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Putative apolipoprotein B-100 in the freshwater turtle Chrysemys picta: effects of estrogen and progesterone
Comp. Biochem. Physiol. Vol. 103B,No. 3, pp. 70%713, 1992 Printed in Great Britain
0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd
PUTATIVE APOLIPOPROTEIN B-100 IN THE FRESHWATER TURTLE CHRYSEMYS PICTA: EFFECTS OF ESTROGEN AND PROGESTERONE LORELEI E. PEREZ,* DAVID WILLIAMS'~and IAN P. CALLARD*:~ *Department of Biology, Boston University, Boston, MA 02215, U.S.A. and Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04609, U.S.A.; and ~'Department of Pharmacology, State University of New York, Stonybrook, NY 11794, U.S.A. (Tel. 61%353-5087; Fax 617-353-6340)
(Received 19 March 1992; accepted 22 April 1992) Atstract--l. The isolation and purification of a putative apolipoprotein B-100 in the plasma of the freshwater turtle Chrysemys picta is described. 2. The protein was purified through differential ultracentrifugation and subsequent Sepharose 6B column chromatography. 3. The molecular weight of the protein determined by electrophoresis was approximately 350 kDa. 4. An antibody to chicken apolipoprotein B-100 specifically recognizes this 350 kDa protein in Western blots, suggesting its identity with apolipoprotein B-100. 5. An antibody to the putative Chrysemys apolipoprotein B-100-1ikeprotein was developed and used in an ELISA to quantitate protein levels in plasma. 6. Acute estrogen treatment increased levels of apolipoprotein B-100 (7.64 + 0.79 mg/ml plasma) over that of control animals (5.07 _+ 1.74 mg/ml plasma). 7. In contrast, chronic estrogen treatment reduced apolipoprotein B-100 significantly to 2.94 + 0.53 mg/ml plasma (P < 0.05).
ory effects of steroids on vitellogenin versus apolipoprotein B-100/LDL in a non-mammalian species may provide insight into the role of steroids in mammalian lipid metabolism and atherosclerosis. Thus, apolipoprotein B-100 was purified and characterized in the freshwater turtle Chrysemys picta, a species in which vitellogenin has been shown to be under the dual regulation of estrogen and progesterone (Ho et al., 1982).
INTRODUCTION Apolipoprotein B-100, the main protein component of very-low-density lipoprotein (VLDL) and lowdensity lipoprotein (LDL), has been isolated and characterized in several mammals: human (Brown et al., 1972), rat (Herbert et al., 1974), guinea pig (Chapman et al., 1975) and one avian (Williams, 1979; Capony and Williams, 1980). Human and avian studies have shown a role for estrogen in the regulation of apolipoprotein B-100 in the liver. In the domestic hen, estrogen greatly increases the level of apolipoprotein B-100 both/!1 vitro (Luskey et al., 1974; Chan et al., 1976; Capony and Williams, 1980) and synthesis in vitro by hepatocytes (Chart et el., 1980; Boehm et al., 1988). In the human, estrogen is reported to both increase (Wynn et al., 1969; Wallace et al., 1979) and decrease (Vessey, 1969; Oliver, 1970) apolipoprotein B-100 levels significantly. Several recent computer analyses have shown that vertebrate vitellogenins and human apolipoprotein B-100 have significant sequence homology, suggesting relatedness (Baker, 1988; Byrne et el., 1989; Perez et al., 1991). Vitellogenin, a major egg yolk protein, like apolipoprotein B-100, is synthesized in the liver and is regulated by estrogen in non-mammalian species (see Tata, 1976; Wallace, 1985; Wahli, 1988, for review). Because of the correlation between LDL/apolipoprotein B-100 and atherosclerosis, and the apparent protective action of estrogen (Furman, 1973), a comparison of the regulat:~To whom correspondence should be addressed.
MATERIALS AND METHODS Freshly caught adult female Chrysemys picta turtles were shipped from Lemberger (Oshkosh, WI) and kept indoors at 22°C in constantly circulating freshwater tanks until used for experiments. Animals were injected i. m. with 1.0 mg/kg body weight estradiol (a dose previously shown to stimulate viteilogenin synthesis in this species; Gapp et al., 1979) in oil and killed by decapitation 7 days later. Blood was collected and serum or plasma isolated in the presence of I mM phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Co. St Louis, MO). In order to determine the effect of progesterone on estrogen-induced apolipoprotein B-100 synthesis, animals were injected i. m. with either estradiol (0.5 mg/kg body weight) in oil for 8 days, progesterone (5 mg/kg body on days 6, 7 and 8), or both. Control animals were injected with oil alone.
Separation of lipoprotein fractions Lipoprotein density classes were isolated essentially as described by Witliams (1979). Briefly, very-low-density lipoprotein (VLDL) was isolated by centrifugation at plasma or serum density for 24 hr at 12°C at 20,000& in a Sorvall 50 Ti rotor. The top 2 ml fraction, containing VLDL, was removed and stored at -20°C.
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Purification of high molecular weight VLDL protein The VLDL sample was diluted in PSP [0.02 M sodium phosphate (pH = 7), 0.15 M NaC1, I00 #g/ml PMSF] and a 7 ml sample was chromatographed on Sepharose 6B (2.6 x 30 cm) equilibrated in PSP at 4°C. Fractions were monitored via absorbance at 280 nm. The leading 70% of the excluded peak was dialyzed against 500 volumes of water with 100 #g/ ml PMSF overnight adjusted to 1% sodium dodecyl sulfate (SDS) and lyophilized. The sample was dissolved in 0.02 M sodium phosphate ( p H = 7 ) , 3% SDS and chromatographed on a Sepharose 6B column equilibrated with 0.02M sodium phosphate, I% SDS, 100#g/ml PMSF at room temperature. The column fractions were monitored by separation on 8.5% SDS-PAGE. The high molecular weight component was concentrated by lyophilization and rerun on Sepharose 6B columns as described.
Eleetrophoresis Protein samples were run on 6.0% SDS--polyacrylamide slab gels with the buffer system of Laemmli (1970). Gels were fixed and stained for 1 hr in 0.2% Coomassie Blue and destained in methanol:acetic acid solution.
Preparation of antibody Antiserum against the high molecular weight VLDL component was raised in New Zealand white rabbits by intradermal administration of 100 #g antigen in an equal
volume of Freund's complete adjuvant (Sigma) as described previously by Vaitukaitis (1971). Animals were bled 10 days after each injection. Booster injections in Freund's incomplete adjuvant (Sigma) were given monthly for 5 consecutive months and serum was tested for reactivity against purified antigen in an ELISA assay (see below). Blood was collected by ear-vein puncture, allowed to clot overnight at 4°C and serum stored at -20°C.
