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Biomedical subjects

A M Scanu

Publications and source records attributed to A M Scanu.

At least 109 records · Page 6Linked to original sources
PubMed1988

Demonstration that the enzyme that converts precursor of apolipoprotein A-I to apolipoprotein A-I is secreted by the hepatocarcinoma cell line Hep G2.

The conversion of the precursor of apolipoprotein A-I (proapoA-I) to apolipoprotein A-I (apoA-I) is known to occur extracellularly by an enzyme that has been shown to be present in plasma. The hepatocarcinoma-derived cell line Hep G2, when grown in culture, secretes proapoA-I. We now show that this cell line also secretes the converting enzyme that correctly processes proapoA-I to mature apoA-I as determined by radio-sequence analyses. The secreted enzyme is inhibited by EDTA and 1,10-phenanthroline, is activated by Ca2+ and is unaffected by both phenylmethylsulfonyl fluoride and diisoprophylfluorophosphate in the same way as the converting enzyme previously described in the plasma. The conversion of proapoA-I to apoA-I effected by this enzyme obeys first-order kinetics and is linear over the first 4 h with a calculated initial velocity of 3.3% conversion per hour. The converting activity is secreted in a time-dependent fashion and parallels the mass of total secreted protein.

Apolipoprotein A-I↗
PubMed1988

Identification of the active-site serine in human lecithin: cholesterol acyltransferase.

Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.

Amino Acid Sequence↗
PubMed1988

Lipoprotein(a): a genetically determined lipoprotein containing a glycoprotein of the plasminogen family.

Lp(a) represents a genetically transmitted class of plasma LDL having apo B-100 linked by a disulfide bridge to a glycoprotein, apo(a). Lp(a) is heterogeneous in size and density. Apo(a) is also heterogeneous in size (molecular weight between approximately 300,000 and 700,000) due probably to the polymorphism of both polypeptide and carbohydrate chains. Recent studies have shown that apo(a) has a striking amino acid sequence homology with plasminogen, a serine protease zymogen that following activation to plasmin enters the fibrinolytic system. Apo(a) is severalfold larger than plasminogen (molecular weight approximately 90,000) and also differs from it because it fails to be activated to plasmin. This is due to the fact that arginine is replaced by serine at the site of cleavage by streptokinase, urokinase, or tissue plasminogen activator. A single gene locus appears to control the Lp(a) polymorphism as well as the concentration of the Lp(a) phenotypes in the plasma. Patients with high plasma levels of Lp(a) have been shown to have an increased incidence of cardiovascular disease but a causal relationship has not been firmly established. The information that is being rapidly acquired on the structure of Lp(a) should facilitate the understanding of the molecular basis of the polymorphism of this genetic variant and of the role that the various Lp(a) phenotypes play in atherosclerosis and thrombosis. The potential physiologic role of Lp(a) remains open to inquiry.

Apolipoproteins↗
PubMed1988

Lipoprotein(a). A potential bridge between the fields of atherosclerosis and thrombosis.

Lipoprotein(a) (Lp[a]) represents a class of plasma lipoprotein particles that have overall characteristics similar to low-density lipoproteins but distinct from them by having apolipoprotein B100 linked to apolipoprotein(a) by disulfide bridge(s). This protein has recently been shown to have a striking amino acid sequence homology with plasminogen, a serine protease zymogen that on activation to plasmin promotes the conversion of fibrinogen to fibrin. The high incidence of Lp(a) in the plasma of patients with cardiovascular disease has been noted by many investigators. The new knowledge being rapidly acquired on the structure of Lp(a) should facilitate the understanding of the mechanism of its atherogenicity and perhaps shed light on its possible physiologic role.

Apolipoproteins A↗
PubMed1988

Modification of low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway.

