Analysis of glycopeptides as borate complexes by polyacrylamide gel electrophoresis

A new method using polyacrylamide gel electrophoresis in a Tris-borate buffer to analyze Pronase-derived glycopeptides is described. Examination of immunoglobulin, Sindbis virus, and ovalbumin-radiolabeled glycopeptides by this system demonstrates a pattern similar to that seen after Bio-Gel-P-6 chromatography and, in addition, exposes a heterogeneity in the immunoglobulin and Sindbis virus glycopeptides not apparent after gel filtration. The resolution of glycopeptides by gel electrophoresis depends on the inclusion of borate ions in the sample, the gel, and the electrophoresis buffer. The borate ions react with neutral sugars, converting them to charged complexes which migrate during electrophoresis. The number of borate ions bound to a glycopeptide is a function of the composition, sequence, and linkages of the carbohydrates. Gel electrophoresis of glycopeptides in a borate buffer has several advantages: (1) The method requires no new equipment or special skills beyond those necessary for conventional polyacrylamide gel electrophoresis; (2) when performed on a slab gel, up to 24 samples can be analyzed simultaneously; and (3) since detection is by radio-autography, small amounts of radiolabeled glycopeptides can be visualized by prolonging the exposure time. These characteristics are advantageous for studies of glycopeptides based on digestion products resulting from incubations with specific exo- and endo-glycosidases. Untreated glycopeptides have been compared on the same gel with glycopeptides sequentially treated with different glycosidases to gain structural information.     

Separation of glycopeptides derived from brain glycoproteins by gel filtration.

Glycopeptides obtained from rat brain by proteolytic digestion with papain have been separated from glycosaminoglycans by means of gel filtration. The glycosaminoglycans appear in the void volume, whereas the glycopeptides are retarded. Glycopeptides of groups A+B (Brunngraber et al., 1973) (MW = 3800-500) C+D (MW = 2000) which were partially resolved by the method, were identified in the elution profile. Nucleic acids, also solubilized by papain, are eluted together with the glycosaminoglycans.     

Purification of bacteriophage DNA by gel filtration chromatography

Two fast and effective methods for high-scale purification of linear phage lambda DNA and circular double-stranded M13 replicative form are presented. A substantial reduction of time is attained by avoiding the long-term CsCl gradient centrifugations and dialysis common to standard procedures. Biologically active DNA preparations, free of chromosomal DNA and RNA, are obtained by including a simple gel filtration chromatography as the last step of purification. Yields are comparable to those from previously described methods.     

Apolipoprotein composition of bovine lipoproteins isolated by gel filtration chromatography

1. Bovine lipoproteins were isolated from plasma by gel filtration and apolipoprotein composition determined by SDS-polyacrylamide gel electrophoresis. 2. Bovine triglyceride-rich lipoproteins contained a novel low mol. wt protein Mr = 22,000 and low mol. wt proteins that may be analogous to non-ruminant apolipoproteins A-I, A-IV, and E. 3. Apolipoprotein C appeared to be a minor constituent of bovine triglyceride-rich lipoproteins. 4. Triglyceride-rich lipoproteins contained two high mol. wt proteins of approx. Mr = 220,000 and 290,000. 5. The predominant bovine low density lipoprotein apolipoprotein was approx. Mr = 290,000, however, greater then 25 proteins were often observed between Mr = 110,000 and 370,000. 6. Bovine high density lipoprotein contained proteins analogous to apolipoprotein A-I and C apolipoproteins. 7. Differences in apolipoprotein profiles between non-lactating and lactating cows were not apparent.     

Heterogeneity of human very low density lipoproteins by gel filtration chromatography

Very low density lipoproteins were separated by gel filtration on Sepharose 4B. A decrease in mean particle diameter and flotation rate was seen with increasing elution volumes. The smaller lipoproteins had relatively more protein and phospholipid and less triglyceride than the larger ones. No differences were noted in the relative contents of the various phospholipids or partial glycerides between small and large lipoproteins. Fatty acid patterns of triglycerides and cholesteryl esters were also similar for the various lipoproteins. Relatively more lecithin containing linoleoyl acyl groups was found in smaller lipoproteins of some subjects. More of the protein of smaller lipoproteins was apo-LDL protein. Apo-HDL peptide was lost from the very low density lipoprotein as a consequence of the gel filtration.     