Enzyme-linked immunosorbent assay (ELISA ) The ELISA test was carried out essentially as described by Enviall and Perlmann (1971). Briefly, U-bottom polyvinyl chloride microplates (Flow Laboratories, Rockville, MD) were coated with 100 #1 of serially diluted plasma in carbonate buffer and incubated overnight at 4°C. After incubation, wells were washed three times with phosphate buffered saline (PBS)-Twecn 20 and blocked with a 1.5% gelatin solution (Bio-Rad, Richmond, CA) for 2 hr at room temperature. The wells were washed and 100 #1 of diluted rabbit anti-high molecular weight VLDL protein antiserum was added to each well. The plates were incubated at room temperature for 2 hr, washed and I00 #1 of goat anti-rabbit antiserum conjugated to alkaline phosphatase (Sigma) was added to each well. The plates were incubated for an additional 2 hr at room temperature, washed and developed with the addition of 100#1 of p-nitrophenylphosphatv solution (Sigma) to each well. The colorimetric readings were recorded at 410 nm ufing a modified microtiter plate spectrophotometer (Dynatvch, Chantilly, VA).
APO B100 205 116 97.4 66
45
29
VLDL Ultracent.
Seph. 6B PSP
Seph. 6B SDS
Fig. l. Purification of a high molecular weight protein in female turtle plasma. VLDL ultracentrifugation (lane 1), Scpharose 6B-PSP column chromatography (lane 2) and Sepharose 6B-SDS column chromatography (lane 3) products were separated on 8.5% SDS-PAGE and stained with Coomassie Blue. A high molecular weight protein, of approximately 350 kDa, was purified using these procedures. VLDL = verylow-density lipoprotein ultracentrifugate; Seph. 6B-PSP = Sepharose 6B-PSP column chromatography eluate; Seph. 6B-SDS = Sopharose 6B-SDS column chromatography elute.
Turtle apolipoprotein B-100
709
350 kD
VLDL Ultra
Seph
LDL Ultra
Seph
Fig. 2. Identification of an apolipoprotein B-100-1ike protein in female turtle plasma. Ultracentrifuged plasma components VLDL (lane 1), Sepharose 6B-PSP purified VLDL (lane 2), LDL (lane 3) and Sepharose 6B-PSP purified LDL (lane 4) and was probed on a Western blot with anti-chicken apolipoprotein B-100 antibody. Only one protein of ca 350 kDa binds the anti-chicken apolipoprotein B-100 antibody.
Western blotting Gels used for Western blotting were equilibrated in methanol-Tris-SDS buffer for 30 mill, mounted in a semi-dry blot system (Fisher BioTechnologies, Pittsburg, PA) and protein was transferred onto a methanol-Tris-SDS equilibrated nitrocellulose filter (Schleicher and Schuell) for 30 min at 15 V. The filter was blocked with 5.0% dried milk in TBST (10mM Tris-HC1, pH=8.0, 150mM NaC1, 0.05% Twecn-20) buffer overnight. The filter was washed three times with TBST and incubated for 2 hr with an appropriate dilution of antiserum (turtle apolipoprotein B-100 or chicken apolipoprotein B-100 (courtesy of Dr David Williams) in 5.0% milk/TBST. The filter was washed and incubated for 30 rain with goat anti- rabbit antibody conjugated to alkaline phosphate (Promega Biotech), subsequently washed three times with TBST and incubated in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Promega Biotech) for visualization of alkaline phosphatase activity.
blots were probed with anti-chicken apolipoprotein B-100 antiserum. The antibody crossreacts specifically to a high molecular weight protein of ca 350 kDa (Fig. 2).
Characteristics of turtle anti-apolipoprotein B-100 Antibody against purified turtle apolipoprotein B-100 was raised in a rabbit. The titer curve of the anti-apolipoprotein B-100 antibody is shown in Fig. 3. The Vm~ of the antibody is at approximately 3.29 log dilution. This dilution was used in subsequent Western blots and assays. Pre-immune serum did not crossreact with purified turtle apolipoprotein B-100 (data not shown). The specificity of the antibody for turtle apolipoprotein B-100 was tested using Western blot assays. The polyclonal antibody reacts only with a 350 kDa protein in isolated turtle VLDL fractions and partially
RESULTS
Isolation and purification of apolipoprotein B-lOO-like protein Figure 1 depicts the purification of a high molecular weight plasma protein from female turtle plasma. Two main proteins are present in ultracentrifugation fractions and Sepharose 6B-PSP elution (lanes 1 and 2; VLDL Ultracent. and Seph. 6B PSP). However, a large protein band, of ca 350 kDa, was apparent. On Sepharose 6B columns equilibrated in phosphateSDS buffer, this protein can be purified to apparent homogeneity from the lower molecular weight component (lane 3; Seph. 6B SDS).
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1,75
O I~1 0
0.75
-0.25
Identification of apolipoprotein B-100 (Fig. 2) Six percent SDS--PAGE gels were loaded with VLDL (lane 1; VLDL Ultra), Sepharose 6B-PSP purified VLDL (lane 2; Seph), LDL (lane 3; LDL Ultra) and Sepharose 6B-PSP purified LDL (lane 4; Seph) f r o m female turtle plasma. After transfer of the proteins to nitrocellulose membranes, the
0 ;
~
;
~
;
~
Log dilution
Fig. 3. Titer curve for anti-turtle apolipoprotein B-100 antibody. Serially diluted antiserum was used in an ELISA against 50 ng of purified apolipoprotein B100. A Vm~ of ca log dilution was obtained, which was the dilution used in subsequent Western blots and assays.
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L. E. I~R~z et al.
(a)
(b) APO BI00
35(~
180
116 84 58 45
APOB100
VLDL
C.B.
W.B.
Fig. 4. Validity of the turtle apolipoprotein B-100 antibody. The validity of the antibody was tested in Western blot analysis of (a) purified apolipoprotein B-100 (lane 1), VLDL ultracentrifugation density fractions (lane 2) and (b) turtle plasma. As can be seen the antibody reacts only with the 350 kDa high molecular weight protein. purified apolipoprotein B-100 (Fig. 4a; Apo BI00 and VLDL). The antibody also reacts solely with a single component of diluted plasma as can be seen in 0.5"
0.4"
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0.3"
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o
0.2"
0.1"
0.0
i
i
2
3
Log Apo B100 (ng/ml)
Fig. 5. Standard curve of the turtle apolipoprotein B-100 ELISA assay. Serially-diluted purified apolipoprotein B-100 was tested in an ELISA against a 3.81 log dilution of anti-turtle apolipoprotein B-100 antibody. The detection limit of the assay was 15.6 ng/ml plasma.
