Role of sialic acid in the microheterogeneity of serum thyroxine-binding globulin. Study by two-dimensional isoelectric focusing

A new technique combining a neuraminidase treatment of thyroxine-binding globulin after serum isoelectric focusing and a second-dimensional isoelectric focusing was developed to study the role of sialic acid in the microheterogeneity of native thyroxine-binding globulin. By showing the change of PI occasioned by the desialylation for each of the bands constituting the thyroxine-binding globulin pattern separately, this procedure clearly demonstrated that the microheterogeneity of the native protein could not be imputed to the varying sialic acid content of the bands only. We suggest that at least three molecules of thyroxine-binding globulin, with probably slight differences in their amino acid composition, are present in the serum, and that different degrees of sialylation ensure greater microheterogeneity of this protein.     

Characterization of ferritin from human placenta. Implications for analysis of tissue specificity and microheterogeneity of ferritins

Mammalian ferritins can be resolved into multiple components by isoelectric focusing, and each tissue contains a characteristic subset of isoferritins. Ferritin isolated from human liver was compared to acidic ferritin isolated from mid-gestational human placenta to define a structural basis for ferritin heterogeneity. Placenta ferritin contained several major bands with isoelectric points in the range of pI = 4.7-5.0 which were more acidic than the predominant isoferritins of human liver. Ferritin from each tissue was resistant to denaturation by 10 M urea and appeared to be identical by electron microscopy. Circular dichroism measurements revealed that placenta ferritin had substantially less ordered secondary structure than liver ferritin. Both types of ferritin contained only two subunits when analyzed by electrophoresis in sodium dodecyl sulfate gels, but isoelectric focusing of dissociated subunits in urea revealed 6-7 different components. In this system, placenta ferritin was enriched in the more acidic subunits and it completely lacked the most basic subunits noted in liver ferritin; placental ferritin had no unique components. Differences in isoelectric points among assembled ferritins from these two tissues appear to result from different proportions of these acidic and basic subunits.     

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.     

Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.     

Isoelectric focusing and isoelectric points of aminoacyl-tRNA synthetases

Isoelectric points and isoelectric focusing behaviour of 10 highly purified eukaryotic aminoacyl-tRNA synthetases from 3 sources, Saccharomyces cerevisiae, Euglena gracilis and Phaseolus vulgaris were examined. The pI-values measured on polyacrylamide gels under native conditions are situated between pH 5.0-7.5. A microheterogeneity was observed for 9 enzymes appearing otherwise homogeneous on gel electrophoresis. A compilation of the isoelectric points of aminoacyl-tRNA synthetases is given and literature data are compared with our experimental results.     

Isoelectric focusing of complex protein mixtures in the nanogram range in microgels (author's transl)]

A method is described for isolectric focusing of complex protein mixtures in 2, 5 or 10 mul capillaries. For one separation only 15- 50 ng of a protein mixture is needed. Isoelectric focusing is finished after 10 min, staining takes 20 min and destaining approximately 30 min. Using defined mixtures of Servalyt from different pH ranges, isoelectric focusing can be adapted to the protein sample to be fractionated. Protein peaks separated by isoelectric focusing can be electrophoretically eluted and for further analysis refractionated directly in a microgradient gel. The resolution power of microisoelectric focusing is as good as that of the wellknown macroprocedure, as is demonstrated by isoelectric focusing of the water soluble proteins from cerebellum and heart, of rat and human serum and of a human oncocytoma of the thyroid gland.     

Resolution of Rat Brain Synaptic Phosphoprotein B-50 into Multiple Forms by Two-Dimensional Electrophoresis: Evidence for Multisite Phosphorylation

Phosphoprotein B-50 was extracted from rat brain membranes by alkaline extraction and purified by ammonium sulphate precipitation and flat-bed isoelectric focusing. The purified protein shows microheterogeneity upon isoelectric focusing in a narrow pH gradient (pH 3.5-5.0). As visualized by two-dimensional gel electrophoresis, B-50 resolved into four clearly separated forms which differ slightly in isoelectric point. The forms are in part mutually convertible by exhaustive phosphorylation (using protein kinase C) and dephosphorylation (using Escherichia coli alkaline phosphatase). Proteolysis with Staphylococcus aureus protease yielded two radioactive peptides. Analysis of their molecular weights and the time course of their formation suggests that B-50 was cleaved at only one specific site. Our data indicate the presence of more than one phosphorylatable site. The possibility that the heterogeneity of B-50 was in part due to a glycoprotein nature of B-50 was studied extensively. However, none of the six different methods used revealed the presence of glyco-moieties in B-50.     

Microheterogeneity of rat glycogen phosphorylase liver-type isozyme

We devised a method of polyacrylamide gel electrophoresis at pH 7.3, modified by omitting base catalyst, N,N,N',N'-tetramethylethylenediamine, in the preparation of separating gels. Using this method, both liver and liver-like types of rat glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyl-transferase, EC 2.4.1.1) were resolved into multiple forms, about 6-10, although either of them was purified to a single protein with the same molecular size on sodium dodecyl sulfate gel electrophoresis. The microheterogeneity of these two types was also confirmed by isoelectric focusing in polyacrylamide gels (pH 5-8). The major isoelectric points of the liver type phosphorylase were between 5.72 and 5.86, but those of the liver-like type were between 5.86 and 5.92, and so the former had slightly but significantly lower isoelectric points than the latter. However, the both types were not distinguished immunologically. The brain and muscle types of rat phosphorylase did not show such a distinct heterogeneity by the same electrophoresis methods.     

