Modification in the purification of the sex steroid-binding protein of human serum by affinity chromatography

A purification procedure for the sex steroid-binding protein of human serum is described. The procedure is significantly superior to that recently published (K. E. Mickelson, D. C. Teller, and P. H. Pétra, 1978, Biochemistry17, 1409–1415) and should replace it for the routine preparation of homogeneous protein in relatively larger quantities. The steps involved diethylaminoethyl-cellulose chromatography, affinity chromatography on 5α-dihydrotestosterone-17α-hexanyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The most important difference between this new procedure and that previously published is the affinity adsorbent with contains the steroid covalently linked at the 17α-position rather than the 17β-position. This modification allows the purification of at least 12 mg of homogeneous protein per preparation with a 63% total yield. The properties of the homogeneous protein are the same as previously described.     

Purification and physico-chemical properties of the sex steroid-binding globulin of human blood

A new technique for purification of the human sex hormone-binding globulin (SHBG) is described. This technique includes affinity chromatography of blood serum on cortisol-Sepharose, (NH4)2SO4 fractionation, gel filtration on a Bio-Gel P-300 column and chromatography on a concanavalin A-Sepharose 4B column. From 21 of retroplacental serum 10 mg of pure SHBG (25% yield) has been obtained. Upon gel filtration SHBG behaved as a biopolymer with Mr of 120,000. The molecular weight of SHBG as determined by electrophoresis was shown to be equal to 50,000. SHBG has a sedimentation constant of s20, w of 4.7S, pI of 5.75, extinction coefficient A1%(280,1cm) = 10,5 and association constants of 4.5 X 10(8) and 3.5 X 10(6) M-1 for 5 alpha-dihydrotestosterone and cortisol, respectively. The amino acid and carbohydrate contents of SHBG were determined.     

Localization of the steroid-binding site of the human sex steroid-binding protein of plasma (SBP or SHBG) by site-directed mutagenesis.

The amino-terminal region of the human sex steroid-binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the steroid-binding site. This was accomplished by constructing and expressing site-directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully-active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M139S was inactive. These results support affinity-labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in steroid binding. The fully active H235S mutant reveals that H235 is not involved in the steroid-binding process.     

Molecular organization of the sex steroid-binding protein (SBP) of human plasma

Several years ago this laboratory presented evidence that SBP is a dimer composed of two subunits having similar molecular weights. The question of whether or not these subunits are identical and therefore products of a single gene remained unanswered. We now report that the two polypeptide chains are identical and that SBP is a homodimer. The experimental approach was to reduce and [14C]alkylate cystine residues in human SBP, digest the product with trypsin or cyanogen bromide and determine the number of unique amino acid sequences around each [14C]carboxymethylcysteine residue. Only four unique sequences were found when all the radioactive peptides were analyzed. Since there are eight half-cystine residues per dimer, the results support a homodimeric structure.     

Immunohistochemical detection of the sex steroid-binding plasma protein in human mammary carcinoma cells

A sex steroid-binding plasma protein-like antigen has been detected in human mammary carcinoma cells. A monospecific antiserum was used, and this protein was located mainly on the cytoplasmic membranes. These results are in agreement with a recent hypothesis according to which steroid hormones could be carried into cells by specific binding plasma proteins.     

Characterization of a cDNA coding for sex steroid-binding protein of human plasma

A cDNA (912 nucleotides) coding for human plasma sex steroid-binding protein (SBP) was characterized from a phage clone previously isolated by screening a Charon 21A human liver cDNA library with rat androgen binding protein (ABP) cDNA. The deduced amino acid sequence from the cDNA indicated that the insert was a partial clone coding for 281 amino acids starting with residue 92 (glycine) encompassing the alternating leucyl residues and the carboxyl-end 373 (histidine) as previously reported [(1986) Biochemistry 25, 7584]. The potential polyadenylation signal sequence ATTAAA is present as part of the 3'-coding region and the stop codon TAA. Both are followed by a short 20 untranslated nucleotides and a poly(A) tract of 49 nucleotides. Significant homologous sequences (76%) at the DNA level exist between human SBP and rat ABP which might suggest the possibility that both evolved from a common primordial gene. Demonstration of the presence of an SBP cDNA in a human liver cDNA library provides the first evidence that liver is the site of SBP biosynthesis.     

