The isolation and characterization of serum albumin from the ostrich (Struthio camelus)

Comp. Biochem. Physiol. Vol. 82B, No. 4, pp. 829 835, 1985

0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd

Printed in Great Britain

THE ISOLATION A N D CHARACTERIZATION OF SERUM A L B U M I N F R O M THE OSTRICH (STRUTHIO CAMELUS) G. BRADLEY, R. J. NAUD~* and W. OELOrSEN Department of Biochemistry, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, Republic of South Africa (Tel: 5311-928) (Received 10 April 1985)

Abstract--1. Ostrich serum albumin (OsSA) was purified by a combination of heat fractionation and

polyethylene glycol precipitation. 2. Equilibrium centrifugation revealed a relative molecular mass of 71,666 for the purified monomer, whereas the presence of a dimeric form was confirmed by means of PAGE and SDS-PAGE analysis. 3. Compared to other species, relatively high levels of proline, glycine, isoleucine and histidine together with lowered amounts of half cystine, phenylalanine and arginine were observed in OsSA. A single N-terminal aspartic acid was identified. 4. Isolated chicken adipocytes revealed a significantly lower in vitro lipolytic responsiveness towards added glucagon when OsSA replaced bovine serum albumin (BSA) in the medium (Kin = 6.359 and 1.135 nM, V,, = 36.70 and 46.72 nmol/hr/#g adipocyte DNA for OsSA and BSA respectively).

INTRODUCTION

MATERIALS AND METHODS

Serum albumin is the most plentiful and the most familiar plasma protein. As the simplest to prepare in a relatively pure form, it has been known for over a century and has been isolated in pure form from a variety of species including human, ox and rat (see Peters, 1975, for a review). The molecule is a single peptide chain of about 580 residues, of which the sequence of amino acids is known for bovine (Brown, 1974) and human (Behrens et al., 1975) serum albumins. Brown has also proposed a model for the primary structure based on its repeating Cys-Cys sequence, which appears to explain many features observed in physicochemical studies. Unlike other plasma proteins which tend to have a single specific function, albumin has been assigned numerous physiological roles (Peters, 1975). It is the principle agent responsible for the osmotic pressure of the blood, for transport of fatty acids and for sequestration and transport of bilirubin. Other functions, less well defined, include conveyance of tryptophan, cystine and various hormones including thyroxine and steroids. Serum albumin also has a nutritive role as a reservoir of amino acids for peripheral tissues. Apart from the earlier reports by Peters et al. (1958) and Schjeide (1963), few if any detailed accounts appear to be available on avian serum albumins. In the present study the isolation and purification of ostrich serum albumin (OsSA) by the rapid and convenient heat-polyethylene glycol (PEG) procedure of Schneider et al. (1975) is reported. The PEG-albumin fraction is evaluated physically and chemically and its behaviour in vitro as a constituent of an isolated avian adipocyte incubation medium has been studied.

*To whom correspondence should be addressed. 829

Chemicals

All standard chemicals obtained from commercial sources were of analytical reagent grade and were used without further purification. Glucagon (crystalline) was obtained from Sigma Chemical Co. Krebs-Ringer bicarbonate buffer, pH 7.4 (KRB) was freshly prepared from cold stock solutions before each experiment and was gassed for at least l0 rain with 95% 02-5% CO2 before adding 4g/100 ml of either bovine serum albumin, fraction V (BSA), obtained from Miles Research Division, Cape Town, or OsSA, isolated in the present study. Both proteins, before being used, were finally purified according to the procedure of Ramachandran et aL (1972). For adipose tissue digestion these buffers were used as such (digestion buffer) but for incubation of adipocytes (incubation buffer), 0.01% Lima bean trypsin inhibitor (Sigma Chemical Co.) was added as well. All BSA and OsSA containing buffers were cleared by centrifugation (35,000g for 15rain) before use and contained no added glucose. Chickens

For the preparation of adipoeytes, male Cornish Game x White Rock chickens were used, 5-13 days posthatching. Serum preparation

Whole ostrich blood was collected in polypropylene bottles at the ostrich abbatoir in Oudtshoorn and was transported on ice. Upon arrival at the laboratory the blood was centrifuged without delay at 13,700 g for 10 rain at 4°C. The serum was stored at -20°C until used. Albumin isolation

The method of isolation was the heat-PEG fractionation procedure of Schneider et al. (1975). Chromatography on DEAE-Sephadex A-50

The freeze-dried PEG-albumin fraction (1 g) was submitted to ion exchange chromatography on a column (76 x 2.6 cm) of DEAE-Sephadex A-50. The column was initially eluted with 0.025 M ammonium acetate buffer (pH

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G. BRADLEYet al.