In previous studies we reported that polymorphonuclear cell (PMN) elastase cleaves apoB-100 of human plasma low density lipoprotein (LDL) into seven or eight large Mr fragments (1, Polacek, D., R.E. Byrne, G.M. Fless, and A.M. Scanu. 1986. J. Biol. Chem. 261: 2057-2063). In the present studies we examined the interaction of native and elastase-digested LDL (ED-LDL) with primary cultures of human monocyte-derived macrophages (HMD-M). For this purpose LDL was digested with purified PMN elastase, re-isolated by ultracentrifugation at d 1.063 g/ml to remove the enzyme, and radiolabeled with 125I. At all LDL concentrations in the medium, the degradation of 125I-labeled ED-LDL was 1.5- to 2.5-fold greater than that of 125I-labeled native LDL, and for both lipoproteins species it was further enhanced by prior incubation of the cells in autologous lipoprotein-deficient serum (ALPDS). ED-LDL incubated with HMD-M in a medium containing [14C]oleate stimulated cholesteryl [14C]oleate formation 2- to 3-fold more than native LDL. In competitive degradation experiments, unlabeled ED-LDL did not inhibit the degradation of 125I-labeled acetylated LDL, whereas it caused a 90% inhibition of the degradation of 125I-labeled native LDL. At 4 degrees C, the binding of both 125I-labeled native and 125I-labeled ED-LDL was specific and of a high affinity. At saturation (Bmax), the binding of 125I-labeled ED-LDL was 2-fold higher (68 ng/mg cell protein) than that of 125I-labeled native LDL (31 ng/mg), with Kd values of 6.5 x 10(-8) M and 2.1 x 10(-8) M, respectively. A possible explanation of the binding data was provided by electrophoretic analyses suggesting that ED-LDL was twice the size of native LDL and thus potentially capable of delivering proportionately more cholesterol to the cells. Taken together, the results indicate that 1) digestion of LDL by purified PMN elastase results in a greater mass of ED-LDL (relative to native LDL) being degraded per unit time by HMD-M; 2) uptake of ED-LDL occurs via the LDL receptor; and 3) LDL digested by PMN elastase undergoes a physical change that may be responsible for its unique interactions with HMD-M. We speculate that if this process were to occur in vivo during an inflammatory process, macrophages could acquire excess cholesterol and be transformed into foam cells which are considered to be precursors of the atherosclerotic process.

Animals↗
PubMed1988

Genetically determined hypercholesterolemia in a rhesus monkey family due to a deficiency of the LDL receptor.

A family of rhesus monkeys comprising a sire, a dam, and four male offspring were fed a cholesterol-free Purina Chow diet for several months. The sire, 431-J, and two of the offspring, B-8204 and B-8806, had persistent plasma cholesterol levels in the range of 100-130 mg/dl, whereas the dam, 766-I, and the two other offspring, B-1000 and B-7643, exhibited a marked hypercholesterolemia in the 250-300 mg/dl range associated with an elevation of plasma LDL and apoB. When fed for 12 weeks a diet containing 12.5% lard and 0.25% cholesterol, sire, dam, B-1000 and B-7643 exhibited a marked hypercholesterolemia (500-800 mg/dl range), whereas B-8204 and B-8806 developed only a modest hypercholesterolemia (200-250 mg/dl). All animals were Lp[a]+. Skin fibroblasts from each animal and from control cells were grown in 10% fetal calf serum, transferred to 10% lipoprotein-deficient serum for 48 hr, and then incubated at 4 degrees C or 37 degrees C with 125I-labeled Lp[a]-free LDL. The fibroblasts from dam and offspring B-1000 and B-7643 bound and internalized 125I-labeled LDL less efficiently than control cells. Mathematical analyses of the 4 degrees C binding data indicated that there were no significant differences in LDL binding affinity between test and control cells suggesting that cells from the animals with a spontaneous hypercholesterolemia had a decreased number of LDL receptors. This conclusion was supported by the results of ligand and immunoblot analyses carried out on cell lysates separated by gradient gel electrophoresis. We conclude that a genetically determined LDL receptor deficiency was responsible, in part, for the spontaneous hypercholesterolemia observed in three out of the six family members and that this deficiency accounted for the hyperresponsiveness to a dietary fat and cholesterol challenge by the dam and the two offspring, B-1000 and B-7643. The hyperresponsiveness noted in the sire that had no evidence for LDL-receptor deficiency illustrates that factors other than the LDL receptor were responsible for the hypercholesterolemia attending the fat challenge.

Animals↗
PubMed1988

Plasma cholesterol synthesis using deuterated water in humans: effect of short-term food restriction.

Our purpose was to develop methods in humans to determine the fractional synthetic rate (FSR) of plasma pool free cholesterol using the rate of deuterium incorporation from body water. The sensitivity of this method was examined by measuring FSR after periods of fasting and feeding. Five healthy men with normal lipoprotein levels were given a prepared diet containing 40% of calories as fat and a polyunsaturated/saturated fatty acid ratio of 0.25 for 8 days, except for day 7 when they were given only drinking water. Beginning after the supper meal on day 6, they received no food until 8 AM on day 8 when they consumed meals as normal. Over days 7 and 8 the subjects were given prime and constant deuterium oxide orally to maintain body water deuterium enrichment at about 0.05 atom % excess. Plasma samples were obtained at 0 hours (day 7, 8 AM) and at 12, 24, 36, and 48 hours thereafter. Free cholesterol was extracted, purified by thin-layer chromatography, and combusted to water. The water was reduced to H2 and analyzed for deuterium enrichment by isotope ratio mass spectrometry. Analytic precision of this system was determined as 3.5 0/00 (parts per mil) vs Standard Mean Ocean Water. Deuterium enrichment of plasma water for the group during the 48-hour deuterium oxide administration period was 3143 0/00 +/- 310 0/00 (mean +/- SEM). Cholesterol deuterium enrichment for the group during the 12-hour period of fasting (10.9 0/00 +/- 4.1 0/00) was not different from that during feeding (14.2 0/00 +/- 6.2 0/00).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Proapolipoprotein-converting enzymes and high-density lipoprotein early events in biogenesis.

