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PubMed1975

The molecular dimensions of mitogenic factor from guinea pig lymph node cells.

The molecular dimensions of a mitogenic factor from guinea pig lymph node cells were investigated by means of gel filtration, sucrose density gradient ultracentrifugation, and CsCi isopycnic ultracentrifugation. The mitogenic factor was produced by stimulation of immune lymph node cells from inbred guinea pigs, strain 2 or 13, with the specific antigen, ovalbumin; and mitogenic activity was assayed by measuring incorporation of tritiated thymidine by target lymph node cells from syngeneic animals not immune to ovalbumin. The D-20,w of the mitogenic factor was estimated to be (9.9 plus and minus 0.2) times 10-7 cm2/sec by gel filtration on Sephadex G-100, and the apparent S20,w was estimated to be 2.4 plus and minus 0.2 S by sucrose density gradient ultracentrifugation. The buoyant density was determined to be 1.324 plus and minus 0.006 g/ml by CsCi isopycnic ultracentrifugation, and from this value the partial specific volume of mitogenic factor was estimated to be 0.71. From these data the molecular weight of the mitogenic factor was calculated to be 20,000, and the frictional ratio was calculated to be 1.2.

Animals↗

PubMed1988

Falsely high high-density lipoprotein triglyceride values by the heparin-manganese precipitation method.

Recent evidence indicates that high-density lipoprotein triglyceride (HDL-Tg) may be a predictor of coronary artery disease. We examined three methods for HDL-Tg measurement, comparing results obtained by measurement of Tg in the supernate after heparin-manganese chloride (heparin-Mn) precipitation of EDTA-treated plasma (I) with results obtained after preparative ultracentrifugation (II and III). In II, we used heparin-Mn precipitation of low-density lipoprotein (LDL) from the infranate after ultracentrifugation at d 1.006 to remove very-low-density lipoprotein (VLDL). In III, we performed sequential flotation ultracentrifugation at d 1.006 and 1.063, then measured Tg in the d greater than 1.063 fraction. Method I gave significantly higher HDL-Tg results than II and III, which gave essentially identical results. The difference in results between I and II was not caused by the presence of heparin or manganese chloride, because these were used in both methods. Prior removal of VLDL in II and III resulted in lower HDL-Tg values, and subsequent removal of LDL by precipitation or ultracentrifugation did not alter final HDL-Tg values. The higher values obtained in I were the result of the presence of VLDL-rich unsedimented precipitate in the supernate.

Chemical Precipitation↗

PubMed1984

Characterization of the interaction of human C4b-binding protein with physiological ligands.

The binding of C4b to C4b-binding protein (C4BP) was demonstrated at physiological ionic strength by analytical ultracentrifugation. The sedimentation rate of C4BP gradually increased from 9.4 S to a maximum of 18.5 S with increasing C4b concentration. The stoichiometry of different C4BP X C4b complexes was calculated from the sedimentation-velocity data. A linear relationship was established between the number of C4b bound per C4BP and the sedimentation rate of the complex. In order to define further the C4BP-C4b interaction, sucrose density gradient ultracentrifugation was also used. Trace amounts of 125I-C4BP were centrifuged through 12 sucrose density gradients, each of which contained a different concentration of C4b throughout the gradient. The sedimentation rate of the C4BP increased with increasing C4b input to a maximum of 19.5 S. These binding data, in conjunction with the stoichiometry measurements determined in the analytical ultracentrifuge, were analyzed by the methods of Scatchard and Hill. At physiological ionic strength, C4BP exhibited four binding sites for C4b, each having an association constant of 1.2 X 10(7) M-1. A Hill coefficient of 1.1 was calculated, indicating that the four binding sites were independent. At reduced ionic strength, two additional sites were detected. The sedimentation coefficient of C4BP(C4b)6 was 24 S. The hydrodynamic data suggest that after four C4b molecules have bound to C4BP, the binding of additional C4b is sterically hindered. This interpretation implies that all six binding sites on C4BP are identical. C4BP also bound C4(H2O) (the product resulting from spontaneous hydrolysis of the thiol ester bond in native C4) and weakly bound C4c, but had no measurable affinity for native C4 or C4d at physiological ionic strength. A low-affinity interaction between C3b and C4BP was also demonstrated in the analytical ultracentrifuge. The C4BP X C3b complex was specific because C4BP mediated the cleavage of C3b by Factor I to C3bi with concomitant dissociation of the complex.