5.2) until 100 ml had been collected. The eluting buffer was then changed to 0.025 M ammonium acetate (pH 4.5). Absorbance of fractions was monitored at 280 nm.

200 mg of fraction 2 (see Results and Discussion section) was submitted to gel filtration on a column (I00 × 2.6 cm) of Sephadex G-100 with 0.05 M ammonium acetate buffer (pH 7) as eluant.

square of the radial distance (r 2) along the cell axis at each of the three concentrations of ostrich PEG-albumin. The sedimentation coefficient (s20,w) was determined from the plot of log x (displacement) vs time in seconds at each of the three concentrations. The diffusion coefficient (D20.w) was only determined at the highest PEG-albumin concentration (0.538 mg/ml) in a synthetic boundary cell and was calculated from the plot of the change in the square of radial distance (At 2) vs time (in seconds).

High Pressure Liquid Chromatography (HPLC)

Amino acid analyses

Five milligrams of fraction 2B, obtained from Sephadex G-100 chromatography, was submitted to HPLC on a Beckman model 344 CRT-based gradient liquid chromatograph, using a Waters/~-Bondapak C-18 reverse phase (RP) column (30 x 0.39 cm) with a linear gradient of npropanol (15-30% over 60min) in 0.01 M trifluoroacetic acid (TFA). Fractions (0.5 ml) were collected and monitored at 280 nm and the pooled peaks were freeze-dried.

Samples of ostrich PEG-albumin fraction were hydrolyzed in the presence of either 5.7 M HCI or 4 M methane sulfonic acid in evacuated pyrex tubes for 20 hr at 100°C. With HC1, 10#1 of 5% (w/v) phenol was also added to protect tyrosine. Hydrolysates were examined with a Beckman 118 BL amino acid analyzer according to the procedure of Spackman et al. (1958).

Chromatography on Sephadex G-IO0

Isoelectric focusing Electrofocusing was carried out on 25 mg of PEGalbumin fraction as described by Naud6 and Oelofsen (1977). Pooled fraction 3 was freed of ampholines and sucrose by filtration through a Sephadex G-25 column in 0.1 M acetic acid.

Gel electrophoresis The different albumin fractions obtained were investigated for homogeneity by means of polyacrylamide gel electrophoresis (PAGE) according to the procedure of Tamura and Ui (1972). PAGE was performed at pH 8.9 for 15 min at 2 mA/tube followed by 1~hr at 7 mA/tube. The gels (75 x 5 mm) contained 10% (w/v) acrylamide and were maintained at 20°C during electrophoresis. Protein bands were made visible by staining with 0.5% (w/v) Amido Schwarz 10B in 7% (w/v) acetic acid followed by electrophoretic destaining. The relative molecular mass (Mr) of the PEG-albumin fraction was determined by sodium dodecylsulphate (SDS) disc gel electrophoresis according to Swank and Munkres (1971), using aldolase, BSA, hen egg albumin and chymotrypsinogen A as M r standards. Preparative PAGE 100/~g of PEG-albumin fraction was applied to each of seven 10% PAGE columns and electrophoresis was performed at pH 8.9 as described above. One gel was stained using Coomassie brilliant blue G-250 as described by Braatz and Mclntire (1977). After 2rain the protein bands were visible. The six unstained gels were then aligned without delay with the stained gel and the segments corresponding to stained bands were excised with a clean razor blade. After overnight elution in water the eluates were freeze-dried. Each of the two eluted fractions were then resubmitted to PAGE under identical conditions as before, and the gels were stained with Coomassie brilliant blue G-250. Ultracentrifugation studies The relative molecular mass (M,) was determined by low speed sedimentation equilibrium centrifugation in a Beckman model L2-75B preparative ultracentrifuge fitted with a UV scanning attachment (280nm filter) and using an aluminium-filled epon double-sector cell (12 mm) with plain quartz windows in a modified An-D (An-A-P) rotor at 20"C. Ostrich PEG-albumin fraction (0.538mg/ml) in 0.02 M phosphate buffer (pH 7) containing 0.1 M KC1 was submitted to centrifugation. The experimental procedure for the determination of Mr, Szo,w and D20,wwas that described by Chervenka (1970). Each of the Mr and s20,~ determinations were also repeated at albumin concentrations of 0.333 and 0.167mg/ml. Assessment of homogeneity and calculation of M r was based on the plot of log A2s0 vs the