The early events in high-density lipoprotein biogenesis involve the extracellular action of two converting enzymes affecting the cleavage of the prosegment of either proapolipoprotein A-I or proapolipoprotein A-II and the generation of mature apolipoprotein (apo) A-I and apo A-II, the main apolipoprotein of high-density lipoproteins. These two converting enzymes differ from each other in mechanism of action and specificity. The observation that they can be secreted by human hepatocarcinoma G2 cells in culture provides an experimental basis for examining the possible coordination between the synthesis and secretion of these two converting enzymes and the events attending the production and cellular export of apo A-I and apo A-II.

Apolipoprotein A-I↗
PubMed1987

Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen.

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr approximately equal to 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from two tryptic fragments show homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.

Amino Acid Sequence↗
PubMed1986

Antigenic relatedness between human lecithin-cholesterol acyltransferase and phospholipases of the A2 family.

A monoclonal antibody, B10, generated against pure human lecithin-cholesterol acyltransferase (EC 2.3.1.43) caused the inhibition of the esterolytic and cholesterol esterifying activities of the enzyme. This antibody also reacted with a number of pancreatic and snake venom phospholipases A2 species but not phospholipase A1. A concentration-dependent inhibition of phospholipase A2 was also seen in the presence of B10. Treatment of lecithin-cholesterol acyltransferase or B10-reacting phospholipases with phenacyl bromide, a reagent known to interact with the active site of phospholipase A2, inhibited both their esterolytic activity and their capacity to bind to B10. A dimeric phospholipase A2 species with a known occluded active site did not cross-react with B10. Thus, lecithin-cholesterol acyltransferase and some enzymes of the phospholipase A2 family share a common antigenic determinant which is probably located near or at their esterolytic active site.

Animals↗
PubMed1986

Physicochemical properties of apolipoprotein(a) and lipoprotein(a-) derived from the dissociation of human plasma lipoprotein (a).

Chemical reduction of human plasma lipoprotein(a) (Lp(a)) yielded two water-soluble products which were separated by rate zonal ultracentrifugation. Apolipoprotein(a) (apo(a)) was completely recovered from the bottom of the gradient, whereas lipoprotein(a-) (Lp(a-)), which contained all of the lipids and apo-B100 of Lp(a), floated. By the techniques of circular dichroism and viscometry Lp(a-) was identical to low density lipoprotein (LDL). Lp(a-) was slightly larger in mass than autologous LDL and contained proportionally more triglyceride. The difference in mass between Lp(a) and Lp(a-) was accounted for by the loss of 2 molecules of apo(a) from the Lp(a) particle. The molecular weight of reduced and carboxymethylated apo(a) was 281,000 as determined by sedimentation equilibrium in 6 M guanidine HCl. By circular dichroism the structure of apo(a) was mostly random (71%) with the remainder representing 8% alpha-helix and 21% beta-sheet; its intrinsic viscosity, 28.3 cm3/g, was consistent with an extended flexible coil. The amino acid composition was characterized by an unusually high content of proline (11.4 mol %) as well as tryptophan, tyrosine, arginine, threonine, and a low amount of lysine, phenylalanine, and isoleucine. Apo(a) contained 28.1% carbohydrate by weight represented by mannose, galactose, galactosamine, glucosamine, and sialic acid in an approximate molar ratio of 3:7:5:4:7, respectively. Overall, the structure of Lp(a) appears to be consistent with a rigid spherical LDL-like core particle which, as a consequence of its association with a flexible glycoprotein such as apo(a), favors the entrapment of significant amounts of hydrodynamically associated solvent. Furthermore, the Lp(a-) remnant generated by the removal of apo(a) from Lp(a) was similar in structure but not identical to autologous LDL.

Adult↗
PubMed1986

In vitro proteolysis of human plasma low density lipoproteins by an elastase released from human blood polymorphonuclear cells.