Carrier Proteins↗

PubMed1982

Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.

Studies in recent years have suggested that measurement of high density lipoprotein (HDL) subclasses may provide significant information beyond that provided by measurement of total HDL. However, conventional methodology for separation of HDL subclasses involves various types of ultracentrifugation that are time-consuming, costly, and not suitable for many clinical or epidemiological studies. We have developed a simple precipitation method for the separation of HDL subclasses in human plasma. After precipitation of apoB-containing lipoproteins with heparin-Mn2+, HDL2 is precipitated by addition of dextran sulfate (mol wt 15,000). HDL2 cholesterol is calculated as the difference between total HDL cholesterol (heparin-Mn2+ supernatant) and HDL3 cholesterol (dextran sulfate supernatant). HDL2 determined by this method correlated well with results obtained by preparative ultracentrifugation (n = 295, r = 0.91) and analytical ultracentrifugation (n = 17, r = 0.92). In the original method final concentration of dextran sulfate was 0.09 g/dl; however further studies indicated that 0.13 g/dl is a more suitable concentration. The chemical compositions of HDL2 and HDL3 isolated by the precipitation method were very similar to those of HDL2 and HDL3 isolated by preparative ultracentrifugation. The concentration of HDL2 cholesterol was 40% higher in normal women than in normal men. In men with coronary heart disease, total HDL was decreased by 28%, HDL2 was decreased by 44%, while HDL3 was 19% lower. A similar pattern of change was found in women with coronary heart disease. In other conditions where total HDL either increased or decreased, the change in HDL2 was always proportionately greater than the change in total HDL. HDL3 showed relatively less change, and in some instances its concentration was unchanged. Thus HDL2 is the more variable component and may be a more meaningful index of altered HDL metabolism.

Chemical Phenomena↗

PubMed1977

Characteristics of soluble immune complexes prepared from oligovalent DNP conjugates and anti-DNP antibodies.

The stability of soluble immune complexes was investigated after isolation by gel filtration and sucrose gradient ultracentrifugation. Soluble immune complexes were formed between specific goat anti-dinitrophenol (DNP) antibodies and DNP conjugated to a large (19 S) carrier, namely bovine thyroglobulin. The composition and molecular weight of these complexes were determined by ultracentrifugation on calibrated sucrose density gradients and the use of different isotopic markers for antigen and antibody. A good separation of immune complexes containing one, two, or three antigen molecules per complex was obtained by ultracentrifugation while gel filtration was less effective. Ultracentrifugational analysis of fractions isolated by these two procedures showed that large immune complexes containing more than one antigen were relatively labile, whereas small immune complexes containing one antigen were stable. The stability of large immune complexes was dependent on dilution. Because dilution affects the size and composition of soluble immune complexes, it is important to emphasize that for the investigation of a causal relationship between the biological properties and the size and composition of immune complexes, analysis of the immune complexes should be performed in the same dilutions in which they will be used experimentally.

Animals↗

PubMed1978

Hormonal regulation of cAMP-independent protein phosphokinase activities: thyroxine and cortisol control of enzymes from rat liver cytosol.

