Int. J. Biochem. Vol. 20, No. 12, pp. 1343-1349, 1988 0020-71 IX/88 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1988 Pergamon Press plc

THE ISOLATION, CHARACTERIZATION A N D AMINO TERMINAL SEQUENCE OF THE VITAMIN D-BINDING

PROTEIN (GROUP SPECIFIC COMPONENT) FROM MOUSE PLASMA

JAMES L. BORKE l, ROBERT D. LITWILLER l, MICHAEL P. BELL 2, DAVID N. FASS 1, DAVID J. MCKEAN 2 and RAJIV KUMAR k2'3

~Department of Medicine, Divisions of Nephrology and Hematology, Mayo Clinic and Foundation, Rochester, MN 55905, U.S.A., 2Department of Immunology, Mayo Clinic and Foundation, Rochester, MN 55905, U.S.A. and 3Department of Biochemistry and Molecular Biology, Mayo Clinic and

Foundation, Rochester, MN 55905, U.S.A. [Tel. (507)284-4343]

(Received 18 March 1988)

Abstrmet--1. In order to establish a homologous system in which to study the interaction of mouse vitamin D-binding protein (MVDBP) with mouse T-cell lymphocytes, we purified MVDBP from mouse plasma.

2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that purified MVDBP had an apparent relative molecular weight of 49,000.

3. Previous work in our laboratory has shown that purified rat vitamin D-binding protein (RVDBP) has an apparent relative molecular weight of 52,000.

4. The amino terminal amino acid sequence of MVDBP is shown below and compared with that of RVDBP.

MVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysAsnGluLeuAlaMetLeuGlyLysGlu RVDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCys~Glu LeuSerT~rThrLeuGly LysAsp

AspPhe AspPhe

While 21 out of 24 residues (87.5%) of the amino terminus of MVDBP are the same as those in RVDBP, residues 14, 17, 18 and 22 (underlined) are different.

5. The sedimentation coefficient of the protein, determined by sucrose density gradient ultra- centrifugation, is 3.8 for MVDBP and 4.1 for the rat VDBP.

6. The MVDBP purified in this study exhibits only one isoform on isoelectric focusing; the isoelectric point was 4.87 as determined on pH 4.~6.5 isoelectric focusing gels (IEF).

7. The binding of vitamin D 3, 25-hydroxyvitamin D 3 and three other analogs was investigated with a charcoal dextran assay.

8. The concentration of 25-hydroxyvitamin D 3 at which 50% of protein bound radiolabeled 25-hydroxyvitamin D 3 was displaced from MVDBP was found to be 6.8 x 10-9 M. A value of 5 × 10 -9 M was previously reported for the corresponding rat protein.

9. Amino acid composition analysis also showed the mole percent of each residue in MVDBP was very similar to that of RVDBP. The observed properties of mouse VDBP suggest a high degree of homology with the vitamin D-binding protein of rat.

INTRODUCTION

In B-cell lymphocyte plasma membranes, membrane immunoglobulin (Ig) moves in the lipid bilayer. Upon binding specific antibodies, Ig shows a lateral mobil- ity with patch and cap formation (Petrini e t al., 1983). Intracellular vitamin D-binding protein (VDBP) also becomes associated with Ig during this process (Pe- trini et al. , 1983). Other studies have shown that VDBP also undergoes a specific high affinity inter- action with monomeric actin (Haddad, 1982; Van Baelen et al. , 1980). This evidence suggests that VDBP may connect the membrane Ig of B-cells with actin and the cytoskeleton in the B-cell cytoplasm.

Human peripheral blood T-cell lymphocytes have been shown to contain receptors for the Fc port ion of immunoglobulin (Ig) (Itoh and Kumagai, 1980; Lydyard and Fanger, 1982). These receptors appear to play an important role in the biological function of these cells and may be involved in their inter- actions with B-cells (Moretta et al., 1982).

Petrini et al. have shown that the Fc receptor bound portion of IgG in the human peripheral blood T-cell also co-caps with VDBP (Petrini et al., 1985). This study suggests that VDBP may play a role in T-cells similar to membrane VDBP in B-cells. This study also implies that a more generalized function of membrane VDBP might be the mediation of inter- actions between specific bound ligands (besides Ig) and the cytoskeleton.

