Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing

Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe0-transferrin, Fe1-transferrin and Fe2-transferrin region. At the level of Fe0-transferrin and Fe1-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.     

Horizontal polyacrylamide gradient gel electrophoresis for the simultaneous phenotyping of transferrin, post-transferrin, albumin and post-albumin in the blood plasma of cattle

A simple method of horizontal polyacrylamide gel electrophoresis was described for the simultaneous phenotyping of transferrin, post-transferrin, albumin and post-albumin in the blood plasma of cattle. A step gradient gel of 8, 4, 12 and 14 % acrylamide concentration was used. The method enabled the detection of a new protein polymorphism in the post-transferrin region. Two alleles were observed. The transferrin phenotypes involving D ₁ and D ₂ alleles were clearly separated. The resolution of the post-albumin fractions was also better than described by earlier methods.     

Equine marker genes. Polymorphism for transferrin alleles, TfF1 and TfF2, in Thoroughbreds

The equine transferrin F variant is distinguishable into two types, F1 and F2, on alkaline polyacrylamide gel electrophoresis. Gene frequencies in 63 related Thoroughbreds are 0.39 and 0.19 for TfF¹TfF², respectively. In contrast the frequencies for these two alleles in 375 related Standardbreds is 0.00 and 0.59.     

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.     

Evidence for a third allele at the β-lactoglobulin (β-Lg) locus of sheep milk and its occurrence in different breeds

Summary. Milk samples from 189 Merinoland Sheep, 145 Black Faced Mutton Sheep, 89 East Friesian Milk Sheep, 36 Rhön, 36 Pleven, 23 Tsigaja, 25 Black Razka and 86 Hungarian Merino x Pleven (F1) sheep were analysed by polyacrylamide gel electrophoresis under acid conditions and isoelectric focusing in ultrathin layer polyacrylamide gels with carrier ampholytes. Six different β-lactoglobulin (β-Lg) phenotypes (A, AB, B, AC, BC and C) were observed by both methods. The occurrence of three codominant alleles (β-Lg^A, β-Lg^B, β-Lg^c) at an autosomal locus (β-Lg) was supported by family and population data on genetic equilibrium. Differences in gene frequencies between the breeds were observed.     

HPLC Analysis of Neurophysin Proteins from Neurosecretory Granules

Abstract: Neurosecretory granules were obtained from neurolobes of porcine pituitary glands. From the granules, highly purified neurophysins were prepared by HPLC. According to polyacrylamide gel electrophoresis, isoelectric focusing, N- and C-terminal amino acid residue determination, and amino acid composition, the neurophysins I₁ I₂, and II were identical to the neurophysins obtained from whole posterior lobes. Since degradation could not have occurred, we conclude that neurophysin I₁ and I₂ originated in the neurosecretory granules.     

Biochemical studies of taste sensation. XI. Isolation, characterization and taste ligand binding activity of plasma membranes from catfish taste tissue

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.     

Phenotyping of pig α1B-glycoprotein (PO2) and haemopexin by 1D polyacrylamide gel electrophoresis and immunoblotting

Summary. Described is an alternative procedure for the phenotyping of pig α₁B-glycoprotein (PO2) and haemopexin. The procedure is based on the separation of serum samples by horizontal polyacrylamide gel electrophoresis, passive blotting onto a nitrocellulose (NC) sheet, and immunochemical detection using a mixture of a primary antibody (rabbit anti-pig α₁B or anti-pig haemopexin) and a peroxidase-labelled secondary antibody. Several NC copies can be obtained from a single gel and these can be developed with different monospecific antisera.     

Genetic polymorphism of horse serum protein 3 (SP3)

Summary. Two-dimensional agarose gel (pH 8.6)-horizontal polyacrylamide gel (pH 9.0) electrophoresis of horse serum samples, followed by general protein staining, revealed genetic polymorphism of an unidentified protein tentatively designated serum protein 3 (SP3). The SP3 fractions appeared distinctly when a 14% concentration of acrylamide was used in the separation gels. The 2-D mobilities of SP3 fractions were quite similar to that of albumin. Family data were consistent with the hypothesis that the observed SP3 phenotypes were controlled by four co-dominant, autosomal alleles ( D,F,I,S ). Evidence was provided that the F allele can be further divided into two alleles ( F ₁ and F ₂); the mobilities of F₁ and F₂ variants were very similar. Each of the SP3 alleles gave rise to one fraction and each of the heterozygous types showed two fractions. More than 600 horses representing five different breeds (Swedish Trotter, North-Swedish Trotter, Thoroughbred, Arab and Polish Tarpan) were typed for SP3, and allele frequency estimates were calculated. SP3 was highly polymorphic in all breeds studied.     

Rapid resolution of transferrin C subtypes through isoelectric focusing with 2-mercaptoethanol

The technique of choice currently used for the detection of serum transferrin molecular polymorphism is isoelectric focusing on polyacrylamide slab gels. However, this procedure is unsatisfactory for routine purposes, since a long pretreatment of the serum with iron-donor compounds or neuraminidase is necessary in order to obtain a complete resolution of the transferrin molecule. A very fast and highly economical standardized procedure for transferrin typing which enables a fair molecular resolution within only 3 1/2 h is reported. Protracted pretreatment of serum with neuraminidase or with iron-donor compounds can be totally avoided. An ultrathin layer of polyacrylamide gel is employed for the run, using pH ranges of 4-6.5 or 5-7. A short pretreatment of serum with a 13% solution of 2-mercaptoethanol is performed before the samples are placed on the gel. This technique has been used to perform transferrin typing in 396 cord serum samples from newborn infants of Arezzo (Tuscany), without occurrence of artifacts or the appearance of extra bands in transferrin patterns.     