Western blots (Fig. 4b; W. B.), where only the 350 kDa protein is selected (Fig. 4b; C. B.). ELISA for turtle apolipoprotein B-100 Figure 5 shows a composite standard curve from six assays. The detection limit of the assay was 15.6 ng/ ml plasma. The standard curve after log probit transformation was linear, with an R 2 for 6 assays of 0.923. The accuracy of the assay, as measured by the coefficient of variance along the standard curve, ranged from 7.2% at 31.3 ng/ml plasma to 1.6% at 1000 ng/ml plasma. The precision of the assay was tested by adding a plasma sample of known apolipoprotein B-100 content (100 ng) to different standard dilutions of vitellogenin (500, 250 and 62.5 ng). The resultant ELISA estimates were 1.26-, 1.13- and 1.1 lfold greater, respectively, than the expected values. The overestimation of the higher values which fell at 500-1000 ng concentration respectively is a reflection of the non-linear behavior of female plasma at higher concentrations. Effects o f estrogen on apolipoprotein B-100 synthesis (Fig. 6) In vehicle treated females (control), apolipoprotein B-100 levels of ca 5.07 _ 1.32 (n = 3) mg/ml plasma were found. Similar values were obtained in animals treated with a single injection of estrogen (7.64 + 0.79; n = 4), progesterone (5.26 + 0.61; n = 3) or a combination of estrogen and progesterone (5.84 _+ 0.48; n = 3). In contrast, chronic treatment of female turtles with estrogen (0.5 mg/kg body weight for 8 days) resulted in a significant decrease of apolipoprotein
Turtle apolipoprotein B-100
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control
E2-chronic
E2-acute
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Fig. 6. Effects of estrogen and progesterone on apolipoprotein B-100 synthesis. The effects of steroids was tested in female turtles using the ELISA for turtle apolipoprotein B-100. After acute (1.0 mg/kg body weight on day 0) or chronic (0.5 mg/kg body weight for 8 days) estrogen treatment had significantly different levels from that of control animals. Treatment with estrogen and progesterone or progesterone alone had no effect. B-100 in the plasma of female turtles (2.94 _+0.53; n = 8; P < 0.05) vs controls. DISCUSSION
In this paper, we describe the identification and purification of a high molecular weight (350 kDa) protein (putative apolipoprotein B-100) from the plasma of estrogen-treated female turtles using the methods of Williams (1979) for isolation of cockerel apo-VLDL-B. In all species studied, including the turtle, apolipoprotein B-100 has been shown to resolve into one component on Sepharose 6B columns in the presence of SDS (Williams, 1979). On the basis of electrophoretic mobility the estimated molecular weight of the putative turtle apolipoprotein B-100 is 350 kDa, similar to that of the chicken (360 kDa; Williams, 1979). The crossreactivity of anti-chicken apolipoprotein B-100 with putative turtle apolipoprotein B-100 on Western blots suggests significant structural homology of these two proteins. Given the phylogenetic descent of birds from reptiles and the apparent molecular conservation of VLDL/LDL apoproteins, this is not unexpected. On the basis of other studies in our laboratory (Perez et al., 1992), it would appear that apolipoprotein B-100 is highly conserved from elasmobranchs to birds. Thus, we have shown that anti-chicken apolipoprotein B-100 also crossreacts with putative elasmobranch apolipoprotein B-100 (Perez et al., 1992). Since anti-human apolipoprotein B-100 crossreacts well with other mammals, yet poorly with avian VLDL (8.0%) and not at all with VLDL from teleost and snake (Goldstein et al., 1977), significant changes in protein structure and/or immunogenic domains may have occurred with mammalian evolution. However, no crossreactivity studies of non-mammalian antibodies CBPB 103/3~N
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to mammalian apolipoprotein B-100 have been done. High levels of apolipoprotein B-100 in turtles facilitated the subsequent isolation and purification of the protein since 5 ml of plasma yielded ca 2.0 mg apolipoprotein B-100. However, acute estrogen treatment did not elevate apolipoprotein B-100 levels in female turtles, unlike the male bird in which apoprotein B levels were elevated ca 500-fold (to 12.5 mg/ml blood) after a single injection of DES (Capony and Williams, 1980). This difference may be due to the high basal levels in female turtles ca 5.0mg/ml plasma) compared to male birds (ca 26.2 #g/ml blood) which possibly prevents a further response. It will be of interest to measure apolipoprotein B-100 in immature, reproductively inactive and castrate (low estrogen) female turtles. However, we have also noted that apolipoprotein B-100 levels are high in normal males (l-2mg/ml, unpublished observations). These observations suggest that apolipoprotein B-100 regulation in the turtle may be quite different from the bird but values of apolipoprotein B-100 in reproductively active female birds would be of interest. The physiological significance of the reduction in turtle apolipoprotein B-100 after chronic estrogen treatment is in question until further studies are presented. In human studies, the data on the effect of estrogens on apolipoprotein B-100 are conflicting: both upregulation (Zorilla et al., 1968; Hazzard et al., 1971; Wynn et al., 1969; Stokes, 1971) and down-regulation (Oliver, 1970; Vessey, 1969) of apolipoprotein B-100 levels have been reported, but no solid quantitative data exists. The effects of estrogen on apolipoprotein B-100 levels may depend upon stage of reproduction as well as dose. Thus, in post-menopausal or ovari¢ctomized women, lower doses (physiological levels) of estrogens have been shown to increase very-lowdensity lipoprotein (VLDL) apolipoprotein B-100 production (Weinstein et al., 1978; Weinstein et al., 1979). However, at higher, or pharmacological doses, estrogens are hypolipidemic, decreasing apoprotein-B containing plasma lipoproteins (Kushwaha and Hazzard, 1981). In addition, studies in pre-menopausal women suggest that estrogens may up-regulate the synthesis of VLDL and LDL apoprotein B (Schaefer et al., 1983; Kekki and Nikkila, 1971; Kissebah et al., 1974). In the experimental paradigm we used, progesterone either alone or in combination with estrogen had no effect on apolipoprotein B-100 levels in the female turtle. Studies in the baboon (Kushwaha et al., 1990) and human (Tikkanen and Nikkila, 1986; LaRosa, 1988), suggest that progestins antagonize the actions of estradiol on apolipoprotein B-100 synthesis. In the baboon, estrogens act to increase metabolism of apolipoprotein B-100, and progestins both decrease metabolism and increase production of apolipoprotein B-100. Moreover, ovariectomy in baboons abolishes the effects of progesterone, suggesting that estrogen may be necessary for the actions of progesterone (Kushwaha et al., 1990), possibly through receptor induction. Similar results are seen in humans, where progestins act to decrease estrogen-elevated plasma VLDL and trigiyceride levels (Tikkanen et al., 1986). Clearly it is premature to
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speculate on any role of progesterone in apolipoprotein B-100 synthesis/secretion until a full evaluation o f the role of estrogen has been conducted as indicated above. In summary, an apolipoprotein B-100-1ike protein has been isolated from the plasma of the female freshwater turtle (Chrysemys picta). The antibody to this protein was used to develop an E L I S A for quantitation of plasma apolipoprotein B-100 in vitro. Plasma levels of apolipoprotein B-100 in reproductively active female turtles were high and chronic estrogen injection significantly reduced apolipoprotein levels. Progesterone had no effect either alone or in combination with estrogen, In order to properly evaluate the role of steroids in the regulation of apolipoprotein B-lO0 in the turtles, it will be necessary to conduct studies in immature, non-reproductive and castrate females, as well as normal and castrate males. Nonetheless, further comparison of apolipoprotein B-lO0 and vitellogenin regulatory systems and their role in lipid transport in mammalian and non-mammalian species are likely to illuminate an important and poorly-understood area of metabolism. Acknowledgement--Supported by NIH grant IR01RR06633 to IPC and an NSF Pre-doctoral fellowship to LEP.