Recent interest in VDBP has been generated be- cause of conflicting data on a proposed association of this protein with the degree of susceptibility to ac- quired immunodeficiency syndrome (AIDS) (Daiger et al., 1987; DeLange et al., 1987; Eales et al., 1987; Gilles et al., 1987; Thymann et al., 1987). While the mouse does not serve as a direct model for this disease, many important questions relating to lym- phocyte function are easily studied in mice. As part of an initial effort to study the interaction of mouse T-cell lymphocytes with VDBP in a homologous system, we purified the vitamin D-binding protein

1343

1344 JAMES L. BOgKE et al.

f rom m o u s e p l a sma and de t e rmined its a m i n o acid c o m p o s i t i o n , a m i n o te rmina l sequence, and l igand b ind ing proper t ies .

METHODS

General

Mouse plasma was obtained from C57BL/10 mice from Dr Chella David's colonies at Mayo Clinic. Protein concen- tration was determined by the method of Bradford (1976). [3 H-26,27] 25-hydroxyvitamin D3, with a specific activity of 23 Ci/mmol, was obtained from New England Nuclear (Boston, Mass.). All HPLC was performed on a Waters HPLC (Milford, Mass.) with a Kratos Spectroflow 783 detector (Ramsey, N.J.) attached in series with a Flo-One beta radioactive flow detector (Radiomatic Instrument Co., Tampa, Fla).

Actin affinity chromatography

MVDBP was isolated by the method of Haddad et al., with minor modifications (Haddad et al., 1984). Briefly, monomeric actin was covalently attached to an Affigel-15 (BioRad Laboratories, Richmond, Calif.) support gel. The column was rinsed several times with a depolymerizing solution consisting of 0 .5mM ATP, 0.2raM DTT and 0.3 mM CaC12 in 0.01 M Tris. The column was eluted with 3 M guanidinium chloride. The column eluates containing the MVDBP were concentrated and the guanidinium solu- tion was exchanged with water using an Amicon PM 10 filter (Amicon, Danvers, Mass.).

Hydroxylapatite chromatography

Isolated MVDBP was purified by high pressure liquid chromatography (HPLC) on a 7.5 × 100 mM Biogel HPHT hydroxylapatite column (BioRad Laboratories, Richmond, Calif.). The lyophilized MVDBP preparation was re- suspended in 0.01 M phosphate buffer, pH 7.3 and applied to the column. The system was run in an isocratic manner for 10 min in 0.01 M phosphate buffer and then a linear gradient was developed to 0.3 M phosphate pH 7.3 over 15 rain. The flow rate was 1 ml/min and absorbance was monitored at 280 nm. Peaks were collected and lyophilized.

Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE)

Lyophilized peaks from the hydroxylapatite chro- matography were first heated to 100°C for 5 min in 50 mM Tris base containing 5% fl-mercaptoethanol, 2% sodium dodecyl sulfate, 10% glycerol and 0.1% bromophenyl blue. These samples were then analyzed on 7% SDS-PAGE (Laemmli, 1970). This was done first to assess protein purity and later to determine apparent molecular weight by com- parison to marker proteins of known molecular weights.

Binding of 25-hydroxyvitamin D 3 to M V D B P

Lyophilized peaks of purified MVDBP from hydroxy- lapatite chromatography were analyzed for protein content (Bradford, 1976). 0.2 pmol of [3H]25-hydroxyvitamin D 3 in 10ul ethanol was added to 0.5#g MVDBP in lml of 0.05 M KH2PO 4 buffer (pH 7.4) or allowed to react with the same amount of [all] 25-hydroxyvitamin D 3 in the presence of a 2500-fold excess of non-radiolabeled 25-hydroxyvitamin D 3. Samples treated either way were chromatographed on the Biogel HPHT hydroxylapatite column.

Amino acid sequence analysis

Automated amino terminus sequence analyses were per- formed on either a Applied Biosystem 470A sequencer (Hewick et al., 1981) or a Beckman 890 sequencer (Hun- kapiller and Hood, 1978). Analyses were performed on either the native material or material that had been reduced with dithiothreitol and carboxymethylated with 14C-iodo-

acetamide (Itoh and Kumagai, 1980). Several analyses were performed with the amount of protein applied ranging between 0.8 and 2 nmol. Repetitive yields were > 90% on both instruments. Phenylthiohydantoins were identified by HPLC on a Beckman Ultrasphere ODS reverse phase column by the program of Tarr with minor modifications (Tarr, 1981).

Amino acid composition analysis

Amino acid composition was determined on hydrolysates, which had been treated at 110°C with gaseous HC1 for 24 hr. Amino acid residues were quantified by precolumn deri- vatization with phenylisothiocyanate followed by reversed- phase HPLC (Bidlingmeyer et al., 1984).

