Determination of metallothionein in tissues by cadmium-hemoglobin affinity assay.
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Animals↗
Biomedical subjects
D L Eaton
Publications and source records attributed to D L Eaton.
Mouse strain differences in glutathione S-transferase activity and aflatoxin B1 biotransformation.
Previous studies have suggested that mice are resistant to the carcinogenic effects of aflatoxin B1 (AFB1) and that this resistance is largely the result of expression of an isoenzyme of glutathione S-transferase (GST) with high activity toward AFB1-8,9-epoxide. Significant interstrain differences in cytosolic GST activities toward a variety of substrates have been reported in mice. If such differences exist for the conjugation of AFB1-8,9-epoxide, then there may be significant mouse strain differences in susceptibility to AFB1-induced hepatocarcinogenicity. The hepatic microsomal and cytosolic biotransformation of AFB1 was studied in 8 different strains of mice fed a purified diet. GST-mediated conjugation of AFB1-8,9-epoxide with glutathione and GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide (CHP) were determined with cytosolic fractions from 8-10 pooled livers. Specific activities of cytochrome-P-450-mediated oxidation of AFB1 to aflatoxin Q1 (AFQ1), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1), as well as the reactive intermediate AFB1-8,9-epoxide, were determined with hepatic microsomal fractions from each mouse strain. No striking differences in specific activity between mouse strains were observed for any of the P-450- or GST-mediated enzymatic pathways measured, although some statistically significant differences were found. GST specific activities toward AFB1-8,9-epoxide, CDNB, DCNB, ECA and CHP ranged from 1.5-2.1, 2,830-5,370, 81-144, 38-69 and 32-73 nmol/mg protein/min, respectively. The rate of formation of AFB1-8,9-epoxide ranged from 208 to 465 pmol/mg protein/min. The specific activities of AFQ1,AFM1, and AFP1 formation by microsomes ranged from 36-70, 161-326, and 252-426 pmol/mg protein/min, respectively. Mice fed a standard rodent chow diet showed evidence of microsomal and cytosolic enzyme induction when compared to mice fed a purified diet. The lack of substantial differences in enzyme specific activities between mouse strains suggests that interstrain variations in the hepatocarcinogenic effects of AFB1 in mice should not be large.
Aflatoxin B1↗
Interaction of recombinant apolipoprotein(a) and lipoprotein(a) with macrophages.
Elevated plasma levels of lipoprotein(a), Lp(a), represent a major, inherited risk factor for coronary heart disease, although the mechanism of its action remains unknown. Lp(a) is distinguished from the related LDL particle by the addition of apolipoprotein(a), apo(a). The presence of this large glycoprotein is likely to affect the binding of the particle to the LDL receptor and/or other receptors which may contribute to the atherogenic potential of Lp(a). Here we demonstrate the binding to macrophages of Lp(a) and pure recombinant apo(a) protein, via a specific, high-affinity receptor. This binding could lead to foam cell formation and the localization of Lp(a) to atherosclerotic plaques.
Animals↗
Effect of styrene oxide on rat brain glutathione.
Glutathione (GSH) plays a primary role in protecting cells from oxidative stress and in detoxifying foreign compounds. The functions and regulations of GSH in nervous tissue have not been thoroughly investigated. This study examines the effects of styrene oxide, a reactive metabolite of the neurotoxic solvent styrene, on GSH metabolism in six regions of the rat brain (cortex, cerebellum, medulla-pons, hippocampus, striatum and hypothalamus). Control levels of GSH in brain regions ranged from 1.6 mM in medulla-pons to 2.7 mM in striatum. Styrene oxide (100-400 mg/kg, ip) depleted GSH in a dose- and time-dependent manner in all brain regions studied. Histochemical studies indicated a predominantly glial distribution of GSH and confirmed the depletion of GSH by styrene oxide in brain. Studies with [8(-14)C] styrene oxide revealed no differences in the distribution of styrene oxide/metabolites among brain regions. gamma-Glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis, was not affected by styrene oxide in any brain region, either in vitro or following in vivo administration. Glutathione S-transferase activity in different brain regions, measured using p-nitrostyrene oxide as a substrate, correlated quantitatively with GSH depletion by styrene oxide. Depletion of brain GSH by styrene oxide may contribute to oxidative injury to neuronal and glial cells and may be involved in styrene neurotoxicity.
Animals↗
Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B1-8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide.