Thyroxine control of cAMP-independent histone and casein phosphokinase activities was studied in thyroidectomized rats treated with thyroxine. All activities were evaluated in the presence of a thermostable inhibitor of cAMP-dependent enzymes. Cytosol enzymes can be resolved by sucrose gradient ultracentrifugation into three peaks of histone kinase activity (3.2S, 5S and 7.2S) and two peaks of casein kinases (3.6S and 7.1S). Neither thyroidectomy nor subsequent treatment of operated animals with thyroxine modifies the total histone kinase activity estimated, either in total cytosol or after its fractionation by the sucrose gradient ultracentrifugation. The activity ratios of different peaks were, however, changed. Casein kinase activity was significantly decreased after thyroidectomy (about 50%). Subsequent treatment with thyroxine restored this activity to its initial value. Sucrose gradient ultracentrifugation analysis showed that thyroxine action on the casein kinase activity is very specific. Only molecules that sediment in the 9S region were significantly stimulated by the hormone. Cortisol action on the casein kinase activity was studied in adrenalectomized animals treated with hormone for 24 h. Cortisol decreases the total casein kinase activity by about 30%. Sucrose gradient ultracentrifugation analysis showed that the population of molecules sedimenting at about 9S was the most sensitive to cortisol. The above data show that both thyroxine and cortisol control, in a selective way, the activities of cAMP-independent protein kinases. The same kinase molecules can be under double control by two different hormones that have opposite effects.

Adrenal Glands↗

PubMed1996

Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, band 3.

Solution studies of the cytoplasmic domain (molecular mass approximately 40kDa) of band 3, the anion exchanger from human erythrocyte membranes, previously suggested a dimeric molecule on the basis of the relative techniques of calibrated gel filtration and calibrated preparative ultracentrifugation. This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography and absolute ultracentrifugation techniques. Sedimentation velocity in the analytical ultracentrifuge combined with calibrated gel chromatography give a molecular mass M of (77 +/- 4) kDa, a value confirmed by low-speed sedimentation equilibrium. Velocity sedimentation in the analytical ultracentrifuge gave a single sedimenting species with an s o 20,w of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation constant Kd, with a value from direct fitting of the concentration distribution curves of (2.8 +/- 0.5) microM, confirmed by a value of approximately 3 microM obtained from fitting a plot of molecular weight Mw,app versus cell loading concentration. Hydrodynamic calculations based on the classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of approximately 10:1, consistent with observations from electron microscopy.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1961

Physiocochemical characterization of mouse myeloma proteins: demonstration of heterogeneity for each myeloma globulin.

Physicochemical characterization of mouse myeloma proteins revealed the individuality of each myeloma protein. When the myeloma proteins are considered collectively a wide range of individual properties were represented, including electrophoretic mobilities varying from the gamma to alpha region, hexose contents from 1 to 4 per cent, and ultracentrifugal components from 6.5 to 13 S. The 20 myeloma proteins could be divided into groups, the gamma type and the beta type myeloma globulins, on the basis of physicochemical, as well as immunoelectrophoretic, studies. Two gamma type myeloma proteins (5563, MPC-11) resembled normal gamma globulins, sedimenting as a single 6.5 S peak in the ultracentrifuge, and having a relatively low hexose content (1 per cent). Eighteen beta type mouse myeloma proteins differed from gamma myeloma proteins and, typically, were found on ultracentrifugal analysis to have multiple components with sedimentation coefficients of 6.5, 9, 11, and 13 S, having a higher hexose content (2 to 4 per cent) as well as distinctive chromatographic and starch gel electrophoretic properties. All of the mouse myeloma proteins were heterogeneous and heterogeneity of two types was observed. Polymer formation was responsible for the 9, 11, and/or 13 S components seen on ultracentrifugation of the beta type myeloma proteins. Starch gel electrophoresis revealed this type of heterogeneity as relatively widely separated myeloma protein components, presumably owing to the retardation effect of starch gel on the electrophoretic migration of the larger polymers. Starch gel electrophoresis revealed a different type of heterogeneity for the two gamma type myeloma proteins, each of these being shown to contain 5 or more components differing only in electrophoretic properties. The physicochemical characteristics of the gamma-type and beta-type myeloma proteins in the mouse indicated the close similarity of these proteins to the gamma- and beta-(2A)-myeloma proteins in man.

Animals↗

PubMed1978

A comparison of some immunological characteristics of very low density lipoproteins of normal and hypercholesterolemic rat sera.

