins of apo-SASSAM-2. Subunit bands of ASSAM separated by alkaline-urea polyacrylamide gel electrophoresis and that migrated to the same positions as apoSASSAM-1 and apoSASSAM-2 were labeled by anti-apoSASSAM-1 antiserum. The amino acid compositions of apoSASSAM-1 and apoSASSAM-2 were much the same and closely resembled those of ASSAM and mouse apoA-II. Sequence analysis of apoSASSAM and ASSAM revealed a blocked amino terminus. ApoSASSAM is considered to be a mouse apoA-II and probably transforms to amyloid fibril "ASSAM" in tissues through a process yet to be clarified.

J-chain-like component in 18-S immunoglobulin of the skate Raja Kenojei, a cartilaginous fish

A J-chain was found in the IgM, an 18-S pentameric immunoglobulin, of the skate, Raja kenojei, a cartilaginous fish, by means of gel filtration-column chromatography of reduced and radioalkylated immunoglobulin, followed by alkaline-urea polyacrylamide gel electrophoresis. The J-chain showed an electrophoretic mobility equivalent to that of the light chain, and much slower than that of the J-chain in human IgM. Its electrophoretic banding patterns, however, were different from those of the light chain. Its mol. wt of 17,500 was close to that of mammalian J-chain. The J-chain was not found in another immunoglobulin of the skate, a 9-S dimer held by a noncovalent force. No immunological cross-reactivity was observed between the skate J-chain and human and chicken J-chains. In view of these findings, it was concluded that the J-chain in the skate IgM is both electrophoretically and antigenically considerably different from the J-chain in mammalian IgM.

The lipid transport system in the mouse, Mus musculus: isolation and characterization of apolipoproteins B, A-I, A-II, and C-III.

Four of the principle apolipoproteins of murine serum have been isolated and characterized. On the basis of their physicochemical properties, they are homologous with the human and rat apoA-I, A-II, B, and C-III. The group of apolipoproteins of middle to low molecular weight, i.e., A-I, A-II and C-III, were separated from the protein moiety of high density lipoproteins (HDL) by gel filtration chromatography, followed by electrophoresis in alkaline-urea polyacrylamide gel with electrophoretic elution. Murine apoA-I, the major protein of HDL (60-80%) displayed an Mr of approximately 27,000, and was polymorphic (four prominent isoproteins with isoelectric points in the range of pH 5.5-5.7). The amino acid profiles of mouse, rat, and human apoA-I generally resembled each other, the former being distinguished by a content of one isoleucine residue per mole. Amino terminal sequence analysis revealed marked homology between the mouse, rat, dog, and human proteins; mouse and rat apoA-I differed at residues 9 and 18 with potential dissimilarities at residues 5 and 15, while the murine and canine sequences were distinct at residues 6, 9, 13, 15, and 30. Apolipoprotein A-II was a monomer, exhibiting an Mr approximately 11,000 in SDS gels; in addition, it was polymorphic (three apparent isoproteins with pI in the pH range 5.05-5.2), and resembled its human and rat counterparts in amino acid composition. ApoC-III, an acidic peptide of pI 4.74 and of Mr approximately 9,600, possessed an amino acid composition very like that of the homologous human and rat proteins. The homology of mouse apoC-III with the human protein was confirmed by NH2-terminal sequence analysis, which revealed identical amino acids in six positions (1, 2, 4, 8, 9, and 13). As shown earlier (Camus et al. 1983. J. Lipid Res. 24: 1210-1228), two forms of immunologically reacting apoB predominated in mouse VLDL and LDL. After isolation of these lipoproteins in the presence of 1 mM PMSF, the apparent sizes of the high and low Mr forms, apoBH and apoBL, were in the ranges approximately 400,000-530,000 and approximately 250,000-280,000, respectively, according to the SDS gel system. We observed that inclusion of 1 mM PMSF was essential to retard degradation of the high Mr form apoBH. The murine B proteins were isolated from apoVLDL and apoLDL by gel filtration chromatography on Sephadex G150 in anionic detergent, and displayed apparent Mr values of 460,000 (apoBH) and 250,000 (apoBL) in 3% SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Degradation of serum amyloid A and apolipoproteins by serum proteases.