As part of the studies of the biochemical basis for species differences in biotransformation of the carcinogen aflatoxin B1 (AFB1) and its modulation by phenolic antioxidants, we have investigated the role of mouse liver glutathione S-transferase (GST) isoenzymes in the conjugation of AFB1-8,9-epoxide. Isoenzymes of GST were purified to electrophoretic homogeneity from Swiss-Webster mouse liver cytosol by affinity chromatography and chromatofocusing. The isoenzyme fractions were characterized in terms of activity toward surrogate substrates and immunologic cross-reactivity with antisera to rat GSTs. The major isoenzymes were identified as SW 4-4, SW 3-3, and SW 1-1. The specific activity of SW 4-4 toward AFB1-8,9-epoxide was at least 50- and 150-fold greater than that of SW 3-3 and SW 1-1, respectively. Relatively high activity toward another epoxide carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, was observed with both SW 4-4 and SW 3-3. SW 1-1 had the highest activity toward 1-chloro-2,4-dinitrobenzene (CDNB) whereas SW 4-4 had relatively low CDNB activity. Following pretreatment with 0.75% butylated hydroxyanisole in the diet, the fraction of total GST contributed by SW 1-1 appeared to increase dramatically, whereas in control mice SW 3-3 constituted the predominant isoenzyme. The high GST activity of mouse liver cytosol toward AFB1-8,9-epoxide is apparently due to an isoenzyme that contributes little to the overall cytosolic CDNB activity.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Species susceptibility to aflatoxin B1 carcinogenesis: comparative kinetics of microsomal biotransformation.
The biotransformation of the potential human carcinogen aflatoxin B1 (AFB1) was studied using hepatic microsomes from the rat, mouse, monkey, and human. Initial rates of AFB1 oxidation to aflatoxins Q1, M1, and P1, as well as the reactive intermediate AFB1-8,9-epoxide, were determined using a high performance liquid chromatography assay. The rates of generation of these AFB1 metabolites were investigated at low substrate concentrations (more representative of environmental exposures) and also at high ("saturating") concentrations commonly utilized in studies in vitro. Striking differences in ratios of the metabolites were observed. At an AFB1 concentration of 124 microM, mouse and monkey microsomes had the highest rates of AFB1-8,9-epoxide formation. Primate liver microsomes formed aflatoxin Q1 in large amounts but failed to produce detectable aflatoxin P1. Determination of the rates of formation over initial AFB1 concentrations ranging from 15 to 475 microM revealed that the proportion converted to AFB1-8,9-epoxide increased at lower substrate concentrations in the case of the rat and human microsomes but not with mouse or monkey microsomes. The differences in patterns of metabolite formation with varying concentrations have implications for interspecies comparisons of carcinogenic potency of AFB1.
Aflatoxin B1↗
Apolipoprotein(a) size heterogeneity is related to variable number of repeat sequences in its mRNA.
Plasma apolipoprotein(a) [apo(a)] shows considerable size heterogeneity, existing as discrete glycoprotein isoform variants that range in apparent molecular mass from approximately 400 to 800 kDa. To study the molecular basis of protein size variability, we have isolated liver RNA from individuals with different apo(a) isoforms, and identified apo(a)-specific transcripts using Northern blot analysis. Transcript sizes were shown to be variable (8.0-12 kb) and in all cases were closely correlated with protein masses (590-850 kDa) as determined from immunoblots. Thus, it is almost certain that apo(a) isoform size variation is due to allelic differences in the number of its tandemly repeated sequences of 114 amino acids that resemble kringle four of plasminogen. The high carbohydrate content of apo(a) makes true molecular weight estimations in SDS-PAGE gels difficult. However, a recombinant form of apo(a) containing 17 kringle repeats (calculated molecular mass of 250 kDa) migrates on SDS-PAGE gels only slightly below apoB-100, with an apparent molecular mass of approximately 500 kDa. Since smaller protein isoforms have been observed in the population, this suggests that plasma apo(a) isoforms contain from less than 17 to greater than 30 tandemly repeated kringle units.
Apolipoproteins A↗
Proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content.