N-terminal analysis The N-terminal amino acid of ostrich PEG-albumin was identified by the dansyl procedure (Gray, 1967) on a polyamide sheet (Li et al., 1970). Chicken adipocyte system Chicken adipocytes were prepared by a modification of the method of Rodbell (1964). The chickens were killed by decapitation and the subcutaneous fat, including fat surrounding the gizzard, was removed without delay (1-3 g/chicken) and was placed in physiological saline. After thorough removal of adhering connective tissue the fat pads were sliced into fragments of about 1 x 2 mm. The fragments were washed twice in about 30 ml of fresh saline at 37°C before being suspended in digestion buffer (3 ml/g adipose tissue) in a 50 ml polypropylene tube to which had been added collagenase Type II from C. histolyticum (Sigma Chemical Co., 141 U/mg, 5 mg/g adipose tissue) and deoxyribonuclease 1 from bovine pancreas (Sigma Chemical Co., 0.4 mg/g adipose tissue). Digestion was executed in a gyrotory incubation bath (New Brunswick Scientific Co., Inc.) at 40°C and 100 shakes/min with intermittent mild shaking by hand at 20 min intervals. When digestion (as judged by eye) was about 80-90~o complete (55-65 min) the dispersed cells were filtered through two or three layers of cheesecloth. After floating the cells by very mild centrifugation (+ 5 g), the infranate was removed and the layer of cells was washed three times with incubation buffer (10 ml/wash), maintaining the temperature at about 37°C. The washed cells were suspended in 1.3-2.0 ml of incubation buffer/g of adipose tissue used, depending on the age of the chickens (using higher dilutions for younger birds and vice versa) and 0.4 ml aliquots of cell suspension were distributed into polypropylene incubation tubes (75 x 12 mm), each already containing 0.05 ml of incubation buffer. After triplicate addition of 0.1 ml aliquots of freshly prepared hormonal test solutions in incubation buffer, the tubes were incubated in the gyrotory shaker at 80 shakes/min for 60 min at 40°C in an atmosphere of 95~o O2-5~o CO2. The control tubes received 0.1 ml of incubation buffer instead of hormonal test solution and were incubated along with the hormone treated cells. At the end of incubation the infranates were analysed for glycerol content according to a modification of the procedure of Korn (1955). Samples were prepared for the assay by adding 0.1 ml of distilled water, followed by 0.4 ml of cold 10% (w/v) trichloroacetic acid (TCA) solution to 0.2 ml of infranate. After vortexing, the precipitate was spun down (5000g for 10 min) and the assay was performed on 0.5 ml of the clear supernatant solution as described by Oelofsen and Ramachandran (I 983). For the determination of DNA the layer of cells in each incubation tube, after removal of the infranates for glycerol determination, was freed of lipid and the DNA was extracted by a modification

Ostrich serum albumin

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Tube number Fig. 1. DEAE-Sephadex A-50 chromatography of PEG-albumin fraction. The column (76 × 2.6 cm) was eluted at 40 ml/hr with 0.025 M ammonium acetate (pH 5.2). After 20 fractions (5 ml) were collected, elution was continued with 0.025 M ammonium acetate (pH 4.5). Fractions were monitored for A280

(

of the method used by Langslow and Lewis (1972). The content of each incubation tube was thoroughly dispersed in a small Potter homogenizer using a total volume of 6 ml of a chloroform:methanol (2:1) mixture, which also contained 10% (w/v) TCA. The resultant fine suspension was centrifuged at 3000g for 20 min and the clear supematant was decanted off carefully. The remaining small pellets were washed once with 4ml of a chloroform:methanol (2:1) mixture. To each washed pellet was added 1 ml of a 5% TCA solution in water and the tubes were then incubated in a waterbath at 72°C for 25 rain with constant shaking along with a standard solution of calf thymus DNA (Miles Research Products, Cape Town), which was suitably diluted in 5% TCA for subsequent calibration of the assay. After cooling the samples in cold tap water the DNA content of each tube, including a series of tubes containing l ml of different concentrations of standard DNA solution, was determined according to the procedure of Ceriotti (1952) as modified by Short et al. (1968). All results were expressed in terms of net glycerol production (nmol) per hr per /zg of DNA at the different hormone concentrations used. The data were analyzed using the BMDPAR derivative-free nonlinear regression program (1981 revised version), developed by the Dept. of Biomathematics, University of California, Los Angeles and as converted for use on Burroughs large systems computers by the Computer Unit, University of Warwick, Coventry CV4 7AL, U.K.