REFERENCES
Baker M. E. (1988) Is vitellogenin an ancestor of apolipoprotein B-100 of apolipoprotein B-100 of human LDL and human lipoprotein lipase? Biochem. J. 255, 1057-1060. Boehm K. D., Hood R. L. and Ilan J. (1988) Induction of vitellogenin in primary monolayer cultures of cockerel hepatoeytes. Proc. natn. Acad. Sci. USA 85, 3450-3454. Brown W. V., Levy R. I. and Fredrickson D. S. (1969) Studies of the proteins in human plasma very low density lipoproteins. J. biol. Chem. 244, 5687-5694. Byrne B. M., Gruber M. and Ab G. (1989) The evolution of egg yolk proteins. Pros. Biophys. molec. Biol. 53, 33-69. Capony F. and Williams D. L. (1980) Apolipoprotein B of avian very low density lipoprotein: Characteristic of its regulation in nonstimulated and estrogen-stimulated rooster. Biochemistry 19, 2219-2226. Chart L., Jackson R. L., O'Malley B. W. and Means A. R. (1976) Synthesis of very low density lipoproteins in the cockerel: Effects of estrogen. J. clin. Invest. 58, 368-379. Chan L., Bradley W. A., Jackson R. L. and Means A. R. (1980) Lipoprotein synthesis in the cockerel liver: effects of estrogen on hepatic polysomal messenger ribonucleic acid activities for the major apoproteins in very low and high density lipoproteins and for albumin and evidence for precursors to these secretory proteins. Endocrinology 106, 275-283. Chapman M. J., Mills G. L. and Ledford J. H. (1975) The distribution and partial characterization of the serum apolipoproteins in the guinea pig. Biochem. J. 149, 423-426. Enviall E. and Perlmann R. (1971) Enzyme-linked immunosorbent assay, ELISA. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigen-coated tubes. J. lmmunol. 109, 129-135. Furman R. H. (1973) Coronary heart disease and the menopause. In Menopause and Aging (Edited by Ryan K. J. and Gibson D. C.), p. 39. Washington, D. C. Gapp D. A., Ho S.-M. and CaUard I. P. (1979) Plasma levels of vitellogenin in Chrysemys picta during the annual
gonadal cycle: measurement by specific radioimmunoassay. Endocrinology 104, 784-790. Goldstein S., Chapman M. J. and Mills G. L. (1977) Biochemical and Immunological evidence for the presence of an apolipoprotein B-like component in the serum low density lipoproteins of several animal species. Artherosclerosis 28, 93-100. Hazzard W. R. Bruzell J. D., Notter D. T., Spiger J. J. and Bierman E. L. (1971) Estrogens and triglyceride transport: increased endogenous production as the mechanism for the hypertriglyceridemia of oral contraceptive therapy. Excerpta Med. Intern. Conr. Ser 273, 1006-1012. Herbert P. N. Windmueller H. G., Bersot T. 1. and Shulman R. S. (1974) Characterization of rat apolipoproteins, l. Low-molecular weight proteins of rat plasma high density lipoproteins. J. biol. Chem. 2,49, 5718-5724. Ho S.-M., Kleis S., McPherson R., Heisermann G. J. and Callard I. P. (1982) Regulation of vitellogenesis in reptiles. Herpetologica 38, 40-50. Katzenellenbogen B. S. (1980) Dynamics of steroid hormone receptor action. Ann. Rev. Physiol. 42, 17 35. Kekki M. and Nikkila E. A. (1971) Plasma triglyceride turnover during use of oral contraceptives. Metabolism 20, 878-889. Kissebah A. H., Harrigan P. and Wynn V. (1974) Mechanism of hypertriglyceridemia associated with contraceptive steroids. Horm. metab. Res. 5, 184-190. Kushwaha R. S. and Hazzard W. R. (1981) Exogenous estrogens attenuate dietary hypercholesterolemia and atheroslerosis in the rabbit. Metabolism 30, 359-366. Kushwaha R. S., Foster D. M., Hugh E, Barrett R. and Carey K. D. (1990) Effect of estrogen and progesterone on metabolism of apoprotein B in baboons. Am. J. Physiol. 258, E172--EI83. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the heat bacteriophage T4. Nature (London) 227, 680-685. La Rosa J. C. (1988) The varying aspects of progestins on lipid levels and cardiovascular disease. Am. J. Obstet. Gynecol. 158, t621-1629. Luskey K. L., Brown M. S. and Goldstein J. L. (1974) Stimulation of the synthesis of very low density lipoprotein in rooster liver by estradiol. J. biol. Chem. 249, 5939-5947. Oliver M. R. (1970) Oral contraceptives and myocardial infarction. Br. Med. J. 2, 210-213. Perez L. E., Fenton M. F. and Callard I. P (1992) Vitellogenin--homologs of mammalian apolipoproteins? Comp. Biochem. Physiol. 100B, 821-826. Perez L. E., Schultz D. S. and CaUard I. P. (1992) Isolation of apolipoprotein B from the plasma of the dogfish, Squalus acanthias. Mt. Desert Isl. Biol. Lab. Bull., in press. Schaefer E. J., Foster D. M., Zech L. A., Lindgren F. T., Brewer H. B. Jr., and Levy R. I. (1983) The effects of estrogen administration on plasma lipoprotein metabolism in premenopausal females. J. Endocrinol. Metab. 57, 262-267. Stokes T. and Wynn V. J. (1971) Serum lipids in women on oral contraceptives. Lancet 2, 677-680. Tata J. R. (1976) The expression of the vitellogenin gene. Cell 9, 1 14. Tikkanen M. J., Kuusi T., Nikkila E. A. and Sipinen S. (1986) Post-menopausal hormone replacement therapy: effects of progestins on serum lipids and lipoproteins. Maturitas g, 7-17. Tikkanen M. J. and Nikkila E. A. (1986) Oral contraceptives and lipoprotein metabolism. J. Reprod. Med. 31, 898-905. Vaitukaitis J., Robbins J. B., Nieschlag E, and Ross G. T. (1971) A method for producing specific antisera with small doses of immunogen. J. clin. Endocrinol. 33, 988494.