In vitro incubation of human plasma low density lipoproteins (LDL) with human blood polymorphonuclear cells (PMN) for 1 h at 37 degrees C resulted in an increased (2-4-fold) release into the medium of an enzymatic activity which co-eluted with LDL by column chromatography at physiological ionic strength but dissociated from it in high salt media in an ultracentrifugal field. The release of this enzymatic activity increased with increasing concentration of LDL in the medium and caused the hydrolysis of the LDL apoprotein B100 as indicated by the appearance of 7-8 low molecular weight bands (immunoreactive with anti-LDL) which were not present in the electropherogram of control LDL. The proteolytic activity was identified as an elastase by the following criteria: 1) capacity to hydrolyze the synthetic substrate methoxysuccinyl-Ala-Ala-Pro-Val-4-methylcoumaryl-7-amide known to be specific for the PMN elastase, 2) pattern of apo-B proteolysis identical to that exhibited by pure PMN elastase, 3) inhibition of the proteolysis by the elastase inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-CH2Cl, 4) identity in molecular weight (28,000-30,000) of this activity with a pure preparation of PMN elastase labeled with [3H]diisopropylfluorophosphate. Based on thiobarbituric acid analyses and the lack of effect by vitamin E, oxidative events appeared to play no detectable role in apo-B proteolysis. Since we previously reported (Byrne, R. E., Polacek, D., Gordon, J. I., and Scanu, A. M. (1984) J. Biol. Chem. 259, 14531-14543) that high density lipoprotein-3 promotes the in vitro release of PMN elastase which cleaves apo-A-II, it is apparent that in vitro, both LDL and high density lipoprotein, two of the major plasma lipoprotein classes, can affect the export from PMN of an elastase which exhibits proteolytic action on apo-B and apo-A-II.

Amino Acid Chloromethyl Ketones↗
PubMed1986

Uptake of endogenous cholesterol by a synthetic lipoprotein.

The addition of cholesterol-poor phospholipid liposomes to canine plasma in vivo and in vitro substantially alters the distribution of phospholipids, apoproteins, and, especially, cholesterol. In vivo, intravenously injected phospholipid liposomes remain discrete particles, which are readily distinguished from the normally occurring lipoproteins by their buoyant density and electrophoretic mobility. They acquire unesterified cholesterol from endogenous sources, thereby producing an acute rise in the concentration of this sterol in plasma. The liposomes also accumulate endogenous proteins, one of which is identified as apolipoprotein A-I. In vitro, phospholipid liposomes incubated with plasma acquire unesterified cholesterol and apolipoprotein A-I at the expense of high-density lipoproteins (HDL), the major carrier of cholesterol in normal canine plasma. In exchange, the HDL particles are enriched in phospholipids and become larger. At sufficiently high concentrations, the liposomes nearly completely deplete HDL of its unesterified cholesterol. Thus, there are generated two types of particles, both rich in apolipoprotein A-I and phospholipid, but one (modified HDL) containing mainly esterified cholesterol in its core and the other (modified liposomes) containing mainly unesterified cholesterol at its surface. It is concluded that phospholipid liposomes produce important changes in the distribution of lipids and protein in canine plasma, particularly at the expense of HDL. These changes appear to favor the mobilization of tissue cholesterol into the plasma, and may have application to atherosclerosis.

Animals↗
PubMed1986

Affinity of nonhomologous amphiphilic peptides toward a monoclonal antibody raised against apolipoprotein A-I.

Monoclonal antibodies against human apolipoprotein A-I (apoA-I) were generated by the hybridoma technique. Clone G-10 was selected on the basis of its highest titer. The affinity of this antibody toward a series of synthetic peptides differing in length, amino acid composition, and amphiphilicity was tested by using both the indirect and the competitive enzyme-linked immunosorbent techniques (ELISA). From these measurements we calculated dissociation constants of the complexes of the antibody with apoA-I bound to the surface of the microtiter plate, apoA-I in solution, and any of the several peptides in solution. The dissociation constant (Kd) of the immobilized apoA-I/anti-apoA-I-complex, Kd = 2 x 10(-9) M, was significantly lower than that of the complex resulting from the interaction between anti-apoA-I and either apoA-I in solution or any of the several amphiphilic helical peptides in solution. Peptides devoid of amphiphilic secondary structure were inert. These data are consistent with the proposal that monoclonal G-10 recognizes in antigenic peptides an alpha-helical secondary structure of defined hydrophilic-lipophilic balance and comparatively less the specific amino acid side chains. We propose that the highest contribution to the free energy of binding (8 Kcal/mole) is derived from the docking of the helix to the antibody. It follows that in probing the specificity of a monoclonal antibody the conformation and the physical environment of the interacting antigen must be taken into account.

Amino Acid Sequence↗