Glutathione (GSH) is important in defense against oxygen free radical damage, in detoxification of xenobiotics, and in mitogenesis. The reducing conditions provided by low molecular weight thiols such as 2-mercaptoethanol (ME) have been shown to promote the growth of lymphocytes in culture. We wished to determine the effects of 2-ME on GSH content, and to determine to what extent GSH status affected lymphocyte proliferation. GSH content was quantitated in human peripheral blood lymphocytes (PBL) using a flow cytometric assay with monochlorobimane. This analysis was performed on PBL as well as on the CD4+ T-cell subset, as identified with fluorescent anti-CD4 monoclonal antibodies (mAb). Cells were viably sorted on the basis of their GSH content, and incubated for 3 days with mitogenic concentrations of PHA (for PBL) or anti-CD3 mAb (for CD4+ cells) in the presence of bromodeoxyuridine (BrdU). BrdU/Hoechst cell cycle analysis was then performed on these cells. High GSH sorted cells had a higher percentage of cells capable of entering the cell cycle than low GSH sorted cells. This data indicates that some of the heterogeneity in proliferative capacity within PBL in culture is directly or indirectly related to GSH content. Incubation of cells in 2-ME prevented the loss of GSH that occurs when cells are cultured. 2-ME improved the proliferative capacity of unsorted cells, and of cells sorted for high and low GSH. Acridine orange staining of anti-CD3 mAb stimulated cells sorted for high and low GSH indicated that an early event in cell activation was affected by GSH content.
CD4 Antigens↗
Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice.
Lp(a) lipoprotein purified from human plasma bound with high affinity to isolated bovine LDL receptors on nitrocellulose blots and in a solid-phase assay. Lp(a) also competed with 125I-LDL for binding to human LDL receptors in intact fibroblasts. Binding led to cellular uptake of Lp(a) with subsequent stimulation of cholesterol esterification. After intravenous injection, human Lp(a) was cleared slowly from the plasma of normal mice. The clearance was markedly accelerated in transgenic mice that expressed large amounts of LDL receptors. We conclude that the covalent attachment of apo(a) to apo B-100 in Lp(a) does not interfere markedly with the ability of apo B-100 to bind to the LDL receptor and that this receptor has the potential to play a major role in clearance of Lp(a) from the circulation of intact humans.
Adult↗
A monoclonal-antibody-based enzyme-linked immunosorbent assay of lipoprotein(a).
Lipoprotein(a) [Lp(a)] is a low-density lipoprotein (LDL)-like lipoprotein particle recently described as a risk factor for premature coronary heart disease, stroke, and atherosclerosis. Structurally, Lp(a) is similar to LDL in that it has comparable lipid composition and contains apolipoprotein B-100 (apo B-100). In addition, Lp(a) contains the glycoprotein apolipoprotein(a) [apo(a)], which is disulfide-linked to apo B-100. The recent awareness of a striking correlation between atherosclerosis and concentrations of Lp(a) in plasma prompted our development of an accurate quantitative assay for plasma Lp(a), a monoclonal-antibody-based enzyme-linked immunosorbent assay for Lp(a) that is shown to be sensitive, precise, and highly specific. The response to several isoforms of Lp(a) is linear, and as many as 80 samples can be quantified on one plate. This easily performed assay is suitable for use in the clinical laboratory and for screening large populations.
Antibodies, Monoclonal↗
Simultaneous determination of cytosolic glutathione S-transferase and microsomal epoxide hydrolase activity toward benzo[a]pyrene-4,5-oxide by high-performance liquid chromatography.
Cytosolic glutathione S-transferase (GST) and microsomal epoxide hydrolase (EH) are important detoxification enzymes for many epoxide xenobiotics. We have developed a rapid, simple, and convenient HPLC assay which measures both of these enzyme activities toward benzo[a]pyrene-4,5-oxide (BaPO) in tissue homogenates. Tissue fractions were incubated at 37 degrees C in the presence of 5 mM glutathione. Reactions were initiated by addition of BaPO and terminated by the addition of ice-cold acetonitrile containing 2-methoxynaphthalene as an internal standard. Samples were analyzed directly on a 15-cm C18 reverse-phase column at room temperature, with a ternary solvent program which utilized 0.01% ammonium phosphate buffer (pH 3.5), acetonitrile, and water. The uv absorbance (260 nm) was monitored. Baseline resolution of BaPO, BaPO-GSH, and BaPO-diol and the internal standard was accomplished in 10 min. In rat hepatic S9, production of both BaPO-GSH and BaPO-diol was linear with time and protein up to 15 min and 500 micrograms/ml, respectively. Coefficients of variation for replicate analyses were 2.7 and 3.7% for GST and EH activities in S9, respectively. With fluorescence detection (ex, 241; em, 389 nm), this assay was sensitive enough to measure GST and EH activities in mononuclear leukocytes (MNL). GST and EH activities in 109 human MNL samples were 142 +/- 74 (mean +/- SD; range 21-435) pmol/mg/min and 19 +/- 9 (mean +/- SD; range 3-59) pmol/mg/min, respectively. These results demonstrate the simplicity, high sensitivity, and applicability of this assay for a broad range of tissues.
Animals↗
Effects of modulation of hepatic glutathione on biotransformation and covalent binding of aflatoxin B1 to DNA in the mouse.