Turtle apolipoprotein B-100 Vessey M. P. and Doll R. (1969) Investigation of relation between use of oral contraceptives and thrombo-embolic disease. A further report. Br. Med. J. 2, 651-657. Wahli W. (1988) Evolution and expression of viteUogenin genes. Trends Gene. 4, 227-232. Wallace R. B., Hoover J., Barret-Connor E., Rifkind B. M., Hunninghake D. B., Mackenthuen A. and Heiss G (1979) Lancet 1, 111. Wallace R. A. (1985) Vitellogenesis and oocyte growth in mammalian vertebrates. In Developmental Biology, Edited by Browder L. Vol. I (Oogenesis), pp. 127-177, Plenum Press, New York. Weinstein I., Turner F. C., Soler-Argilaga C. and Heimberg M. (1978) Effect of ethynyl estradiol on serum lipoprotein lipids in male and female rats. Biochim. biophys. Acta 530, 394-401.
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Weinstein I., Soler-Argilaga C., Werner H. V. and Heimberg M. (1979) Effect of ethyl estradiol on the metabolism of [l-t4C] oleate by perfused livers and hepatocytes from female rats. Biochem. J. 180, 265-271. Williams D. L. (1979) Apoproteins of very low density lipoprotein: demonstration of a single high molecular weight apolipoprotein. Biochemistry - 18, 10561063. Wynn V. J., Doar W. H., Mills G. L. and Stokes T. (1969) Fasting serum triglyceride, cholesterol and lipoprotein levels during oral contraceptive therapy. Lancet 2, 756-760. Zorrilla E., Hulse M., Hernandea A. and Gershberg H. (1968) Severe endogenous hypertriglyceridemia during treatment with oestrogen and oral contraceptives. J. clin. Endocrinol. Metab. 28, 1793-1796.
0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd
PUTATIVE APOLIPOPROTEIN B-100 IN THE FRESHWATER TURTLE CHRYSEMYS PICTA: EFFECTS OF ESTROGEN AND PROGESTERONE LORELEI E. PEREZ,* DAVID WILLIAMS'~and IAN P. CALLARD*:~ *Department of Biology, Boston University, Boston, MA 02215, U.S.A. and Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04609, U.S.A.; and ~'Department of Pharmacology, State University of New York, Stonybrook, NY 11794, U.S.A. (Tel. 61%353-5087; Fax 617-353-6340)
(Received 19 March 1992; accepted 22 April 1992) Atstract--l. The isolation and purification of a putative apolipoprotein B-100 in the plasma of the freshwater turtle Chrysemys picta is described. 2. The protein was purified through differential ultracentrifugation and subsequent Sepharose 6B column chromatography. 3. The molecular weight of the protein determined by electrophoresis was approximately 350 kDa. 4. An antibody to chicken apolipoprotein B-100 specifically recognizes this 350 kDa protein in Western blots, suggesting its identity with apolipoprotein B-100. 5. An antibody to the putative Chrysemys apolipoprotein B-100-1ikeprotein was developed and used in an ELISA to quantitate protein levels in plasma. 6. Acute estrogen treatment increased levels of apolipoprotein B-100 (7.64 + 0.79 mg/ml plasma) over that of control animals (5.07 _+ 1.74 mg/ml plasma). 7. In contrast, chronic estrogen treatment reduced apolipoprotein B-100 significantly to 2.94 + 0.53 mg/ml plasma (P < 0.05).
ory effects of steroids on vitellogenin versus apolipoprotein B-100/LDL in a non-mammalian species may provide insight into the role of steroids in mammalian lipid metabolism and atherosclerosis. Thus, apolipoprotein B-100 was purified and characterized in the freshwater turtle Chrysemys picta, a species in which vitellogenin has been shown to be under the dual regulation of estrogen and progesterone (Ho et al., 1982).
INTRODUCTION Apolipoprotein B-100, the main protein component of very-low-density lipoprotein (VLDL) and lowdensity lipoprotein (LDL), has been isolated and characterized in several mammals: human (Brown et al., 1972), rat (Herbert et al., 1974), guinea pig (Chapman et al., 1975) and one avian (Williams, 1979; Capony and Williams, 1980). Human and avian studies have shown a role for estrogen in the regulation of apolipoprotein B-100 in the liver. In the domestic hen, estrogen greatly increases the level of apolipoprotein B-100 both/!1 vitro (Luskey et al., 1974; Chan et al., 1976; Capony and Williams, 1980) and synthesis in vitro by hepatocytes (Chart et el., 1980; Boehm et al., 1988). In the human, estrogen is reported to both increase (Wynn et al., 1969; Wallace et al., 1979) and decrease (Vessey, 1969; Oliver, 1970) apolipoprotein B-100 levels significantly. Several recent computer analyses have shown that vertebrate vitellogenins and human apolipoprotein B-100 have significant sequence homology, suggesting relatedness (Baker, 1988; Byrne et el., 1989; Perez et al., 1991). Vitellogenin, a major egg yolk protein, like apolipoprotein B-100, is synthesized in the liver and is regulated by estrogen in non-mammalian species (see Tata, 1976; Wallace, 1985; Wahli, 1988, for review). Because of the correlation between LDL/apolipoprotein B-100 and atherosclerosis, and the apparent protective action of estrogen (Furman, 1973), a comparison of the regulat:~To whom correspondence should be addressed.