Sucrose gradient ultracentrifugation

Linear gradients from 4 to 20% sucrose in 0.05 M potas- sium phosphate, pH 7.5, were prepared as previously de- scribed (Bouillon et al., 1980). Ultracentrifugation was carried out at 200,000 g for 24 hr at 4°C using a SW41 rotor in a Beckman L8-7OM centrifuge. Two 16.7 ,ug aliquots of purified protein were added to individual gradients. One of these aliquots contained MVDBP that had been previously incubated with [3H]25-hydroxyvitamin D 3. The other ali- quot contained protein that was incubated with [3H]25-hydroxyvitamin D 3 plus a 2500-fold excess non- radiolabeled 25-hydroxyvitamin D 3. Gradients containing marker proteins with known sedimentation coefficients were run at the same time. Samples from the gradient were analyzed for radioactivity by scintillation counting and for protein by the low ultraviolet spectroscopic method of Ehresmann et al. (1973).

lsoelectrie ./bcusing

Determination of the isoelectric point (pI) of purified MVDBP was carried out on precast pH4-6.5 poly- acrylamide gels (LKB Instruments, Bromma, Sweden). Gels were run at 4°C under constant power for 2 hr with current limit set at 30 W and voltage limit adjusted every 15 min, from a beginning value of 600 V to a final value of 2500 V. The gels were fixed in 20% trichloroacetic acid and stained with Coomassie Blue. The isoelectric point (pI) of MVDBP was determined by graphic comparison of MVDBP to soybean trypsin inhibitor (pI =4.55), bovine milk fl-lactoglobulin A (pI = 5.13) and bovine erythrocyte car- bonic anhydrase B (pl = 5.85) on the same gel.

Displacement of radiolabeled 25-hydroxyvitamin D 3 by vita- min D, analogs

The ability of vitamin D3, 25-hydroxyvitamin D 3, 1,25-dihydroxyvitamin D 3, 24,25-dihydroxyvitamin D 3, 25,26-dihydroxyvitamin D 3 and lct-hydroxyvitamin D 3 to compete with [3H]25-hydroxyvitamin D 3 for binding to MVDBP was determined as previously described for RVDBP (Kumar et al., 1979). Briefly, 0.2pmol [3 H]25-hydroxyvitamin D 3 in 10 p l of ethanol was added to 0.5 #g MVDBP in l ml of 0.05 M KH2PO 4 buffer, pH 7.4. Incubation was carried out in the presence of increasing amounts of the above non-radiolabeled sterols reconstituted in 50#1 ethanol. Unbound sterol was then adsorbed on dextran-coated charcoal, and the supernatants were assayed by scintillation counting.

RESULTS

The final yield o f purif ied M V D B P f rom 5 0 m l o f p l a sma af ter act in affinity and hydroxy lapa t i t e c h r o m a t o g r a p h y was 1.6 mg. A n a p p a r e n t molecu la r weight o f M r = 49,000 was d e t e r m i n e d by the mobi l - ity o f a single b a n d o f purif ied M V D B P relative to molecu la r weight ma rke r p ro te ins on 7% S D S - P A G E (Fig. 1). A value o f M r = 52,000 was

A B

116K

97.4K

66K

45K

29K

49 K

Fig. 1. Molecular weight determination of mouse VDBP by 7% sodium dodecylsulfate-polyacrylamide gel electro-

phoresis.

1345

-I- .¢.

A B C D E

4.55

5.13

5.85

4.87

Fig. 4. Isoelectric point of mouse VDBP on pH 445.5 polyacrylamide gel. Lanes A, B and C, pI marker proteins;

Lane D, empty; Lane E, mouse VDBP.

1346

Vitamin D-binding protein from mouse plasma 1347

E 100

L ̂ Q ~ 5 o

5 (

E 10C

/k ! I I I

.a _ __L _ ~ _ , ..J I L I

t~ 5c

~ o ~

~ so 0

O0 5 10 15 20 25 30 3 4~ Time, minutes

F i g . 2. R e v e r s e p h a s e H P L C o f m o u s e V D B P i n c u b a t e d w i t h [ 3 H ] 2 5 ( O H ) D 3 (A) , a n d [ 3 H ] 2 5 ( O H ) D 3 p l u s e x c e s s

u n l a b e l e d 2 5 ( O H ) D 3 (B).