Relative to the rat and most other species tested, the mouse is resistant to the carcinogenic effects of aflatoxin B1 (AFB). Previous investigations in our laboratory demonstrated that mouse liver cytosol has 52 times greater hepatic glutathione S-transferase (GST) activity toward the AFB-epoxide, compared with rat liver cytosol. To determine the importance of GST-mediated detoxification of the AFB-epoxide in the mouse in vivo, we examined the effects of glutathione (GSH) depletion on the covalent binding of AFB to hepatic DNA in control and 2(3)-butyl-4-hydroxyanisole (BHA)-treated mice. Male Swiss-Webster mice were fed control or 0.75% BHA diet for 14 days. Depletion of hepatic GSH was accomplished with D,L-buthionine-S-sulfoximine (BSO, 0.6 g/kg in saline) and diethyl maleate (DEM, 0.75 ml/kg), administered by ip injection at 2 and 1.5 hr, respectively, prior to administration of 3H-AFB (0.25 mg/kg, ip in DMSO). The combined BSO-DEM treatment depleted hepatic GSH by 97 and 70% in control and BHA-treated mice, respectively. In mice receiving the control diet, GSH depletion was associated with a 30-fold increase in the covalent binding of AFB to hepatic DNA. AFB-DNA binding in mice treated with dietary BHA alone was reduced to 54% of control. In BHA-treated mice, BSO-DEM treatment increased AFB-DNA binding by 62%. Dietary BHA increased hepatic S-9 mediated activation of AFB to the AFB-epoxide by eightfold in both control and BSO-DEM mice. BHA also increased GST activity toward the AFB-epoxide by 52 and 68% in control and BSO-DEM mice, respectively. The BSO-DEM treatment alone had no significant effect on the in vitro biotransformation of AFB. These results support the hypothesis that GST is the principal determinant of species differences in susceptibility to AFB-induced hepatocarcinogenicity. The results also support the hypothesis that BHA may protect against the toxic and carcinogenic effects of xenobiotics in part by preventing the depletion of hepatic GSH.
Aflatoxin B1↗
Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene.
Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus.
Adult↗
Use of inductively coupled plasma-atomic emission spectroscopy (ICP-AES) in screening for trace metal exposures in an industrial population.
A heterogeneous group of asymptomatic industrial workers potentially exposed to various toxic metals was screened for trace metal exposure. Each was examined and then completed an occupational health history questionnaire and had hair and blood samples analyzed for 10 metals via the inductively coupled plasma-atomic emission spectroscopy technique. No industrial hygiene survey was performed. The concentrations of trace elements found in our cohort were comparable to previously published levels for nonoccupationally exposed groups. This finding is not unexpected in light of the nature of the population studied and the screening technique. The levels in blood and hair were not well correlated, and subsets of the population with high metal burdens could not be identified on the basis of self-reported occupational exposure histories.
Adult↗
Modification of aflatoxin B1 biotransformation in vitro and DNA binding in vivo by dietary broccoli in rats.
Cruciferous vegetables have been shown to have anticarcinogenic effects in animals but biochemical mechanisms have not been completely elucidated. The effects of dietary broccoli on in vivo DNA binding of the hepatocarcinogen aflatoxin B1 (AFB) and in vitro formation of the putative carcinogenic intermediate, AFB-8,9-epoxide, as well as detoxification of the epoxide by conjugation with glutathione (GSH), were examined in this study. Animals were fed a purified diet, a purified diet plus 25% freeze-dried broccoli, or standard rodent chow for 3 wk. In vivo binding of AFB to hepatic DNA was determined. Biotransformation of AFB in vitro (microsomal oxidation to AFB-8,9-epoxide, as well as hydroxylated metabolites, and cytosolic GSH conjugation of AFB-8,9-epoxide generated in situ) was measured by an HPLC method that allows specific and direct determination of AFB metabolites and thus, the rates of their formation. Microsomal mixed-function oxidase and epoxide hydrolase activities and cytosolic glutathione S-transferase activities were also measured with commonly used surrogate substrates. The rate of cytosolic conjugation of AFB-8,9-epoxide was increased 2.8-fold by the broccoli diet and 2.2-fold by the chow diet. These changes were not fully reflected by increases in activity with surrogate substrates. The chow diet did not affect epoxide hydrolase activity nor glutathione S-transferase activity toward 3,4-dichloronitrobenzene or benzo[a]pyrene 4,5-oxide, whereas these activities were significantly increased by the broccoli diet. Microsomal formation of AFB-8,9-epoxide was unaffected by the dietary treatments, whereas formation of aflatoxin M1 was increased. The chow diet, but not the broccoli diet, increased the amount of aflatoxin Q1 formed from AFB. Binding of AFB to DNA in vivo was significantly lower in the broccoli group but not in the chow-fed animals. These results indicate that broccoli contains substances that cause a reduction in the binding of AFB metabolites to DNA, possibly through the induction of glutathione S-transferase(s). Broccoli and rodent chow differ in their constituents that increase levels of xenobiotic biotransformation enzymes relative to a purified diet. The results also indicate the limitations of reliance on measurements of biotransformation pathways using surrogate substrates instead of carcinogenic compounds of interest.