Identification of NaK-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids

Elevated plasma levels of factors with cardiac glycoside-like activity have been implicated in the response to volume expansion in animals and in the pathogenesis of certain human diseases. We recently described four fractions (IR1, EI1, EI2, EI3) from normal human plasma that inhibit NaK-ATPase, displace ouabain from the enzyme, and exhibit digoxin-like immunoreactivity (Kelly, R. A., O'Hara, D. S., Canessa, M. L., Mitch, W. E., and Smith, T. W. (1985) J. Biol. Chem. 260, 11396-11405). In this report, we identify the active component of these plasma fractions as long-chain nonesterified fatty acids (NEFA) and lysophospholipids. These lipids were present in fractions EI1, EI2, and EI3 in quantities sufficient to account for all of the NaK-ATPase inhibitory activity. The digoxin-like immunoreactivity in fraction IR1 could be attributed to hydrocortisone and other endogenous steroids. To explore the nature of the lipid-NaK-ATPase interactions, we examined the effects of various ATP or sodium concentrations on the NaK-ATPase activity measured in the presence of NEFA. Varying sodium did not affect the inhibition of NaK-ATPase by linoleic acid. At less than 0.15 mM ATP, linoleic acid stimulated NaK-ATPase, but at higher ATP concentrations, the enzyme was progressively inhibited. In summary, NEFA and lysophospholipids, at levels similar to those occurring in human plasma, may account for all of the NaK-ATPase inhibitory activity observed in human plasma fractions. These lipids probably do not directly regulate NaK-ATPase in vivo under normal physiologic conditions, but may alter the sodium pump in disease states characterized by abnormalities in lipid metabolism or plasma protein binding.     

Purification and characterization of the sex steroid-binding protein of rabbit serum. Comparison with the human protein

The sex steroid-binding protein (rSBP) of immature rabbit serum was purified to homogeneity by the sequential use of DEAE-cellulose chromatography, affinity chromatography on 5alpha-dihydrotestosterone-17 beta-succinyl-diaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, agarose (Bio-Gel-A-0.5m) gel filtration, and preparative polyacrylamide gel electrophoresis. The cumulative yield is 13%. Homogeneity of rSBP was shown by the equilibrium sedimentation ultracentrifugation in 6 M guanidine HCl containing 0.1 M mercaptoethanol which yields an average molecular weight of 36,475 +/- 865. Analytical gel electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on agarose yield a molecular weight of 57,000 and 120,000, respectively. The variation is due to a 30% carbohydrate content. The amino acid composition is reported. Comparison of the rabbit and human SBP indicate that they are different in both their molecular and functional properties.     

Quantitative extraction and determination of nonesterified fatty acids in plasma

A method is described for the determination of nonesterified fatty acids in plasma. Extraction is at least 98% efficient, and losses during subsequent stages are corrected for by the use of an internal radioactive standard. The method is suitable for reference purposes rather than for routine determinations. Higher values are obtained than by other methods of analysis, and it is suggested that some plasma fatty acids remain protein-bound after normal methods of extraction.     

Albumin bound nonesterified fatty acids inhibit in vitro lipid peroxidation

Individual nonesterified fatty acids were bound to albumin in vitro and these fatty acid albumin complexes were used to study their effect on lipid peroxidation in liver microsomes. Peroxidation was induced by various methods and malondialdehyde (MDA) was measured as an index of peroxidation. Among the fatty acids tested, albumin-bound monounsaturated fatty acids showed more inhibition of peroxidation as compared to other fatty acids. Increasing the concentration of iron in the peroxidizing system, partially reversed the inhibition by fatty acids. Moreover, albumin-bound fatty acid did not inhibit iron independent peroxidation. This suggests that, like nonesterified fatty acids, albumin-bound fatty acids inhibit peroxidation by chelating the iron. Albumin fatty acid complex, similar to the fatty acid composition present in the circulating albumin, also showed inhibition of peroxidation. These data indicate that nonesterified fatty acids even when bound to albumin are capable of inhibiting peroxidation and circulating albumin, which contains various fatty acids bound to it, may impart some antioxidant effect in addition to other plasma antioxidants.     