The immunological characteristics of a very low density lipoproteins (VLDL) from normal and hypercholesterolemic rat sera were compared using polyspecific antisera to VLDL and high density lipoproteins (HDL) and monospecific antisera to apo-B, apo-C, apo-A-I, and apo-E. Ultracentrifugally isolated VLDL from normal serum were studied by immunodiffusion and found to contain both discrete and associated (with apo-B) apo-C and apo-E, probably in the form of lipid-containing lipoproteins. However, immunoelectrophoresis of whole serum revealed only an associated form of the liporpotein having pre-beta mobility (i.e., VLDL), suggesting that the presence of discrete lipoproteins in isolated VLDL, each containing a single apoprotein family, may represent ultracentrifugal artifacts. Ultracentrifugally isolated VLDL from diet-induced hypercholesterolemic rat serum contained only trace amounts of apo-C and large quantities of apo-E, both of which were totally associated with apo-B. VLDL isolated by ultracentrifugation from perfusate of normal and hypercholesterolemic livers contained only associated lipoprotein complexes made up of apo-B, apo-C, and apo-E in the former but only apo-B and apo-E in the latter. These data suggest that normal VLDL are secreted as lipoprotein complexes containing apo-B, apo-C, and apo-E, which may become destabilized in the circulation. However, VLDL from hypercholesterolemic serum shows a marked diminution in the quantity of apo-C as indicated by the relative incorporation of [3H]leucine in vivo and by polyacrylamide gel electrophoresis of apo-VLDL.

Animals↗

PubMed1980

A comparison of three methods of measuring serum high density lipoprotein cholesterol in diabetics and non-diabetics.

Serum high density lipoprotein (HDL) cholesterol was measured in non-diabetic and diabetic men and women by ultracentrifugation, heparin-manganese chloride precipitation (heparin/Mn++), and sodium phosphotungstate-magnesium chloride precipitation (PT/Mg++). Results of the three methods were closely correlated in both diabetics and non-diabetics. Concentrations were, however, generally lowest by PT/Mg++, intermediate by ultracentrifugation, and highest by heparin/Mn++. These differences were small magnitude except in the case of diabetic patients treated with insulin. In these patients, serum HDL cholesterol levels were significantly greater than those of non-diabetic patients when measured by heparin/Mn++ and ultracentrifugation, but not by PT/Mg++. This was explained by the finding that the discrepancy between PT/Mg++ and ultracentrifugation increased with increasing HDL cholesterol concentration.

Adult↗

PubMed2002

Influence of adsorption and deproteination on potential free thyroxine reference methods.

BACKGROUND: There is a need for consensus concerning reference methods to be used for calibration of commercial free-thyroxine (FT(4)) assays. METHODS: Three different potential reference techniques were investigated for adsorption of T(4) to membrane materials, including any in vitro solid surfaces to which T(4) might adsorb, and for efficient separation of the T(4)-binding proteins from the free hormone fraction. We compared ultrafiltration with different commercial ultrafiltration units, ultrafiltration by dialysis tubing, equilibrium dialysis, and ultracentrifugation. We measured the adsorption to membranes and materials with L-[(125)I]T(4). Separation efficiency was determined by measuring the T(4)-binding protein albumin in the ultrafiltrate and the dialysate as well as in the supernatant from the ultracentrifugation with a double-antibody sandwich ELISA technique. RESULTS: We found a constant relationship between the amount of T(4) adsorbed to the dialysis or ultrafiltration membranes/materials and the initial T(4) concentration in HEPES buffer (protein-free medium). T(4) was considerably less adsorbed from serum than from HEPES buffer (P <0.001). Serum T(4) was less adsorbed upon ultracentrifugation than during dialysis and ultrafiltration (P <0.001). It was difficult to completely separate FT(4) from the binding proteins by ultrafiltration and ultracentrifugation. Separation by ultrafiltration depended on the material used. CONCLUSIONS: No investigated separation technique provides technically and theoretically correct separation of the free fraction of T(4) from the protein-bound fraction. Equilibrium dialysis seems to be the least compromised.

Adsorption↗

PubMed1991

Nucleation of cholesterol crystals from native bile and the effect of protein hydrolysis.