Aflatoxin B1↗
Comparative effects of butylated hydroxyanisole on hepatic in vivo DNA binding and in vitro biotransformation of aflatoxin B1 in the rat and mouse.
To determine the mechanisms which mediate species- and treatment-related differences in susceptibility to aflatoxin B1 (AFB), we conducted a comparative study of the effects of dietary butylated hydroxyanisole (BHA) on the hepatic in vivo DNA binding and in vitro biotransformation of AFB in the rat and mouse. Mice are resistant to the hepatocarcinogenic effects of AFB, and BHA pretreatment has been shown to inhibit the carcinogenic effects of AFB in the highly susceptible rat. Rats and mice were fed a control diet or an identical diet containing 0.75% BHA for 10 days. On the 11th day, one-half of the control and BHA animals were administered [3H]AFB (0.25 mg/kg in dimethyl sulfoxide) via intraperitoneal injection. Animals were killed 2 hr later and covalent binding of AFB to hepatic DNA was determined. The remaining animals were killed for preparation of hepatic subcellular fractions used in in vitro assays. BHA treatment resulted in a decrease in in vivo hepatic AFB-DNA adduct formation in mice to 68% of control, but, in rats, treatment decreased AFB-DNA binding to 18% of control. Furthermore, hepatic AFB-DNA binding in control mice was only 1.2% of that measured in control rats. The rate of in vitro activation of AFB to the epoxide was 3.4-fold greater in control mice relative to control rats. BHA pretreatment increased the activation of AFB in mice 3.3-fold, but had no effect on oxidative metabolism in rats. Control mice had 52 times greater glutathione S-transferase (GST) activity toward the AFB-epoxide, but only 2.6 times greater GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), compared to that of control rats. In mice, BHA did not significantly increase GST activity toward the AFB-epoxide, but increased GST activity toward CDNB 3.1-fold. In rats, BHA increased GST activity toward the AFB-epoxide and CDNB by 3.2- and 2.1-fold, respectively. Epoxide hydrolase activity toward p-nitrostyrene oxide in mice was only 52% of the activity in rats. BHA increased epoxide hydrolase activity 3.8- and 2.5-fold in mice and rats, respectively. These data indicate that mice have high levels of an AFB-epoxide-specific GST activity relative to that of the rat. The rate of formation of the AFB-epoxide and the activity of epoxide hydrolase appear to be relatively unimportant under conditions of high GST activity, whereas elevated GST activity, and thus inactivation of the AFB-epoxide, appears to be the critical component in species- and BHA-induced differences in AFB-DNA adduct formation and, presumably, AFB hepatocarcinogenicity.
Aflatoxin B1↗
Characterization of recombinant human factor VIII.
Recently, complete human factor VIII DNA clones have been obtained and subsequently expressed in baby hamster kidney cells (Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., and Lawn, R. M. (1984) Nature 312, 330-337). The recombinant factor VIII (rVIII) protein secreted from these cells has now been purified allowing its structural analysis and comparison to plasma-derived factor VIII (pdVIII). Analysis of purified rVIII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with relative mobilities (Mr) ranging from 80,000-210,000. The same pattern of polypeptides is also observed for pdVIII resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins associated with rVIII are recognized by pdVIII antibodies in a Western blot. When rVIII and pdVIII are subjected to isoelectric focusing they are resolved into a similar pattern of protein bands. Thrombin, factor Xa, and activated protein C, which modulate factor VIII activity by proteolysis, process rVIII in the same manner they do pdVIII. As is the case for pdVIII, thrombin activation of rVIII coagulant activity correlates with the generation of subunits with Mr of 73,000, 50,000 and 43,000. These subunits appear to form a metal-(perhaps Ca2+) linked complex. EDTA inactivates thrombin-activated rVIII and pdVIII, with the activity being regenerated after the addition of a molar excess of MnCl2. The results suggest that rVIII is structurally and functionally very similar to pdVIII.
Animals↗
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↗