previously reported for RVDBP (Botham et al., 1976; Bouillon et al., 1978). Incubation of radiolabeled 25-hydroxyvitamin D 3 with MVDBP resulted in a single peak of radioactivity, which co-eluted with a single protein peak when chromatographed on an

Table 2. Amino acid composi t ion analysis (moles percent o f amino acids)

Amino acid M V D B P a R V D B P b

Asp 9.3 6.7 Glu 14.2 13.7 Ser 8.3 9.9 Gly 5.2 4.2 His 1.6 1.4 Arg 4.8 4.7 Thr 8.4 7.2 Ala 6.7 6.9 Pro 5.9 6.5 Tyr 3.3 3.7 Val 5.2 4.9 Met 2.5 3.5 lie 3.5 3.1 Leu 10.3 11.7 Phe 4.4 4.4 Lys 6.9 7.6

~Average of two analyses. bAverage of three analyses.

HPHT hydroxyl apatite column [Fig. 2(A)]. Incu- bation of MVDBP with the same concentration of [3 H]25-hydroxyvitamin D 3 and excess cold 25-hydroxyvitamin D 3 showed a protein peak but a greatly reduced co-eluting peak of radioactivity when chromatographed under the same conditions [Fig. 2(B)].

Amino acid sequence analysis of the purified pro- tein gave 24 residues of the amino terminus (Table 1). Twenty-one or 87.5% of these were homologous with RVDBP (Litwiller et al., 1986). The cysteine residue at amino acid number 13 was obtained by reduction of the protein in dithiothreitol and alkylation in the presence of 14C-iodoacetamide. Amino acid com- position analysis on 24-hr hydrolysates also showed a composition very similar to rat vitamin D binding protein (RVDBP) (Table 2).

The sedimentation coefficient of MVDBP was de- termined by sucrose density ultracentrifugation

Table I. Amino terminal amino acid sequence analysis o f mouse vi tamin D-binding protein

Reduced carboxyamidomethyla ted protein Nat ive protein

Cycle Amino acid residues Amino acid residues cpm

1 Leu - - - - 2 Glu - - - - 3 Arg - - - - 4 Gly - - - - 5 Arg - - - - 6 Asp Asp 3,185 7 Tyr Tyr 3,500 8 Glu Glu 365 9 Lys Lys 4,280

10 Asp Asp 2,290 11 Lys Lys 3,875 12 Val Val 2,925 13 - - Cys 12,200 14 Asn Ash 1,505 15 Glu Glu 5,890 16 Leu Leu 4,965 17 Ala Ala 5,330 18 Met Met 2,450 19 Leu Leu 4,270 20 Gly Gly 1,925 21 Lys Lys 1,815 22 Glu - - - - 23 Asp - - - - 24 Phe - - - -

1348 JAMES L, BORKE et al.

9o

80

7O

E =

,tO

3 0

k:'O

10

~ 2 I:- " ~ 0

\ _ o MVDBP bound a[a]25(OH)Da

I I I I I I I I I I 1 2 3 4 5 6 7 8 9 10 11

Cumulative volume

Fig. 3, Determination of sedimentation coefficient for mouse VDBP by sucrose density ultracentrifugation.

I I ~ I I I AVIID 3

= Z4, 2S 40 IOHI2 th

50%B * ~ (O~'i) D=

6 0 • t= (OH) D 3

lOO

I I I I I I "~ I I I lO /~g 1 /zg 100 ng 10 ng 1 ng 100 pg

Fig. 5. Displacement of radiolabeled 25-hydroxyvitamin D 3 from mouse vitamin D-binding protein by various vitamin

D 3 compounds.

(Fig. 3). The sedimentation coefficient for MVDBP was found to be 3.8. The sedimentation coefficient for RVDBP was previously shown to be 4.1 (Bouillon et al., 1978). Isoelectric focusing of the purified protein on pH4qS.5 gels showed a single band with an isoelectric point equal to 4.87 (Fig. 4). This was calculated graphically by comparison to marker pro- teins of known pI.

The concentrations of vitamin D 3 metabolites necessary to displace 50% of radiolabeled 25-hydroxyvitamin D 3 from MVDBP (B-50 values) were determined (Fig. 5). The B-50 value of each metabolite tested is listed in order of decreasing displacement ability: 25-hydroxy- vitamin D 3 (1.32 x 10 8 M), 25,26-dihydroxyvit- amin D 3 (2,49 x 10 8 M), 24,25-dihydroxy- vitamin D 3 (3.85 x 10 -8 M), 1,25-dihydroxyvitamin D 3 (4.75 × 10 -7 M), lc~-hydroxyvitamin D~ (2.05 x 10 6M), and vitamin D 3 (4.66 × 106M).