Nucleation time represents the terminal step in in vitro studies examining bile lithogenicity. Because of the concern that residual microcrystals, left after ultracentrifugation, may be responsible for the rapid nucleation time of gallbladder bile from patients with cholesterol gallstones, we have included a final filtration step. However, we found this procedure to considerably lengthen the nucleation time of abnormal biles. In view of the central importance of the nucleation assay we compared the effect of three commonly used gallbladder bile pre-treatment regimes (designed to remove endogenous crystals) on nucleation time. They were: a) immediate filtration of bile (0.22 micron filter); b) ultracentrifugation; and c) ultracentrifugation followed by filtration. The respective nucleation times were: a) 9.3 +/- 3.7 days, n = 6; b) 2.9 +/- 0.4 days, n = 10; c) 12.8 +/- 2.3 days, n = 11. To determine whether the dramatic change in nucleation time was due to the removal of components other than seed crystals, we examined the mucus content, the total lipid composition of bile, and that of its cholesterol transport components following the different pre-treatments. No significant difference in total lipid, percentage cholesterol carried by the transport components, or their cholesterol/phospholipid ratio were found. Ultracentrifugation alone was sufficient to removal all detectable large molecular weight mucus glycoprotein. Although nucleation time of the abnormal gallbladder samples was extended in the ultracentrifuged/filtered biles, it was still significantly different (P less than 0.01) from that of normal gallbladder biles, confirming an intrinsic difference between abnormal and normal biles, in cholesterol metastability. We also examined the effect of protein digestion on the nucleation time of native biles.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile↗

PubMed1986

Lack of relationship in humans of the parameters of body cholesterol metabolism with plasma levels of subfractions of HDL or LDL, or with apoE isoform phenotype.

The factors involved in regulating parameters of whole body cholesterol metabolism in humans have been explored in a series of investigations. Several physiological variables have been identified (weight, excess weight, plasma cholesterol, and age) that can predict 53-76% of the variation in production rate (PR) and in the sizes of the rapidly exchanging pool of body cholesterol (M1) and of the minimum estimates of the slowly exchanging pool of body cholesterol (M3min) and of total body cholesterol (Mtotmin). Surprisingly, measurements of the plasma levels of HDL cholesterol and of the major HDL apolipoproteins (apoA-I, A-II, and E) did not provide additional information useful in predicting parameters of whole body cholesterol metabolism. A study was therefore conducted to investigate possible relationships of the plasma levels of subfractions of lipoproteins, determined by analytic ultracentrifugation, and of apoprotein E phenotype, with the parameters of whole body cholesterol metabolism. Ultracentrifugal analysis of plasma lipoprotein subfractions was performed at the Donner Laboratory in 49 subjects; all of these subjects were currently undergoing whole body cholesterol turnover studies or had previously had such studies and were in a similar metabolic state as judged by plasma lipid and lipoprotein values. Apoprotein E phenotyping was carried out in 71 subjects. Differences in model parameters were sought among subjects with various apoprotein E phenotypes. Ultracentrifugal LDL subfractions Sof 0-2 (the region of LPa), Sof 0-7 (smaller LDL), Sof 7-12 (larger LDL), Sof 12-20 (IDL), and ultracentrifugal HDL subfractions Fo1.20 0-1.5 (smaller HDL3), Fo1.20 2-9 (larger HDL3 plus HDL2), and Fo1.20 5-9 (larger HDL2 or HDL2b) were examined for correlations with each other and with parameters of whole body cholesterol metabolism.

Adolescent↗

PubMed1982

Identification of multiple subclasses of plasma low density lipoproteins in normal humans.