Polymorphic post-albumin of cattle and horse plasma identified as vitamin D binding protein (Gc protein)

Cattle and horse plasma samples of known post-albumin types were radiolabelled with ¹⁴C-vitamin D₃. These samples were then analysed by polyacrylamide gel electrophoresis, followed by autoradiography. The patterns observed were identical to those of post-albumin variants. The polymorphic post-albumin protein of cattle and horse was thus identified as the vitamin D binding protein and homologous to the Gc protein of human plasma.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

New variants of alpha 1-antitrypsin: comparison of Pi typing techniques.

Four new rare inherited variants (Pi types) of alpha 1-antitrypsin (alpha 1-protease inhibitor) are described. Each variant has been compared with previously reported genetic variants by several techniques used for Pi typing: isoelectric focusing in polyacrylamide gel, starch gel electrophoresis, and agarose gel electrophoresis. Some variants are identical or very similar by one technique but can be clearly distinguished by another technique. Crossed immunoelectrophoresis and gel immunofixation have been used to identify the gel bands as alpha 1-antitrypsin.     

Comparison of Methods for Separating Proteins Using Bisalbuminemia as a Model

Sera from bisalbuminemic chicken-turkey hybrids contain two albumins in equal amounts. These are observed as inherited electrophoretic variants and originate from the respective chicken and turkey parents. Sera from the hybrid birds served as a model system by which fractionating and identification procedures for evaluating serum albumin variants were compared.

The two albumins in the hybrid were isolated with preparative polyacrylamide gel electrophoresis (PAGE) and starch block preparative electrophoresis. Isoelectric focusing of the hybrid albumins resulted in the isolation of the turkey albumin. Interference of ampholinea prevented the complete isolation of the chicken albumin.

The two albumins in the hybrid have identical molecular weights and cannot be identified by sedimentation coefficient, gel filtration behavior, or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Because of the close relatedness the chicken and turkey albumins in the hybrid cross reacted with rabbit anti-hybrid serum as well as with rabbit anti-chicken and anti-turkey sera.     

Blood protein polymorphism in the one-humped camel (Camelus dromedarius) in the Sudan

The blood protein polymorphism of serum albumin, haptoglobin, transferrin, ceruloplasmin and haemoglobin have been studied in 135 samples from one-humped Arabian camel (Camelus dromedarius) of the Sudan by starch gel electrophoresis. Only the serum albumin and haptoglobin systems exhibited polymorphism with the estimated frequencies of 0.0222, 0.2227 and 0.7773 for Alb^v, Hp¹Hp⁰respectively. The frequency of Hp was 0.0325. No electrophoretic variant was observed at transferrin, ceruloplasmin and haemoglobin loci in the camel. The activity of the ceruloplasmin of the camel sera was weak.     

Human alpha 1-antitrypsin genetic polymorphism: PI N subtypes

Three new genetic variants (PI types) of alpha 1-antitrypsin are described. They have been compared to previously described phenotypes by several techniques including narrow pH range isoelectric focusing in ultrathin polyacrylamide gels. In this system, the relevant alpha 1-antitrypsin gel bands, identified by crossed immunoelectrophoresis, focused between PI M2, the most cathodal PI M subtype, and PI P BUD, the most anodal PI P subtype. They were therefore considered to be PI N subtypes. Two of them, PI N GRO and PI N YER, could not be separated by isoelectric focusing, but gave a different pattern in agarose gel electrophoresis. None of the new alleles seemed to be associated with disease. The high resolving power of isoelectric focusing is emphasized with respect to the information it may provide concerning amino acid substitutions, while the use of other techniques proved to be of utmost importance in the differentiation of other variants showing similar isoelectric points.     

Purification and physical characterization of colicin 0 111

A colicin isolated from a strain of Escherichia coli 0 111:B4:H2 has been purified by a combination of molecular sieve chromatography on Sephadex G-200 and ion-exchange chromatography on CM-Sephadex C50. The protein is homogeneous by the criteria of polyacrylamide gel electrophoresis at pH 4.5, 8.5, and 10.0, by dodecyl sulfate acrylamide gel electrophoresis, and by isoelectric focusing. The colicin has a molecular weight of 69,000, a sedimentation coefficient of 4.2 S, and a frictional ratio of 1.49. Isoelectric focusing indicated a pI of 9.50.     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein

A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.     

Characterisation of the α1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 1. Protein staining

The isoelectric points and the molecular weights of the major components of the eight Thoroughbred protease inhibitor (Pi) types have been determined by polyacrylamide gel isoelectric focusing and polyacrylamide gel pore gradient (ISO-DALT) electrophoresis respectively. The major Pi proteins focus in the range pH 3.74–4.43 and have molecular weights ranging from 55 000–72 000 daltons. Using the ISO-DALT method of electrophoresis, protein maps for the eight Thoroughbred Pi types have been presented for the first time. None of the homozygous Pi types are identical except for the types S₁ and S₂ which show partial identity. The results do not necessarily support. Juneja et al.s (1979) contention of two closely linked α1 Pi systems based on molecular weight differences. It is suggested that the traditional nomenclature originally proposed by Braend (1970) be maintained to describe the eight Pi alleles in Thoroughbred horse plasma. The ISO-DALT method provides a sensitive technique which is superior to existing techniques for the analysis of the horse Pi system.