D I S C U S S I O N

The vitamin D carrier function of VDBP in serum has suggested a possible role for this protein in calcium and vitamin D metabolism (Haddad, 1984). Under normal circumstances, however, < 5% of se- rum VDBP is in the holoprotein form, which suggests the possibility of other functions (Haddad and Wal- gate, 1976). Several of these proposed functions involve the immune system.

The role of VDBP in the normal functioning of lymphocytes is unknown. Studies by Van Baden et al. have shown that VDBP has the ability to bind actin monomers and prevent actin polymerization (Had- dad, 1982; Van Baden et al., 1980). Additional studies have shown that VDBP is spatially associated with membrane Ig in B-cell lymphocytes (Petrini et al., 1983) and with IgG Fc receptor on the members of subpopulations of T-cell lymphocytes (Petrini et al., 1985). These studies suggest that VDBP may play a generalized role in receptor-mediated intracellular signal transduction in lymphocyte membranes.

As a first step in the analysis of the function of VDBP, we have performed a preliminary biochemical analysis of VDBP from mouse plasma. The physio- chemical properties of MVDBP were found to be similar, but not identical, to VDBP previously iso-

lated from rat plasma. The relative molecular weight of the mouse protein was found to be slightly less than that of the rat (M r = 49,000 vs Mr = 52,000). This value is also less than human VDBP (M, = 52,000-60,000), rabbit VDBP (M~ = 58,000) and chicken VDBP (M r = 52,000-60,000) (Bouillon et al., 1978). Amino acid composition analysis of MVDBP gave values close to those of RVDBP for all residues. Of the 24 aminoterminal residues of the MVDBP sequence, 21 residues, or 87.5%, were iden- tical to RVDBP. The differences at residues 17, 18 and 22 can be accounted for by a single base change in the genes that encode these proteins. A change of two bases at residue 14 (Asn-~Glu), however, is required in the respective genes. The sedimentation coefficient for MVDBP (3.8) was also very similar to the value previously published for the rat VDBP (4.1). While previous studies have shown that some VDBPs from other species contain multiple isoforms, our study demonstrates a single isoform with pI = 4.87 for MVDBP (Haddad, 1984). The MVDBP purified in our study was from a single mouse strain (C57BL/10). The possibility that additional isoforms exist in other mouse strains was not investigated. The B-50 values for binding affinity, which we report here for vitamin D 3 and five metabolites were found to be similar to the binding affinities found with rat VDBP. As with rat VDBP, vitamin D 3 sterols with 3fl-hydroxy and 25-hydroxy groups bind most efficiently and with similar affinities (25-hydroxy- vitamin D 3 ~ 24(R),25-dihydroxyvitamin D3 = 25(S), 26-dihydroxyvitamin D3). Sterols lacking the C-25 hydroxyl group bind less well (1 ~-hydroxyvitamin D 3 and vitamin D3). 1,25-Dihydroxy-vitamin D3, despite the presence of C-3 and C-25 hydroxyl groups, binds less well, presumably because of the presence of the l~-hydroxyl that might interfere with binding. Amino acid composition analysis showed a similar mole percent of each residue in the rat and mouse proteins.

Mouse lymphocytes have been extensively used for the study of mechanisms of immune system function. Studies have suggested that VDBP may play a role in the function of both B-cell and T-cell lymphocytes (Petrini et al., 1983, 1985). In this study, we have purified and characterized VDBP from mouse plasma; this protein should serve as a useful tool for further studies of VDBP-lymphocyte interaction.

Vitamin D-binding protein from mouse plasma 1349

Acknowledgements--James L. Borke was supported by a NIH Training Grant AM-07013. This work was supported by a NIH Grant DK-25409.

REFERENCES

Bidlingmeyer B. A., Cohen S. A. and Tarvin T. L. (1984) Rapid analysis of amino acids using pre-column deri- vatization. J. Chromat. 336, 93-104.

Botham K. M., Ghazarian J. G., Kream B. E. and DeLuca H. F. (1976) Isolation of an inhibitor of 25-hydroxy- vitamin D3-l-hydroxylase from rat serum. Biochemisry 15, 2130-2135.