Properties and role of ferritin in the hemolymph of the chiton Clavarizona hirtosa

The major iron-binding protein found in the hemolymph of the chiton Clavarizona hirtosa has been purified for the first time and identified as ferritin. This ferritin, which is present at a concentration of approx. 400 μg·ml⁻¹, has a M_r of 28 000 and 25 500, exhibits microheterogeneity with isoelectric values in the range 5.3–6.0, binds 1500–2500 Fe atoms·mol⁻¹ and is immunologically distinct from horse spleen ferritin. The initial rate of iron accumulation by ferritin molecules was determined to be markedly higher than that exhibited by horse spleen ferritin. Taken together, these data suggest that ferritin found in the hemolymph serves as a key component of the high-capacity transport system necessary to deliver iron to the rapidly mineralizing tissue of the radula in these molluscs.     

Analysis of the microheterogeneity of the glycan chain of rat transferrin

To investigate the microheterogeneity of the glycan chain of rat transferrin, either the protein moiety was labeled with 125I or the sialyl residues with 3H. The molecule was then subjected to Con A chromatography. Three components were obtained. Each was enzymatically desialylated and sialyl/protein molar ratios were calculated. The native protein as well as the 3 components were also subjected to isoelectric focusing. The results indicated that rat transferrin may have 3 types of glycan chain: The major type (60%) corresponds to a molecular species with triantennary branching, while 30% consists of molecules with biantennary and 10% with tetraantennary branching. The last species has not been previously described.     

Immunochemical aspects, molecular and kinetic properties of multiple forms of acetyl-CoA acetyltransferase from rat liver mitochondria.

Acetyl-CoA acetyltransferase (EC 2.3.1.9) from rat liver mitochondria, which catalyzes the first step in the biosynthesis of ketone bodies, exists in two forms, designated transferase A and transferase B. Both transferases showed immunochemical cross-reactivity, but are immunologically unrelated to cytosolic acetyl-CoA acetyltransferase activity and the mitochondrial acetyl-CoA acyltransferase from rat liver. The transferases A and B were estimated to have molecular weights of 151 000 in the absence and 40 000 in the presence of sodium dodecyl sulfate. They differ with respect to charge states and multiplicity of forms as indicated by isoelectric focusing. Transferase A appeared in two forms with isoelectric points of 8.4 and 9.1, whereas transferase B represents a stable protein state with an isoelectric point of 9.0. Kinetic analysis of the reactions leading to acetoacetyl-CoA synthesis revealed saturation curves with multiple intermediary plateaus, indicating a complex kinetic behaviour. The data presented are interpreted as representing a microheterogeneity of forms of the mitochondrial acetyl-CoA acetyltransferase. The kinetic properties exhibited suggest a role for this microheterogeneity in the regulation of ketogenesis.     

Characterization of alpha 2-macroglobulin from plasma of cystic fibrosis patients and controls

The physical, kinetic, and isoelectric focusing properties of native alpha 2-macroglobulin from cystic fibrosis and control plasmas were studied. No differences were found in the esterolytic activity levels of control, obligate heterozygote, and cystic fibrosis plasmas. Stability studies indicated that both control and cystic fibrosis alpha 2-macroglobulin retained full activity for at least 8 months at -20 degrees C, a week at 0-4 degrees C, 11 hr at 50 degrees C, and showed no differences in thermostability at several preincubation temperatures. The microheterogeneity of native alpha 2-macroglobulin was studied by column isoelectric focusing of five control and five cystic fibrosis plasmas. The number and pI values of the isoelectric forms between pH 4.5-8.0 were quite similar for both groups even though consistently less cystic fibrosis alpha 2-macroglobulin activity was recovered after isoelectric focusing.     

Demonstration of heavy and light protomers of human testosterone-estradiol-binding globulin