MATERIALS AND METHODS Freshly caught adult female Chrysemys picta turtles were shipped from Lemberger (Oshkosh, WI) and kept indoors at 22°C in constantly circulating freshwater tanks until used for experiments. Animals were injected i. m. with 1.0 mg/kg body weight estradiol (a dose previously shown to stimulate viteilogenin synthesis in this species; Gapp et al., 1979) in oil and killed by decapitation 7 days later. Blood was collected and serum or plasma isolated in the presence of I mM phenylmethylsulfonyl fluoride (PMSF; Sigma Chemical Co. St Louis, MO). In order to determine the effect of progesterone on estrogen-induced apolipoprotein B-100 synthesis, animals were injected i. m. with either estradiol (0.5 mg/kg body weight) in oil for 8 days, progesterone (5 mg/kg body on days 6, 7 and 8), or both. Control animals were injected with oil alone.
Separation of lipoprotein fractions Lipoprotein density classes were isolated essentially as described by Witliams (1979). Briefly, very-low-density lipoprotein (VLDL) was isolated by centrifugation at plasma or serum density for 24 hr at 12°C at 20,000& in a Sorvall 50 Ti rotor. The top 2 ml fraction, containing VLDL, was removed and stored at -20°C.
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Purification of high molecular weight VLDL protein The VLDL sample was diluted in PSP [0.02 M sodium phosphate (pH = 7), 0.15 M NaC1, I00 #g/ml PMSF] and a 7 ml sample was chromatographed on Sepharose 6B (2.6 x 30 cm) equilibrated in PSP at 4°C. Fractions were monitored via absorbance at 280 nm. The leading 70% of the excluded peak was dialyzed against 500 volumes of water with 100 #g/ ml PMSF overnight adjusted to 1% sodium dodecyl sulfate (SDS) and lyophilized. The sample was dissolved in 0.02 M sodium phosphate ( p H = 7 ) , 3% SDS and chromatographed on a Sepharose 6B column equilibrated with 0.02M sodium phosphate, I% SDS, 100#g/ml PMSF at room temperature. The column fractions were monitored by separation on 8.5% SDS-PAGE. The high molecular weight component was concentrated by lyophilization and rerun on Sepharose 6B columns as described.
Eleetrophoresis Protein samples were run on 6.0% SDS--polyacrylamide slab gels with the buffer system of Laemmli (1970). Gels were fixed and stained for 1 hr in 0.2% Coomassie Blue and destained in methanol:acetic acid solution.
Preparation of antibody Antiserum against the high molecular weight VLDL component was raised in New Zealand white rabbits by intradermal administration of 100 #g antigen in an equal
volume of Freund's complete adjuvant (Sigma) as described previously by Vaitukaitis (1971). Animals were bled 10 days after each injection. Booster injections in Freund's incomplete adjuvant (Sigma) were given monthly for 5 consecutive months and serum was tested for reactivity against purified antigen in an ELISA assay (see below). Blood was collected by ear-vein puncture, allowed to clot overnight at 4°C and serum stored at -20°C.
Enzyme-linked immunosorbent assay (ELISA ) The ELISA test was carried out essentially as described by Enviall and Perlmann (1971). Briefly, U-bottom polyvinyl chloride microplates (Flow Laboratories, Rockville, MD) were coated with 100 #1 of serially diluted plasma in carbonate buffer and incubated overnight at 4°C. After incubation, wells were washed three times with phosphate buffered saline (PBS)-Twecn 20 and blocked with a 1.5% gelatin solution (Bio-Rad, Richmond, CA) for 2 hr at room temperature. The wells were washed and 100 #1 of diluted rabbit anti-high molecular weight VLDL protein antiserum was added to each well. The plates were incubated at room temperature for 2 hr, washed and I00 #1 of goat anti-rabbit antiserum conjugated to alkaline phosphatase (Sigma) was added to each well. The plates were incubated for an additional 2 hr at room temperature, washed and developed with the addition of 100#1 of p-nitrophenylphosphatv solution (Sigma) to each well. The colorimetric readings were recorded at 410 nm ufing a modified microtiter plate spectrophotometer (Dynatvch, Chantilly, VA).
APO B100 205 116 97.4 66
45
29
VLDL Ultracent.
Seph. 6B PSP
Seph. 6B SDS
Fig. l. Purification of a high molecular weight protein in female turtle plasma. VLDL ultracentrifugation (lane 1), Scpharose 6B-PSP column chromatography (lane 2) and Sepharose 6B-SDS column chromatography (lane 3) products were separated on 8.5% SDS-PAGE and stained with Coomassie Blue. A high molecular weight protein, of approximately 350 kDa, was purified using these procedures. VLDL = verylow-density lipoprotein ultracentrifugate; Seph. 6B-PSP = Sepharose 6B-PSP column chromatography eluate; Seph. 6B-SDS = Sopharose 6B-SDS column chromatography elute.
Turtle apolipoprotein B-100
709
350 kD
VLDL Ultra
Seph
LDL Ultra
Seph
Fig. 2. Identification of an apolipoprotein B-100-1ike protein in female turtle plasma. Ultracentrifuged plasma components VLDL (lane 1), Sepharose 6B-PSP purified VLDL (lane 2), LDL (lane 3) and Sepharose 6B-PSP purified LDL (lane 4) and was probed on a Western blot with anti-chicken apolipoprotein B-100 antibody. Only one protein of ca 350 kDa binds the anti-chicken apolipoprotein B-100 antibody.
Western blotting Gels used for Western blotting were equilibrated in methanol-Tris-SDS buffer for 30 mill, mounted in a semi-dry blot system (Fisher BioTechnologies, Pittsburg, PA) and protein was transferred onto a methanol-Tris-SDS equilibrated nitrocellulose filter (Schleicher and Schuell) for 30 min at 15 V. The filter was blocked with 5.0% dried milk in TBST (10mM Tris-HC1, pH=8.0, 150mM NaC1, 0.05% Twecn-20) buffer overnight. The filter was washed three times with TBST and incubated for 2 hr with an appropriate dilution of antiserum (turtle apolipoprotein B-100 or chicken apolipoprotein B-100 (courtesy of Dr David Williams) in 5.0% milk/TBST. The filter was washed and incubated for 30 rain with goat anti- rabbit antibody conjugated to alkaline phosphate (Promega Biotech), subsequently washed three times with TBST and incubated in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Promega Biotech) for visualization of alkaline phosphatase activity.
blots were probed with anti-chicken apolipoprotein B-100 antiserum. The antibody crossreacts specifically to a high molecular weight protein of ca 350 kDa (Fig. 2).