Refolding of fusion ferritin by gel filtration chromatography (GFC)

Fusion ferritin (heavy chain ferritin, F_H+light chain ferritin, F_L), an iron-binding protein, was primarily purified from recombinantEscherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, andl-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclussion bodies (Ibs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.     

Rat small-intestinal galactosidases. Separation by ion-exchange chromatography and gel filtration

1. The chromatography of rat small-intestinal beta-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure beta-galactosidase activity (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid beta-galactosidase, hydrolysing lactose and the hetero-beta-galactosides at about the same rate, and one more neutral beta-galactosidase, hydrolysing lactose much more rapidly than the hetero-beta-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.     

Interpretation of the stokes radius of macromolecules determined by gel filtration chromatography

From a comparison of the gel chromatographic properties of large randomly-coiled polypeptides in 6 M guanidine hydrochloride and of large globular proteins, we found that the distribution coefficient was more closely correlated with the intrinsic viscosity-based Stokes radius than with the translational frictional coefficient-based Stokes radius. This means that the effect of the hydrodynamic flow of dissolved molecules during gel chromatography should be considered. The ratio of transport of solute by bulk flow as compared with that by net diffusion (i.e., Brownian motion) is large under some conditions. On the other hand, we consider that the distribution coefficient obtained in static equilibrium experiments should be determined by the translational frictional coefficient-based Stokes radius, since the solvent does not flow. On this basis, we discuss the meaning of the Stokes radius and the separation mechanism of macromolecules by gel filtration.     

Structural analysis of glycopeptides by polyacrylamide gel electrophoresis

Three different well-characterized preparations of proteoglycan subunits were analyzed by high-performance liquid chromatography on a silica-based material bonded with an amide phase. The biochemical integrity of the proteoglycan subunits was retained during this procedure. The high sensitivity coupled with the increased speed of high-performance liquid chromatography will permit rapid analysis and comparisons of very small specimens.     

Isolation of small agranular synaptic vesicles of rat brain by gel filtration chromatography

I attempted to isolate synaptic vesicles by gel filtration. The rat brain synaptic vesicles in a synaptosomal lysate were collected by ammonium sulfate salting-out and fractionated on a Sephacryl S-500 with a mean exclusion size of 200 nm. Peak I at the void volume contained large vesicular membranes and coated vesicles besides synaptic vesicles; Peak II consisted almost entirely of small agranular synaptic vesicles of 40-50 nm diameter; and Peak III comprised soluble proteins. Western blotting revealed that components of 72 kDa in peaks I and II reacted with an anti-H(+)-ATPase A-subunit antibody [Moriyama et al. (1995) FEBS Lett. 367, 233-236]. When examined for Mg(2+)-ATPase activity, peak I showed specific activity of 4.52 ( micromol ATP hydrolyzed/mg protein/30 min), while that of peak II was as low as 0.22. As estimated from the inhibition by bafilomycin A(1) [Bowman et al. (1988) PROC: Natl. Acad. Sci. USA 85, 7972-7976], the percentage of H(+)-ATPase as to total Mg(2+)-ATPase, 18-22%, was unchanged, indicating no accumulation of the H(+)-ATPase in peak II even on the chromatography. In brief, the small agranular synaptic vesicles in peak II showed little or no Mg(2+)-ATPase activity, although they reacted with the H(+)-ATPase antibody. The reason for this is obscure. Mg(2+)-ATPase might not be a constituent of small agranular synaptic vesicles of rat brain.     

Binding of FKBP23 to BiP in ER shown by gel filtration chromatography

FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal domain with Ca2+ binding sites. Previously, we reported that FKBP23 specifically binds to BiP, the main protein of the molecular chaperone Hsp70 in ER lumen, and the binding is interrelated with the Ca2+ concentration. In this work we have found the existence of the complex FKBP23/BiP by separation of an ER extract using gel filtration chromatography (GFC), and that the existence of this complex is Ca2+-interrelated. This result further verified the Ca2+-interrelated binding of these two proteins in vivo.     

Purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase by gel filtration chromatography.

A procedure is presented for the large-scale purification of rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12). Sephadex G-100 chromatography was more effective than repeated recrystallizations for removing heme impurities, hence these proteins appear to cocrystallize. The column purified enzyme has full enzymatic activity according to dehydrogenase, esterase, and acetyl phosphatase assays.