Density gradient ultracentrifugation of low density lipoproteins (LDL) from 12 normal subjects showed multiple, distinct isopycnic bands. Densitometric scanning of the gradient tubes revealed that each band could be assigned to one of four density intervals and that the boundaries of these intervals were consistent among all the subjects. Analytic ultracentrifuge flotation (S(f)(0)) rates were assigned to the four density intervals, and there was a strong correlation between peak S(f)(0) rate and peak isopycnic banding position (R(f)) of the LDL in the 12 subjects. The S(f)(0) value corresponding to the boundary between the two most buoyant LDL density subgroups was 7.5. This value is close to that previously demonstrated to define two LDL subdivisions (S(f)(0) 0-7 and S(f)(0) 7-12) that were discriminated by differing concentrations in men and women, and differing statistical relationships with levels of HDL and VLDL in a normal population. Further delineation of distinct subspecies of LDL was afforded by electrophoresis in 2-16% gradient polyacrylamide gels. Densitometric scans of protein-stained gels revealed multiple peaks, and particle diameters were assigned to these peaks using calibration markers. Particles of diameter >/= 280 A included both IDL and Lp(a), the latter defined by pre-beta mobility on agarose electrophoresis and density > 1.050 g/ml. LDL particles with diameters 220-272 A could be grouped into seven size intervals defined by modes in the distribution of gradient gel electrophoretic peaks in LDL from a group of 68 healthy men and women. Particle diameters of the major peaks in each of seven density subfractions decreased with increasing density of the fractions. However, particles within each of the size groups were distributed across a range of densities. Use of a lipid-staining procedure allowed identification of electrophoretic bands in whole plasma which corresponded to those seen in isolated LDL, eliminating the possibility that ultracentrifugation was responsible for formation of the subspecies detected by the gradient gel procedure. The application of density gradient ultracentrifugation and gradient gel electrophoresis provides a means of characterizing LDL from normal humans in terms of multiple distinct subpopulations which may also prove to have differing metabolic and pathologic properties.-Krauss, R. M., and D. J. Burke. Identification of multiple subclasses of plasma low density lipoproteins in normal humans.

Adult↗

PubMed1997

CR1, CD35 in synovial fluid from patients with inflammatory joint diseases.

OBJECTIVE: To investigate synovial fluid (SF) for the presence of CR1 and to study its relationship to SF leukocytes and to serum levels of soluble CR1 (sCR1) in patients with rheumatic diseases. METHODS: Synovial fluids were collected from 35 patients with rheumatoid arthritis (RA) and 26 patients with other inflammatory joint diseases. Total CR1 in the SF and serum were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) that recognized both soluble and transmembrane forms of CR1. The characteristics of CR1 in SF were analyzed by ultracentrifugation and by a second ELISA specific for transmembrane CR1. RESULTS: CR1 was found in all SF samples tested (range 5-281 ng/ml). SF CR1 was higher in patients with RA (mean +/- SD 81 +/- 66 ng/ml) than in those with other inflammatory joint diseases (31.8 +/- 23.8 ng/ml) (P < 0.001). Serum sCR1 was not significantly increased in the patients compared with the normal subjects. There was no correlation between serum sCR1 and SF CR1. In 44% of the patients, the SF CR1 level was higher than the serum sCR1 level. A fraction (30-80%) of SF CR1 was pelleted by ultracentrifugation and, unlike serum sCR1, it reacted in an ELISA specific for transmembrane CR1. Thus, SF contained 2 forms of CR1: a membrane-associated and a soluble form, which was confirmed by sucrose density-gradient ultracentrifugation. SF CR1 levels correlated directly with the number of SF total leukocytes and polymorphonuclear leukocytes (PMN). These 2 forms of CR1 were also found in the supernatant of in vitro-activated PMN from normal subjects. SF CR1 exhibited the capacity to act as a cofactor for the factor I degradation of C3b. CONCLUSION: CR1 is found in the SF of patients with joint inflammation. The data suggest that SF CR1 originates from the infiltrating leukocytes, which shed both a soluble and a membrane-associated form. Whether SF CR1 participates in the local regulation of complement activation remains to be examined.

Arthritis↗

PubMedDate not supplied

Mucin glycoprotein content of human pigment gallstones.