Characteristics of turtle anti-apolipoprotein B-100 Antibody against purified turtle apolipoprotein B-100 was raised in a rabbit. The titer curve of the anti-apolipoprotein B-100 antibody is shown in Fig. 3. The Vm~ of the antibody is at approximately 3.29 log dilution. This dilution was used in subsequent Western blots and assays. Pre-immune serum did not crossreact with purified turtle apolipoprotein B-100 (data not shown). The specificity of the antibody for turtle apolipoprotein B-100 was tested using Western blot assays. The polyclonal antibody reacts only with a 350 kDa protein in isolated turtle VLDL fractions and partially
RESULTS
Isolation and purification of apolipoprotein B-lOO-like protein Figure 1 depicts the purification of a high molecular weight plasma protein from female turtle plasma. Two main proteins are present in ultracentrifugation fractions and Sepharose 6B-PSP elution (lanes 1 and 2; VLDL Ultracent. and Seph. 6B PSP). However, a large protein band, of ca 350 kDa, was apparent. On Sepharose 6B columns equilibrated in phosphateSDS buffer, this protein can be purified to apparent homogeneity from the lower molecular weight component (lane 3; Seph. 6B SDS).
2.75 •
1,75
O I~1 0
0.75
-0.25
Identification of apolipoprotein B-100 (Fig. 2) Six percent SDS--PAGE gels were loaded with VLDL (lane 1; VLDL Ultra), Sepharose 6B-PSP purified VLDL (lane 2; Seph), LDL (lane 3; LDL Ultra) and Sepharose 6B-PSP purified LDL (lane 4; Seph) f r o m female turtle plasma. After transfer of the proteins to nitrocellulose membranes, the
0 ;
~
;
~
;
~
Log dilution
Fig. 3. Titer curve for anti-turtle apolipoprotein B-100 antibody. Serially diluted antiserum was used in an ELISA against 50 ng of purified apolipoprotein B100. A Vm~ of ca log dilution was obtained, which was the dilution used in subsequent Western blots and assays.
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L. E. I~R~z et al.
(a)
(b) APO BI00
35(~
180
116 84 58 45
APOB100
VLDL
C.B.
W.B.
Fig. 4. Validity of the turtle apolipoprotein B-100 antibody. The validity of the antibody was tested in Western blot analysis of (a) purified apolipoprotein B-100 (lane 1), VLDL ultracentrifugation density fractions (lane 2) and (b) turtle plasma. As can be seen the antibody reacts only with the 350 kDa high molecular weight protein. purified apolipoprotein B-100 (Fig. 4a; Apo BI00 and VLDL). The antibody also reacts solely with a single component of diluted plasma as can be seen in 0.5"
0.4"
'•t,'-
0.3"
LO O ,q,
o
0.2"
0.1"
0.0
i
i
2
3
Log Apo B100 (ng/ml)
Fig. 5. Standard curve of the turtle apolipoprotein B-100 ELISA assay. Serially-diluted purified apolipoprotein B-100 was tested in an ELISA against a 3.81 log dilution of anti-turtle apolipoprotein B-100 antibody. The detection limit of the assay was 15.6 ng/ml plasma.
Western blots (Fig. 4b; W. B.), where only the 350 kDa protein is selected (Fig. 4b; C. B.). ELISA for turtle apolipoprotein B-100 Figure 5 shows a composite standard curve from six assays. The detection limit of the assay was 15.6 ng/ ml plasma. The standard curve after log probit transformation was linear, with an R 2 for 6 assays of 0.923. The accuracy of the assay, as measured by the coefficient of variance along the standard curve, ranged from 7.2% at 31.3 ng/ml plasma to 1.6% at 1000 ng/ml plasma. The precision of the assay was tested by adding a plasma sample of known apolipoprotein B-100 content (100 ng) to different standard dilutions of vitellogenin (500, 250 and 62.5 ng). The resultant ELISA estimates were 1.26-, 1.13- and 1.1 lfold greater, respectively, than the expected values. The overestimation of the higher values which fell at 500-1000 ng concentration respectively is a reflection of the non-linear behavior of female plasma at higher concentrations. Effects o f estrogen on apolipoprotein B-100 synthesis (Fig. 6) In vehicle treated females (control), apolipoprotein B-100 levels of ca 5.07 _ 1.32 (n = 3) mg/ml plasma were found. Similar values were obtained in animals treated with a single injection of estrogen (7.64 + 0.79; n = 4), progesterone (5.26 + 0.61; n = 3) or a combination of estrogen and progesterone (5.84 _+ 0.48; n = 3). In contrast, chronic treatment of female turtles with estrogen (0.5 mg/kg body weight for 8 days) resulted in a significant decrease of apolipoprotein
Turtle apolipoprotein B-100
1°t E M _=
[ 8
•" m
4
o ,¢ 2
control
E2-chronic
E2-acute
E+P
P4
Fig. 6. Effects of estrogen and progesterone on apolipoprotein B-100 synthesis. The effects of steroids was tested in female turtles using the ELISA for turtle apolipoprotein B-100. After acute (1.0 mg/kg body weight on day 0) or chronic (0.5 mg/kg body weight for 8 days) estrogen treatment had significantly different levels from that of control animals. Treatment with estrogen and progesterone or progesterone alone had no effect. B-100 in the plasma of female turtles (2.94 _+0.53; n = 8; P < 0.05) vs controls. DISCUSSION
In this paper, we describe the identification and purification of a high molecular weight (350 kDa) protein (putative apolipoprotein B-100) from the plasma of estrogen-treated female turtles using the methods of Williams (1979) for isolation of cockerel apo-VLDL-B. In all species studied, including the turtle, apolipoprotein B-100 has been shown to resolve into one component on Sepharose 6B columns in the presence of SDS (Williams, 1979). On the basis of electrophoretic mobility the estimated molecular weight of the putative turtle apolipoprotein B-100 is 350 kDa, similar to that of the chicken (360 kDa; Williams, 1979). The crossreactivity of anti-chicken apolipoprotein B-100 with putative turtle apolipoprotein B-100 on Western blots suggests significant structural homology of these two proteins. Given the phylogenetic descent of birds from reptiles and the apparent molecular conservation of VLDL/LDL apoproteins, this is not unexpected. On the basis of other studies in our laboratory (Perez et al., 1992), it would appear that apolipoprotein B-100 is highly conserved from elasmobranchs to birds. Thus, we have shown that anti-chicken apolipoprotein B-100 also crossreacts with putative elasmobranch apolipoprotein B-100 (Perez et al., 1992). Since anti-human apolipoprotein B-100 crossreacts well with other mammals, yet poorly with avian VLDL (8.0%) and not at all with VLDL from teleost and snake (Goldstein et al., 1977), significant changes in protein structure and/or immunogenic domains may have occurred with mammalian evolution. However, no crossreactivity studies of non-mammalian antibodies CBPB 103/3~N