Bouillon R., Van Baelen H., Rombants W. and DeMoor P. (1978) The isolation and characterization of the vitamin D-binding protein from rat serum. J. biol. Chem. 253, 4426-4431.

Bouillon R., Van Baelen H., Tan B. K. and DeMoor P. (1980) The isolation and characterization of the 25-hydroxyvitamin D-binding protein from chick serum. J. biol. Chem. 255, 10925 10930.

Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248 254.

Daiger S. P., Brewton G. W., Rios A. A., Mansell P. and Reuben J. M. (1987) Letters to the Editor. New Engl. J. Med. 317, 631~532.

DeLange G. G., VanEede P. H., TenVeen J. H., Reesink H. W. and Lelie P. N. (1987) Letters to the Editor. Lancet 2, 282-283.

Eales L. J., Nye K. E., Parker J. M., Weber J. N., Forster S. M., Harris J. R. W. and Pinching A. J. (1987) Association of different allelic forms of group specific component with susceptibility to and clinical man- ifestation of human immunodeficiency virus infection. Lancet 1, 999-1002.

Ehresmann B., Imbault P. and Weil J. H. (1973) Spec- trophotometric determination of protein concentration in cell extracts containing tRNA's and rRNA's. Analyt. Biochem. 54, 454-463.

Gilles K., Lonie L., Newman B., Crandall J. and King M. (1987) Letters to the Editor. New Engl. J. Med. 317, 630-631.

Haddad J. G. (1982) Human serum binding protein for vitamin D and its metabolites (DBP): evidence that actin is the DBP binding component in human skeletal muscle. Archs Biochem. Biophys. 213, 538-544.

Haddad J. G. (1984) Vitamin D: Basic and Clinical Aspects, pp. 383-395. Martinus Nijhoff, Boston.

Haddad J. G. and Walgate J. (1976) 25-Hydroxyvitamin D

transport in human plasma: isolation and partial charac- terization of calcifidol binding protein. J. biol. Chem. 251, 4803-4809.

Haddad J. G., Kowalski M. A. and Sanger J. W. (1984) Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins). Biochem. J. 218, 805-810.

Hewick R. M., Hunkapiller M. W., Hood L. E. and Dreyer W. J. (1981) A gas-liquid solid phase peptide and protein sequenator. J. biol. Chem. 256, 7990-7997.

Hunkapiller M. W. and Hood L. E. (1978) Direct micro- sequence analysis of polypeptides using an improved sequenator, a nonprotein carrier (polybrene), and high pressure liquid chromatography. Biochemistry 17, 2124-2133.

ltoh K. and Kumagai K. (1980) Effect of tunicamycin and neuraminidase on the expression of Fc-IgM and IgG receptors on human lymphocytes. J. Immunol. 124, 1830-1836.

Kumar R., Cohen W. R., Silva P. and Epstein F. H. (1979) Elevated 1,25-dihydroxyvitamin D plasma levels in nor- mal human pregnancy and lactation. J. din. Invest. 63, 342-344.

Laemmli U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature, Lond. 227, 6804585.

Litwiller R., Fass D. and Kumar R. (1986) The amino acid sequence of the NH2-terminal portion of rat and human vitamin D binding protein: evidence for a high degree of homology between rat and human vitamin D binding protein. Life Sci. 38, 2179-2184.

Lydyard P. M. and Fanger M. W. (1982) Characteristics and function of Fc receptors on human lymphocytes. Immunology 47, 1-17.

Moretta L., Mingari M. C. Moretta A. and Fauci A. S. (1982) Human lymphocyte surface markers. Seminars Hemat. 19, 273-284.

Petrini M., Emerson D. C. and Galbraith R. M. (1983) Linkage between surface immunoglobulin and cyto- skeleton of B lymphocytes may involve Gc protein. Nature 306, 73.

Petrini M., Galbraith R. M., Emerson D. L., Nel A. E. and Arnaud P. (1985) Structural studies of T lymphocyte Fc receptors. J. biol. Chem. 260, 1804-1810.

Tarr G. E. (1981) Rapid separation of amino acid phenyl- thiohydantoins by isocratic high-performance liquid chromatography. Analyt. Biochem. 111, 27-32.

Thymann M., Dickmeiss E., Svejgaard A., Pedersen C., Bygbjerg I. and Faber V. (1987) Letters to the Editor. Lancet 1, 1378.

Van Baelen H., Bouillon R. and DeMoor P. (1980) Vitamin D-binding protein (Gc-globulin) binds actin. J. biol. Chem. 255, 2270-2272.