Free fatty acids alter the electrophoretic mobility of serum lipoproteins

When normal human serum was analysed after addition of free fatty acids (FFA: oleic acids), the lipoproteins showed abnormal mobility on agarose-gel electrophoresis. β- and α-lipoproteins from the serum with a FFA concentration of 2.04 μEq/ml or more migrated with a mobility showing a more negative charge than native ones. Although the ultracentrifugal profile of the lipoprotein cholesterol was not altered with the excess-FFA, FFA were detected in the lipoprotein fractions as well as in free proteins. The lipoproteins isolated from the excess-FFA aerum also showed abnormal mobility on agarosegel electrophoresis.These results suggest that, in the serum containing excess-FFA, FFA attach themselves to the lipoproteins and alter their charge.     

Separation and size determination of human serum lipoproteins by agarose gel filtration

A method is described for the separation of the three major classes of human serum lipoproteins by gel filtration on columns of 4 and 6% agarose gel. After calibration of the columns, the elution volumes of the lipoproteins were used to calculate the molecular sizes and molecular weights of these macromolecules. The technique was employed to demonstrate aggregation of low density lipoprotein following partial delipidation, partial proteolysis, or mild heat denaturation. Agarose gel filtration shows promise as a useful method for the isolation, purification, and characterization of lipoproteins.     

Separation of subclasses of human serum high density lipoproteins by zonal ultracentrifugation.

An improved one-step method for the preparative separation of three subfractions of high-density lipoproteins from normal human serum has been developed. It employs the method of rate zonal ultracentrifugation in a z-60 rotor using a discontinuous NaBr gradient in the density range of 1.0-1.4. The density gradients were monitored directly by a flow-through density meter allowing the direct read-out of the actual densities in the process of filling and emptying the rotor. The separation of the three density fractions from 5 to 15 ml serum was achieved during a single 12 hours run at 59.000 rpm. The three fractions showed characteristically different patterns on polyacrylamide gel electrophoresis and differences in their lipid and protein composition.     

Studies on pig serum lipoproteins. I. Separation and properties of low-density lipoproteins.

The low-density lipoproteins in pig serum were separated into two subclasses (LDL1 and LDL2) by 2 to 7% pore size gradient gel electrophoresis. Preparative gel electrophoresis in 2 to 4% gradient gel made it possible to isolate these components as distinct entities. After delipidation by chromatography on Sepharose 4B in the presence of SDS, both apo-LDL1 and apo-LDL2 were found to have a molecular weight of 2.6X10(5). However, when these apoproteins were incubated in 10% sodium dodecyl sulfate, fragmentation occurred and the minimum fragment molecular weight was estimated to be 2.4X10(4). No essential difference was found in the amino acid compositions or fragmentation patterns of the apoproteins. However, the amounts of carbohydrates in the two apoproteins were different (7.09% in apo-LDL1 and 5.08% in apo-LDL2). The carbohydrate composition was 0.8% sialic acid, 2.38% N-acetyl-glucosamine, and 4.01% neutral sugars in apo-LDL1 and 0.5, 1.75, and 2.83% in apo-LDL2, respectively. In both apoproteins, mannose, galactose, and fucose were present in almost the same molar ratio of 4-5 : 2-3 : 1.     

Separation of macromolecular components of plant cell walls: electrophoretic methods

Qualitative quantitative and preparative electrophoretic methods of separating polymeric substances derived from plant cell walls are described. Analyt     

Characterization of apolipoprotein B-containing lipoproteins separated by preparative free flow isotachophoresis