Mucin glycoproteins, a secretory product of the gallbladder, are thought to contribute to the matrix or nucleus of gallstones. Human black pigment stones originate in the gallbladder and have as their major constituent calcium bilirubinate, as well as inorganic salts and small amounts of cholesterol. The object of this study was to estimate the amount of glycoprotein in black pigment stones and to isolate gallbladder mucin from dissolved stones. Black pigment stones containing 18 to 65% calcium bilirubinate were first dissolved in 12.5 mM EDTA/0.1 N NaOH and decolorized, then subjected to glycoprotein assay. The mean glycoprotein content of eight stones was 12.4%. In separate experiments, pigment stones were partially dissolved by brief exposure to EDTA/NaOH to minimize glycoprotein breakdown, and the glycoproteins isolated by gel filtration and ultracentrifugation. Pigment stones contained two glycoprotein fractions on Sepharose 4B; a high molecular weight mucin glycoprotein in the void volume and a lower molecular fraction in the included volume. Mucin was further purified by density gradient ultracentrifugation in cesium chloride. Three separate mucin fractions had an average buoyant density of 1.48 gm per ml which is typical for these glycoproteins. Bile pigment was associated with high molecular weight mucin even after extensive dialysis, gel filtration, and density gradient ultracentrifugation. The identity of mucin was further established by beta-elimination of glycoproteins in alkaline borohydride which yielded galactosaminitol from cleavage of O-glycosidic bonds. Our results indicate that mucin glycoproteins are present in significant concentrations in human black pigment stones and can be purified from stones solubilized in EDTA/NaOH. The association of bile pigment with gallbladder mucin, even after extensive purification, is consistent with the hypothesis that mucin contributes to the matrix of pigment gallstones.

Bilirubin↗

PubMed2001

Identification of a candidate integrin-fraction associated with the activated form of the PDGF-receptor.

The activated PDGF-receptor has been shown to coimmunoprecipitate together with alpha v beta 3 integrin out of the 15,000g soluble supernatant of non-ionic detergent cell lysates. I have now further characterized this complex by ultracentrifugation analysis. The ultracentrifugation-conditions were chosen so that the phosphorylated form of the PDGF-receptor was pelleted out of the 15,000g soluble supernatant. Together with the tyrosine-phosphorylated PDGF-receptor small amounts of integrins, cytoskeletal- and extracellular matrix proteins were recovered in the pelleted material. The results show that (i) the candidate-fraction of integrins interacting with the activated PDGF-receptor is small compared to the overall integrin population in the cell lysate, (ii) several proteins known to be present in focal adhesions such as FAK, talin, and vinculin are absent from the integrin-growth factor receptor complexes, while on the other hand (iii) a tyrosine-phosphorylated protein migrating at 120 kDa was highly enriched in the ultracentrifugation-pellet, and finally (iv) non-ionic detergent cell lysates appear to contain quantitatively small fractions of complexed proteins that are qualitatively distinct from their total cellular population. Thus, the separation of protein-complexes from the total cellular proteom may be instrumental for the investigation of cellular protein complexes in general.

Cell Fractionation↗

PubMed1980

Interaction of human plasma high density lipoprotein HDL2 with synthetic saturated phosphatidylcholines.

The interaction of human plasma high density lipoprotein HDL2 (d 1.063-1.125 g/ml) with sonicated dispersions of synthetic saturated phosphatidylcholines, dipalmitoyl- (diC16PC), dimyristoyl- (diC14PC), didodecanoyl- (diC12c), didecanoyl- (diC10PC), and dioctanoyl- (diC8PC) L-alpha phosphatidylcholine, was investigated. Incubation (4.5 hr, 37 C) of HDL2 with diC14PC, diC12PC, diC10PC and diC8PC followed by gradient gel electrophoresis of preparative ultracentrifugation resulted in a redistribution of apolipoprotein A-I (apoA-I). The extent of redistribution depended on the molar ratio of the phospholipid to HDL2 in the incubation mixture. Redistributed apoA-I occurred as lipid-free apoA-I and/or as complexes of apoA-I and/or as compelxes of apoA-I with phosphatidylcholine. Increasing the length of time of ultracentrifugation of the interaction mixtures did not increase the extent of redistribution. No redistribution of apoA-I was detected following incubation and gradient gel electrophoresis or preparative ultracentrifugation of mixtures of HDL2 with dispersions of diC16PC.

Apolipoprotein A-I↗