Human testosterone-estradiol-binding globulin (hTeBG) was purified from pregnancy serum by sequential ammonium sulfate precipitation, affinity chromatography, and hydroxylapatite chromatography. An overall purification of 2800-fold was achieved with a 27% total yield. Apparent homogeneity of the final product was shown by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate (SDS). The equilibrium dissociation constant (Kd) at 4 degrees C for 5 alpha-dihydrotestosterone (DHT) was estimated to be 1.94 +/- 0.95 X 10(-9) M. Analysis of the purified protein revealed microheterogeneity with regard to size on polyacrylamide gel in the presence of SDS and to charge on isoelectric focusing gels. The apparent molecular weight of native hTeBG determined by gradient gel electrophoresis was 115,000. SDS-polyacrylamide gel electrophoresis indicated that hTeBG is comprised of two molecular weight components of 53,000 and 46,000, which are designated as heavy (hTeBGH) and light (hTeBGL) protomers, respectively. Photolysis of purified hTeBG with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one [( 3H]delta 6-testosterone) resulted in specific labeling of its binding sites. Analysis of photolabeled products by SDS-polyacrylamide gel electrophoresis revealed two radioactive products with electrophoretic mobilities identical to those of the hTeBGH and hTeBGL. The ratio of hTeBGH to hTeBGL was about 10:1. The H and the L protomers were separated and examined by peptide mapping using protease V8 and chymotrypsin. Comparison of the fragmentation patterns produced by these proteases revealed that hTeBGH and hTeBGL components were nearly identical. Removal of sialic acid or carbohydrate residues from hTeBG did not affect the presence of two molecular components. Isoelectric focusing of native hTeBG demonstrated three isoelectric variants with pIs at 4.75, 4.85 and 4.90. After treatment with neuraminidase and other glycosidases, only two isoelectric species were observed with more alkaline pIs. Although purified hTeBG appeared heterogeneous with regard to size and charge, it was remarkably homogeneous in its ability to absorb to Concanavalin A-Sepharose. We conclude that hTeBg, like the androgen binding proteins of the rabbit and rat, is a dimer whose monomer exhibits two protomeric forms.     

A rapid micromethod for apolipoprotein E phenotyping directly in serum

A new method for the apolipoprotein E phenotyping has been developed. The method is based on isoelectric focusing of either delipidated or guanidine-HC1-treated serum or plasma in a horizontal slab gel system followed by immunoblotting using either polyclonal or monoclonal anti-apolipoprotein E antibodies as first antibody. Apolipoprotein E phenotyping with this method in 200 serum samples that had been stored at -20 degrees C for more than one year gave exactly the same results as obtained with the conventional method based on isoelectric focusing of delipidated very low density lipoproteins isolated from fresh serum followed by protein staining. Compared with the conventional method, the present method is less laborious because ultracentrifugation to isolate VLDL is not needed; it is suitable for large scale screening purposes; it needs only a few microliters of serum or plasma, and can easily be performed with samples with low concentrations of apolipoprotein E.     

A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.     

Microprocedure to determine the polymorphic forms of acid α1-glycoprotein in plasma : Application to depressive patients treated with amitriptyline

The polymorphic forms of α₁-acid glycoprotein have been determined by isoelectric focusing of small samples of whole plasma, without prior isolation of the protein. The results obtained by this technique confirm the microheterogeneity of this glycoprotein, which is not due to artefacts. Densitometric measurements of the polymorphic forms of this protein, which binds antidepressive drugs, have been performed in twelve depressive patients.     

Ox spleen ferritin: an isoelectrofocusing and crossed immunoelectrofocusing study

Ox spleen ferritin was purified and its purity checked by two-dimensional immunoelectrophoresis and polyacrylamide plate electrophoresis. Microheterogeneity was shown with a preparation of purified ferritin by isoelectric focusing. The protein was separated into at least 6 fractions; two large fractions in the 4.50-4.55 pH range and another 4 in the 4.65-4.80 interval. Microheterogeneity was confirmed in purified preparations by crossed immunoelectrofocusing. Seven fractions were observed, the most acid ones (4.50-4.55) also being the most abundant. In the crossed IEF procedure, exactness in the isoelectrophoretic separation time is important in that excessive time may impair the resolution potential.     

A Double-Isotope Procedure for Examining Protein Microheterogeneity: Multiple Forms of Fast-Transported Glycoproteins and Sulfoproteins Possess a Common Polypeptide Chain

Several fast-transported proteins that appear as single bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolve into multiple spots during isoelectric focusing. A method was devised for determining if such microheterogeneity in net charge indicates that individual polypeptides have been posttranslationally modified to differing extents. Dorsal root ganglia were pulse-labeled with [35S]methionine and either [3H]leucine or [3H]proline, proteins fast-transported into peripheral sensory axons were separated by two-dimensional gel electrophoresis, and isotope incorporation ratios of proteins associated with individual gel spots were determined. When four microheterogeneous glycoproteins were analyzed, each protein "family" showed markedly similar isotope ratios for its three to seven characteristic spots. Such ratios differed between families by almost twofold. In addition, a group of nonglycosylated, sulfate-containing proteins was identified as a family on the basis of the similar isotope incorporation ratios of its component spots. These results suggest that protein microheterogeneity can result from variable sulfation of tyrosine residues as well as from variation in sialic acid-containing oligosaccharide side-chains. More generally, the method can be utilized to test for protein microheterogeneity in cases where the amounts of protein are too low to permit peptide mapping analysis and where the nature of the charge-altering modification is unknown.     

Developmental Change in Microheterogeneity of Serum Transferrin of Chickens

Developmental change in microheterogeneity of chicken serum transferrin (Tf) was investigated by polyacrylsmide-gel isoelectric focusing, direct immunofixation, and densitometry. Three main Tf species (Tf-0, Tf-1, and Tf-2, which have 0, 1, and 2 sialic acid residues per molecule, respectively) were resolved and their relative ratios were determined. As development proceeded, a relative increase occurred in the most acidic species (Tf-2) with decreases in the less acidic ones (Tf-0 and Tf-1).