Preparative free flow isotachophoresis (ITP) was used for the fractionation of apoB-containing lipoproteins (d less than 1.063 g/ml) from fasting and postprandial sera derived from normolipidemic individuals. According to their net electric mobility, four major particle groups (I-IV) have been recognized. The fast-migrating particles in group I, which correspond predominantly to very low density lipoproteins (VLDL), are rich in triglycerides, free cholesterol, phosphatidylcholine, and apoE and C apolipoproteins. This group expresses nonspecific binding to fibroblasts but binds to HepG2 cells with high affinity (KD = 3.6 micrograms/ml, Bmax = 37 ng) to a single class of binding sites. The particles migrating in group II, which are related to intermediate density lipoproteins (IDL), are richer in cholesteryl esters and apoB than those in group I. They interact specifically with a single site on fibroblasts (KD = 7.8 micrograms/ml, Bmax = 54 ng) while on HepG2 cells two binding sites, one with a higher (KD = 3.5 micrograms/ml, Bmax = 22 ng) and one with a lower affinity component (KD = 16.9 micrograms/ml, Bmax = 53 ng), are involved. The particles migrating in groups III and IV correspond to low density lipoproteins (LDL). The protein moiety of both fractions consists almost exclusively of apoB. Group III represents cholesteryl ester-rich LDL particles, while the particles in group IV contain smaller amounts of cholesteryl esters. The lipoproteins of both groups are ligands for apoB,E-receptors. However, the particles in group IV interact with fibroblasts with the highest affinity (KD = 2.3 micrograms/ml, Bmax = 58 ng) and with the biphasic HepG2 cell binding sites with the lowest affinity of all analyzed groups (KD1 = 11.2 micrograms/ml, Bmax1 = 58 ng, KD2 = 68 micrograms/ml, Bmax2 = 170 ng). When apoB-containing lipoproteins were isolated from postprandial sera of the same individuals, significant changes in the lipid composition were observed only in particle groups I and II, where the triglyceride and phospholipid content was enhanced. Group I particles from postprandial serum bind to HepG2 cells with a higher affinity (KD = 2.5 micrograms/ml) than group I particles from fasting serum. Postprandial group II particles bind with the same affinity to the biphasic HepG2 cell receptor as fasting group II particles, while the affinities of postprandial group III (KD1 = 4.1 micrograms/ml, KD1 = 47 micrograms/ml) and group IV particles (KD1 = 3.9 micrograms/ml, KD2 = 38 micrograms/ml) to the high affinity binding site of the biphasic receptor are enhanced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunoelectrophoretic analysis of mink sera lipoproteins fractionated by preparative ultracentrifugation]

Using the method of preparative ultracentrifugation, four lipoprotein fractions were obtained differing in hydrate density. By means of immunoelectrophoretic analysis of the fractions, it is found that mink sera (except chilomicrons) contains at least five lipoproteins; two of them, lipoproteins 2 and 3, bind the major bulk of lipids. Lipoprotein 1 is of very low density. Lipoprotein 2 mostly belongs to the fraction with density less than 1.100 g/ml, although antigenically identical molecules with higher density were identified. Lipoprotein 3 is much more heterogeneous antigenically, electrophoretically and in its density (from 1.006 to 1.210 g/ml and higher). This protein seems to contain several structurally similar, though not identical, apoproteins. Lipoproteins 4 and 5 are very dense (more than 1.210 g/ml) and are weakly stained with Sudan black. These data, together with the results obtained in our previous studies, indicate that lipoproteins 2 and 4 are marked with genetically alloantygenic determinants.     

A rapid electrophoretic technique for identification of subunit species of apoproteins in serum lipoproteins

The denaturing solvent tetramethylurea (TMU) delipidates and quantitatively liberates the apoproteins of human serum high-density lipoprotein (HDL) in soluble form while virtually the whole apoprotein of human lowdensity lipoprotein (LDL) is precipitated. A fraction of the apoprotein of very low density lipoprotein (VLDL) which appears to represent its content of LDL-like protein (apo B) is precipitated by this reagent, while the remaining apoprotein species are liberated in soluble form.The dissociation of the soluble apoproteins from lipid by TMU obviates the need for time-consuming delipidation by organic solvents, permitting immediate electrophoretic analysis in polyacrylamide gels. Bands are observed with mobilities corresponding to those of all the major soluble polypeptide species isolated from serum lipoproteins by ion-exchange chromatography. The apparent distribution of these elements in the different classes of lipoproteins is in agreement with findings of studies employing chromatographic methods. The predominant apoprotein of HDL, which has been identified immunochemically in VLDL, appears to comprise less than 1% of the apoprotein of VLDL from normal serum.     

Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by chromatographic and electrophoretic methods

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by oxo-compounds, particularly glucose, ribose, glyoxal and glutardialdehyde, have been investigated using a set of modern chromatographic and electrophoretic separation methods. High-performance liquid chromatography (HPLC) alternatively with UV spectrophotometric (diode array) or mass spectrometric (MS) detection, polyacrylamide gel electrophoresis (PAGE) with Coomassie brilliant blue staining detection, and capillary zone electrophoresis (CZE) with UV spectrophotometric detection have been employed for the investigation of the chemical and structural changes of BSA caused by its reaction with the above oxo-compounds exhibiting different degree of reactivity. The extent of modifications was found to be dependent on the nature of the oxo-compound used and progressed in the glucose相似文献   

Rapid preparative isolation of concentrated low density lipoproteins and of lipoprotein-deficient serum using vertical rotor gradient ultracentrifugation

In order to study cellular metabolism of low density lipoproteins (LDL), ultracentrifugal methods have been used to isolate the lipoproteins. The use of vertical rotor ultracentrifugation very quickly produces small quantities of diluted lipoproteins per gradient, as well as small volumes of lipoprotein-deficient serum. We present modifications to this method in order to prepare routinely more concentrated LDL and a sufficient volume of lipoprotein-deficient serum in a relatively short time with minimal cost and handling.