Mechanism of insulin-stimulated clearance of plasma nonesterified fatty acids in humans

Insulin increases plasma nonesterified fatty acid (NEFA) clearance in humans, but whether this is independent of change in plasma NEFA appearance is currently unknown. Nine nondiabetic men (age: 28+/-3 yr, body mass index: 27.2+/-1.7 kg/m2) underwent euglycemic clamps to maintain low (LINS) vs. high (HINS) physiological insulin levels for 6 h. An intravenous infusion of heparin+Intralipid (HI) was performed during 4 of the 6 h of the clamps (in the last 4 h at LINS and in the first 4 h at HINS), whereas saline infusion (SAL) was administered in the remaining 2 h to modulate plasma NEFA levels independently of plasma insulin levels. Four experimental conditions were obtained in each individual: LINS with saline (LINS/SAL) and with HI infusion (LINS/HI) and HINS with saline (HINS/SAL) and with HI infusion (HINS/HI). Plasma palmitate appearance during HINS/SAL was lower than during the three other experimental conditions (P<0.05). In contrast, plasma linoleate appearance, as expected, was increased by HI independently of insulin level (P<0.02). Plasma palmitate clearance during HINS/SAL was higher than LINS/SAL and LINS/HI (P<0.008), and this increase was blunted during HINS/HI. We observed a linear decrease in plasma palmitate clearance with increasing plasma NEFA appearance independent of insulin levels. Plasma NEFA levels increased exponentially with increase in plasma NEFA appearance. We conclude that insulin stimulates plasma NEFA clearance by reducing the endogenous appearance rate of NEFA. The relationship between plasma NEFA level and appearance rate is nonlinear.     

The amino acid sequence of the sex steroid-binding protein of rabbit serum

The amino acid sequence of the sex steroid-binding protein (SBP or SHBG) of rabbit serum, specific for binding testosterone and 5 alpha-dihydrotestosterone, was determined using a complementary combination of mass spectrometric and Edman degradation techniques. The monomeric unit of the homodimeric protein is a single chain glycopeptide of 367 amino acid residues, with N-linked oligosaccharide side chains at Asn-345 and Asn-361 and disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355. The polypeptide molecular weight of the monomer calculated from the sequence is 39,769. The molecular weight of the homodimer including 9% carbohydrate is 87,404. The sequence contains a relatively hydrophobic segment between Trp-241 and Leu-282, which includes many leucine residues in an alternating pattern. An amino acid sequence repeat is also located within that segment. Both of these patterns are present in human SBP and in the androgen-binding protein of rat epididymis. The sequence data indicate that the previously reported microheterogeneity of rabbit SBP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reflects variants generated by differential glycosylation of the monomer rather than different gene products. Seventy-nine percent of the amino acids of rabbit SBP are identical to those of human SBP; rabbit SBP thus joins human SBP and rat androgen-binding protein in one gene family that is distinct from the steroid hormone receptor superfamily. It appears that the problem of binding sex steroid hormones has been solved independently in two different gene families that contain completely different steroid-binding domains. Since the nonhomologous steroid-binding domains of both families of proteins recognize essentially the same steroid structure, it will be interesting to determine the structural basis of the two different protein designs that lead to similar steroid-binding specificity.     

Transport of K+ and other cations across phospholipid membranes by nonesterified fatty acids

The rate of change of internal pH and transmembrane potential has been monitored in liposomes following the external addition of various cation salts. Oleic acid increases the transmembrane movement of H⁺ following the imposition of a K⁺ gradient. An initial fast change in internal pH is seen followed by a slower rate of alkalinization. High concentrations of the fatty acid enhance the rate comparable to that seen in the presence of nigericin in contrast to the effect of FCCP (carbonyl cyanide p-(tri-fluoromethoxy)phenyl hydrazone) which saturates at an intermediate value. The ability of nonesterified fatty acids to catalyze the movement of cations across the liposome membrane increases with the degree of unsaturation and decreases with increasing chain length. Li and Na salts cause a similar initial fast pH change but have less effect on the subsequent slower rate. Similarly, the main effect of divalent cation salts is on the initial fast change. The membrane potential can enhance or inhibit cation transport depending on its polarity with respect to the cation gradient. It is concluded that nonesterified fatty acids have the capability to complex with, and transport, a variety of cations across phospholipid bilayers. However, they do not act simply as proton/cation exchangers analogous to nigericin nor as protonophores analogous to FCCP. The full cycle of ionophoric action involves a combination of both functions.The authors would like to thank P. Nicholts (Brock University, Canada) for helpful discussions. M.A.S. received a Science and Engineering Research Council studentship and C.E.C. acknowledges the award of a King's College London fellowship followed by a Medical Research Council Training Fellowship.