We have investigated the protease activity, present in human serum, that digests the serum amyloid A (SAA) protein. SAA radiolabeled with 125I was incubated at 37 degrees C with serum and plasma and analyzed for degradation products by alkaline urea-polyacrylamide gel electrophoresis and gel filtration chromatography. Serum initially digested SAA to intermediates of 3000-5000 in molecular weight, and these were further degraded to smaller peptides with prolonged incubation. SAA was not degraded by plasma anticoagulated with ethylenediaminetetraacetic acid (EDTA) or heparin. Recalcification of plasma anticoagulated with EDTA led to the generation of protease activity against SAA whereas EDTA plasma defibrinated with thrombin was inactive. We employed both nonselective and selective protease inhibitors and synthetic substrates for kallikrein and plasmin to further characterize the serum protease. These studies demonstrated that degradation of SAA is not directly attributable to enzymes involved in coagulation, kinin formation, or fibrinolysis, but the unidentified protease may be activated by one of the clotting factors. The specificity of the SAA degradation was demonstrated in experiments with three of the well-characterized apolipoproteins. Apolipoproteins A-I, C-I, and C-III-1, which also associate with the plasma high-density lipoproteins, were not degraded by serum although they were good substrates for purified thrombin and plasmin.

Isolation, characterization and comparative aspects of the major serum apolipoproteins, B-100 and AI, in the common marmoset, Callithrix jacchus

The two major apolipoproteins of marmoset serum have been isolated and characterized, and on the basis of physicochemical and immunological criteria are homologous with the human AI and B-100 proteins. Marmoset apolipoprotein AI was the principal protein of high-density lipoproteins (HDL) and was purified by gel filtration chromatography and electrophoresis in alkaline-urea polyacrylamide gel followed by electrophoretic elution. Purified marmoset apolipoprotein AI displayed an M r of approx. 27000, was polymorphic (five forms) on isoelectric focussing, with p I values in the range 4.8–5.0, and migrated similarly to human apolipoprotein AI in alkaline-urea gels. An overall resemblance was seen in the amino acid composition of marmoset apolipoprotein AI and that of its human counterpart with the notable exception that marmoset AI contained 1 isoleucine residue/mole. An immunological reaction of partial identity between the human and monkey proteins was seen upon immunodiffusion of their HDLs against antiserum to human apolipoprotein AI. Marmoset B-100 was the predominant apoprotein of VLDL and LDL, resembling the human protein in its elution profile on gel filtration chromatography in anionic detergent, and in its high apparent M r (approx. 520 000). The marmoset and human B-100 proteins were alike in amino acid composition and carbohydrate content. Moreover, their immunological behaviour with an antiserum to marmoset apolipoprotein B showed them to share certain antigenic determinant(s). We conclude that the physicochemical properties of the principle apolipoproteins of Callithrix jacchus, a New World primate, markedly resemble those of the human AI and B-100 proteins, suggesting therefore that they may function similarly in lipid transport and metabolism. Counterparts to human apolipoproteins AII, E, CII and CIII have also been tentatively identified.

Studies on subunit components of immunoglobulin M from a bony fish, the chum salmon ( Oncorhynchus keta)

Immunoglobulin M (IgM) was isolated from serum of the chum salmon ( Oncorhynchus keta) by means of ion-exchange chromatography followed by gel filtration. The purified chum salmon IgM had a mol. wt of 730,000 and a tetrameric structure. However, a fraction of tetrameric IgM was considered to be non-covalently associated molecules. The amino acid composition was determined for the chum salmon μ-and L-chains and was found to be similar to that reported for other teleost fish μ- and L-chains. No J-chain-like component could be identified in the salmon IgM by either alkaline urea polyacrylamide gel electrophoresis or by the immunological cross-reaction with antisera to the human and chicken J-chain. The immunoglobulin cross-reactivity between the chum salmon μ-chain and that of 22 other fishes was tested, and only μ-chains from the family Salmonidae revealed cross-reactivity.