711
to mammalian apolipoprotein B-100 have been done. High levels of apolipoprotein B-100 in turtles facilitated the subsequent isolation and purification of the protein since 5 ml of plasma yielded ca 2.0 mg apolipoprotein B-100. However, acute estrogen treatment did not elevate apolipoprotein B-100 levels in female turtles, unlike the male bird in which apoprotein B levels were elevated ca 500-fold (to 12.5 mg/ml blood) after a single injection of DES (Capony and Williams, 1980). This difference may be due to the high basal levels in female turtles ca 5.0mg/ml plasma) compared to male birds (ca 26.2 #g/ml blood) which possibly prevents a further response. It will be of interest to measure apolipoprotein B-100 in immature, reproductively inactive and castrate (low estrogen) female turtles. However, we have also noted that apolipoprotein B-100 levels are high in normal males (l-2mg/ml, unpublished observations). These observations suggest that apolipoprotein B-100 regulation in the turtle may be quite different from the bird but values of apolipoprotein B-100 in reproductively active female birds would be of interest. The physiological significance of the reduction in turtle apolipoprotein B-100 after chronic estrogen treatment is in question until further studies are presented. In human studies, the data on the effect of estrogens on apolipoprotein B-100 are conflicting: both upregulation (Zorilla et al., 1968; Hazzard et al., 1971; Wynn et al., 1969; Stokes, 1971) and down-regulation (Oliver, 1970; Vessey, 1969) of apolipoprotein B-100 levels have been reported, but no solid quantitative data exists. The effects of estrogen on apolipoprotein B-100 levels may depend upon stage of reproduction as well as dose. Thus, in post-menopausal or ovari¢ctomized women, lower doses (physiological levels) of estrogens have been shown to increase very-lowdensity lipoprotein (VLDL) apolipoprotein B-100 production (Weinstein et al., 1978; Weinstein et al., 1979). However, at higher, or pharmacological doses, estrogens are hypolipidemic, decreasing apoprotein-B containing plasma lipoproteins (Kushwaha and Hazzard, 1981). In addition, studies in pre-menopausal women suggest that estrogens may up-regulate the synthesis of VLDL and LDL apoprotein B (Schaefer et al., 1983; Kekki and Nikkila, 1971; Kissebah et al., 1974). In the experimental paradigm we used, progesterone either alone or in combination with estrogen had no effect on apolipoprotein B-100 levels in the female turtle. Studies in the baboon (Kushwaha et al., 1990) and human (Tikkanen and Nikkila, 1986; LaRosa, 1988), suggest that progestins antagonize the actions of estradiol on apolipoprotein B-100 synthesis. In the baboon, estrogens act to increase metabolism of apolipoprotein B-100, and progestins both decrease metabolism and increase production of apolipoprotein B-100. Moreover, ovariectomy in baboons abolishes the effects of progesterone, suggesting that estrogen may be necessary for the actions of progesterone (Kushwaha et al., 1990), possibly through receptor induction. Similar results are seen in humans, where progestins act to decrease estrogen-elevated plasma VLDL and trigiyceride levels (Tikkanen et al., 1986). Clearly it is premature to
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speculate on any role of progesterone in apolipoprotein B-100 synthesis/secretion until a full evaluation o f the role of estrogen has been conducted as indicated above. In summary, an apolipoprotein B-100-1ike protein has been isolated from the plasma of the female freshwater turtle (Chrysemys picta). The antibody to this protein was used to develop an E L I S A for quantitation of plasma apolipoprotein B-100 in vitro. Plasma levels of apolipoprotein B-100 in reproductively active female turtles were high and chronic estrogen injection significantly reduced apolipoprotein levels. Progesterone had no effect either alone or in combination with estrogen, In order to properly evaluate the role of steroids in the regulation of apolipoprotein B-lO0 in the turtles, it will be necessary to conduct studies in immature, non-reproductive and castrate females, as well as normal and castrate males. Nonetheless, further comparison of apolipoprotein B-lO0 and vitellogenin regulatory systems and their role in lipid transport in mammalian and non-mammalian species are likely to illuminate an important and poorly-understood area of metabolism. Acknowledgement--Supported by NIH grant IR01RR06633 to IPC and an NSF Pre-doctoral fellowship to LEP.
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Turtle apolipoprotein B-100 Vessey M. P. and Doll R. (1969) Investigation of relation between use of oral contraceptives and thrombo-embolic disease. A further report. Br. Med. J. 2, 651-657. Wahli W. (1988) Evolution and expression of viteUogenin genes. Trends Gene. 4, 227-232. Wallace R. B., Hoover J., Barret-Connor E., Rifkind B. M., Hunninghake D. B., Mackenthuen A. and Heiss G (1979) Lancet 1, 111. Wallace R. A. (1985) Vitellogenesis and oocyte growth in mammalian vertebrates. In Developmental Biology, Edited by Browder L. Vol. I (Oogenesis), pp. 127-177, Plenum Press, New York. Weinstein I., Turner F. C., Soler-Argilaga C. and Heimberg M. (1978) Effect of ethynyl estradiol on serum lipoprotein lipids in male and female rats. Biochim. biophys. Acta 530, 394-401.
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Weinstein I., Soler-Argilaga C., Werner H. V. and Heimberg M. (1979) Effect of ethyl estradiol on the metabolism of [l-t4C] oleate by perfused livers and hepatocytes from female rats. Biochem. J. 180, 265-271. Williams D. L. (1979) Apoproteins of very low density lipoprotein: demonstration of a single high molecular weight apolipoprotein. Biochemistry - 18, 10561063. Wynn V. J., Doar W. H., Mills G. L. and Stokes T. (1969) Fasting serum triglyceride, cholesterol and lipoprotein levels during oral contraceptive therapy. Lancet 2, 756-760. Zorrilla E., Hulse M., Hernandea A. and Gershberg H. (1968) Severe endogenous hypertriglyceridemia during treatment with oestrogen and oral contraceptives. J. clin. Endocrinol. Metab. 28, 1793-1796.