Separation and Properties of Pig-Serum Lipoproteins

Janado, Masanobu; Martin, William G.; Cook, W. H.

doi:10.1139/o66-137

Cited by 82 publications

(26 citation statements)

References 6 publications

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“…Low density lipoprotein and its two fractions LDL, and LDL2 were isolated from pig serum as previously described. 8 ) Then approximately 20 ml of the appropriate LDL (4 to 7 ~~ concentration) solution was placed in a 500 ml flask, 450 ml of peroxide-free ether added, and the mixture stirred with a magnetic stirrer. Agitation was adjusted to keep the lipoprotein suspension well distributed in the lower part of the ether phase, but without allowing it to reach the air-ether interphase at the top.…”

Section: Methodsmentioning

confidence: 99%

“…He separated a fraction (called az lipoprotein) from pig serum in the density range 1.063 to 1.074 that may have been a mixture since differential centrifugation in a comparable density range is necessary to isolate LOLl' Ultracentrifugal and immunoelectrophoretic examination of his a2 lipoprotein showed only one component, but LOLl and LOL 2 cannot be resolved ultracentrifugally in 0.1 M NaCI. 8 ) Immunoelectrophoresis showed only one protein but this could also be interpreted as two ultracentrifugal fractions carrying the same protein.…”

Section: _ ---------Of About 3 and 6 S)mentioning

confidence: 95%

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Evidence for Protein Subunits in Low Density Lipoprotein from Pig Serum

Janado¹,

Martin²

1973

Agricultural and Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Section: _ ---------Of About 3 and 6 S)mentioning

confidence: 95%

Evidence for Protein Subunits in Low Density Lipoprotein from Pig Serum

Janado¹,

Martin²

1973

Agricultural and Biological Chemistry

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“…Low-density lipoprotein was purified from porcine serum as described previously (7). Collodion bags retaining proteins larger than molecular weight 25,000 (cat.No.…”

Section: Materials Porcine Adrenocortical Cathepsinsmentioning

confidence: 99%

Activation of cathepsin D by polyanionic compounds

1996

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Summary: We examined the nature of the activation of cathepsin D by polyanionic compounds. Tripolyphosphate, a model compound for polyanions, decreased the K m value of porcine cathepsin D for bovine serum albumin without affecting V . Halfmaximal activation was achieved at 0.2 mM free lripolyphosphate. Electrophoretic mobility of cathepsin D decreased as the tripolyphosphate concentration was increased, and the enzyme had no net charge at 50 mM triP. The concentration for the halfmaximal mobility change of cathepsin D (0.18 mM) was similar to that for half-maximal activation. These results suggest that tripolyphosphate increased affinity of the enzyme for its substrate by cancelling positive charges on cathepsin D and thus decreasing the electrostatic repulsion.Key Words: cathepsin D, polyanion, ATP, tripolyphosphate, lysosome, activation, adrenal cortexThe lysosomal proteinase cathepsin D (EC 3.4.23.5) is activated by polyphosphate compounds such as ATP, GTP, coenzyme A, and sodium tripolyphosphate (triP)(1-3).The extent of activation increases as the number of phosphate in the molecule is increased up to 5. In the presence of triP the pH-activity curve is bimodal, with striking stimulation above pH 4. This is physiologically important, since intralysosomal pH is 4-6 (4). Interestingly, cathepsin D is also activated by phospholipid micelles (5), suggesting that lysosomal membranes activate the enzyme and that intralysosomal protein degradation is active on the surface of the membranes.Since the activation by polyphosphates was not observed with pepsin, another carboxyl proteinase, or with trypsin but was observed with several different substrates including a synthetic peptide, the activators were supposed to act upon the enzyme protein.abbreviations: BSA, bovine serum albumin; triP, tripolyphosphate

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“…Plasma from heparinor EDTA-treated pig blood (1 mg/ml) was separated by centrifugation, and the LDL was isolated by ultracentrifugation in a 60 Ti rotor of a Beckman L5-65B centrifuge (7,8). For the first centrifugation, a density of 1.006 g/ml was achieved with KBr.…”

Section: Introductionmentioning

confidence: 99%

Selective removal of apolipoprotein B-containing serum lipoproteins from blood plasma.

Stoffel¹,

Demant²

1981

Proc. Natl. Acad. Sci. U.S.A.

151

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Studies were undertaken to determine the applicability and effectiveness of immunoadsorption chromatography on anti-low density lipoprotein (LDL) columns as a plasma-cholesterol-lowering procedure. Mass production and isolation of monospecific antibodies against swine LDL (p = 1.006-1.063 g/ml) was carried out by immunization of sheep with swine LDL and selective antibody adsorption from their antisera by chromatography on LDL-Sepharose. The isolated LDL antibodies were then covalently linked to Sepharose CL-4B. Pig plasma LDL was effectively removed in vitro by the anti-LDL-Sepharose beads. In vivo studies were performed in pigs with (i) a plasma-separator membrane permeable to solutes below M, 2,000,000 or (ii) a blood centrifuge interposed into an arteriovenous shunt to separate the corpuscular elements of blood from the plasma. In either case, the plasma was passed through the anti-LDL-Sepharose column and recirculated into the venous part of the shunt. Plasma cholesterol levels were reduced 70-80% by this procedure and rebounded 3-4 days after the extracorporeal immunoadsorption procedure. This continuous plasma separation-immunoadsorption procedure may have broad applicability for the elimination of any plasma component with antigenic properties.Familial hypercholesterolemia (type II hyperbetalipoproteinemia) (1) is characterized by increased plasma cholesterol concentrations and increased plasma low density lipoprotein (LDL) levels. It has been established that this form of hypercholesterolemia is due to a reduced fractional catabolic rate and to a 2-to 3-fold overproduction of LDL (1-3); as a result, there is premature development of atherosclerosis.Three independent studies have been reported (4-6) that were designed to reduce the plasma cholesterol concentrations in patients with familial hypercholesterolemia by nonsurgical methods: plasmapheresis techniques (4, 6) and extracorporeal removal of LDL by means of heparin-agarose beads in blood venous packs (5). These approaches have produced transient reductions in plasma LDL levels.We report a procedure that results in specific removal ofapolipoprotein B (apo B)-containing lipoproteins (VLDL and LDL) by immunoadsorption. This study was undertaken to evaluate (i) the extent to which plasma LDL can be removed in vitro by an anti-LDL-Sepharose immunoadsorbent and (ii) whether and to what extent LDL (and VLDL) can be removed by continuous extracorporeal immunoadsorption in swine. Monospecific LDL antibodies were prepared from the serum of sheep immunized against pig LDL and were covalently linked to Sepharose. When swine plasma was passed through a column of anti-LDLSepharose, LDL was absorbed from the plasma. In vivo studies in pigs were performed by shunting blood from the arterial branch between the common carotid artery and internal jugular vein, separating the blood into a cell concentrate and a plasma fraction by means of a selective plasma-separator membrane technique or a continuous-flow blood cell-separator centrifuge. The plasma fraction wa...

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Separation and Properties of Pig-Serum Lipoproteins

Cited by 82 publications

References 6 publications

Evidence for Protein Subunits in Low Density Lipoprotein from Pig Serum

Evidence for Protein Subunits in Low Density Lipoprotein from Pig Serum

Activation of cathepsin D by polyanionic compounds

Selective removal of apolipoprotein B-containing serum lipoproteins from blood plasma.

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