Myosin phosphorylation in intact platelets.

The phosphorylation state of myosin in intact platelets has been investigated with alkaline urea-polyacrylamide gel electrophoresis. In gels of control cells, a band was found that co-migrated with the dephosphorylated form of isolated platelet myosin 20,000-dalton light chain. Stimulation of the cells by thrombin produced a dose-dependent shift of this band to the same position as that of the phosphorylated light chain. Conversion to the phosphorylated position was both complete and saturable with respect to thrombin concentration. Two-dimensional polyacrylamide gel electrophoresis was used to confirm the identity of this band as the 20,000-dalton myosin light chain. When (32P)PO4-labeled platelets were used, a direct correlation was found between the position of the light chain on the alkaline urea gel and the radioactivity. Our results demonstrate that in resting platelets myosin exists mainly in the dephosphorylated state and that stimulation by thrombin can produce a shift to the totally phosphorylated state.

IgM-like natural hemagglutinin from ratfish serum: Isolation and physico-chemical characterization. ( Callorhynchus callorhynchus)

The ratfish, Callorhynchus callorhynchus, a representative of the Holocephalii possesses a natural seric hemagglutinin characterized by a molecular weight of approximately 960.000 and composed of heavy (MW-71.200) and light (MW=23.000) polypeptides linked by disulphide bonds. Analysis of the polypeptide components, after complete reduction and alkylation, by conventional alkaline urea polyacrylamide gel electrophoresis and gel filtration on Sephadex G-200 in sodium dodecyl sulphate (SDS), did not show the presence of a J-like chain. The neutral sugar analysis of the heavy chain indicated a mannose-to-galactose ratio of 1.25. Phylogenetical implications in relation to proposed theories about the origin of the Holocephalii subclass are discussed.

A comparative study of the cardiac troponin inhibitory factor (TNI) from mammalians

Troponin inhibitory factor, TNI, was prepared by affinity chromatography from different mammalian hearts. (i) Structure. These different TNI have the same M.W. (28000), which is higher than that found in rabbit skeletal muscle (23000). Nevertheless they differ with respect of their charge as shown by alkaline urea polyacrylamide gel electrophoresis using cardiac TNI which has previously been bound to an excess of skeletal troponin Ca2+-binding factor. These changes do not correlate with the PO4 content of TNI. They are associated with structural differences demonstrated by peptide mapping of the unfolded molecule after papain treatment. The structure of cardiac TNI from rat and rabbit differs clearly from that of crow and pig. (ii) Biological activity. These different TNI have the same inhibitory effect on skeletal actomyosin. ATPase, the same content of PO4 and the same ability to be phosphorylated in-vitro by a bovine heart c-AMP-dependent protein kinase.

Improved procedure for the isolation of J-chain from human polymeric immunoglobulins

Highly purified J-chain was isolated from S-sulfonated human polymeric myeloma IgA by gel filtration and DEAE-cellulose chromatography, without using any dissociating agents. The purified J-chain was homogeneous upon immunoelectrophoresis and electrophoresis in agarose gel, alkaline-urea polyacrylamide gel, and sodium dodecyl sulfate polyacrylamide gel. The molecular weight of the J-chain, estimated by electrophoresis in sodium dodecyl sulfate polyacrylamide gel, was found to be 24 000 ± 1200 (S.D.). The J-chain dimerized in non-dissociating solvents, even in the presence of weak dissociating agents, such as 1 M propionic acid. During the isolation of the J-chain, two minor protein components which upon electrophoresis migrated, respectively, faster and slower than the J-chain itself were identified. These two components were antigenically unrelated to each other but both were clearly precipitated with anti-J-chain antisera, giving a reaction of partial identity to the J-chain. They were considered as two different fragments of J-chain, produced by some unknown protease(s).