).

PEG-albumin fraction. The only difference noted was that after R P - H P L C the slower moving band seemed to have increased in intensity relative to the faster component to the extent that it now appeared comparable to the faster band. Furthermore during the Sephadex G-100 and R P - H P L C steps a third minor component moving very slowly in the P A G E system and absent in the original PEG-albumin fraction, also made its appearance. A further attempt to fractionate the PEG-albumin fraction on Biogel P-150, using 0.025 M a m m o n i u m acetate (pH 7) as eluant, once again produced a single peak (profile not shown), the P A G E pattern of which was identical to that of the starting material, except for an increase in intensity of the slower component relative to that of the faster band. The PEG-albumin fraction was isoelectrically homogeneous giving a pHl value of 5.2 (Fig. 3) as compared to a value of 4.7 for both BSA and H S A (Peters, 1975). Twelve milligrams of fraction 3 were obtained and once again on P A G E revealed the presence of the same two major components as before, as well as some additional minor slower components which were absent in the PEG-albumin fraction.

RESULTS AND DISCUSSION The P E G isolation procedure yielded 21 g of purified OsSA (PEG-albumin fraction) per liter of serum used. P A G E analysis of this procedure revealed one major fast moving band (which corresponded in mobility to standard BSA), together with a much slower moving component. When the P E G albumin fraction was submitted to further purification steps, 1 g of this material after D E A E Sephadex A-50 chromatography yielded 296 mg of the major component (fraction 2 in Fig. 1), which in turn after gel filtration of 200rag on a Sephadex G-100 column, yielded 123.4 mg of fraction 2B (Fig. 2). The latter fraction when applied to R P - H P L C appeared to partly resolve into two fractions, 2B1 and 2B2. However, P A G E analysis of all these fractions (2, 2B, 2B1 and 2B2) produced patterns which were essentially identical to that of the original

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Tube number Fig. 2. Sephadex G-100 chromatography of DEAE-Sephadex fraction 2. The column (100 x 2.6cm) was eluted at 20 ml/hr with 0.05 M ammonium acetate (pH 7). Fractions (5 ml) were monitored for A280 ( ).

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Fig. 3. Isoelectric focusing of PEG-albumin fraction in the pH range 3 10. Conditions: 25 mg protein, 5°C, 800 V for 72 hr. The eluted fractions (2 ml) were monitored for A2s0 ( © - - © ) and pH ( A - - A ) .

To determine whether the apparent heterogeneity noted for all the purified OsSA fractions was due to intermolecular association behavior, a preparative separation of the two components in the P E G albumin fraction was performed on P A G E as described by Braatz and McIntire (1977). U p o n reelectrophoresis of the eluted bands each of them once again separated into the same two components which was originally observed in the PEG-albumin fraction. The fast moving component produced the slower one in relatively minor quantity whereas the slower one produced its faster counterpart in about equal intensity upon re-electrophoresis, hence resembling the P A G E patterns observed for the R P - H P L C fractions (2B1 and 2B2). The observed apparent heterogeneity of OsSA fractions would thus seem to represent

polymeric forms of the protein. The presence of dimers and trimmers in purified serum albumin preparations has been reported previously by Bruce Edwards et al. (1969) and by Saifer et al. (1961) for human serum albumin (HSA); by Houston (1971) and by Saifer et al. (1961) for BSA and also by Saifer et al. (1961) for porcine (PSA) and rat serum albumin (RSA). Further confirmation of the apparent association behaviour of OsSA came from S D S - P A G E analysis. F r o m a comparison with standard proteins (Fig. 4) the behaviour of ostrich PEG-albumin on S D S - P A G E revealed the presence of two components with M, values of 78,500 and 149,600 corresponding respectively to the proposed m o n o m e r and dimer. The value so obtained for the proposed mono-

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Ostrich serum albumin Table 1. Centrifugation data on ostrich serum albumin at different concentrations Sample concentration (mg/ml)

M~

S~,w (Svedbergs)

02o w ( x 107 cm'2 sec i)

0.538 0.333 0.167

68819 75278 70902

4.09 3.97 4.03, 4.06

4.61, 5.53

~Using a value of 0.732 for tY, based on amino acid analysis data.

mer is also in reasonable agreement with that obtained from sedimentation equilibrium centrifugation (71,666) and from amino acid analysis (68,471). Our results seem to suggest a strong tendency of purified OsSA to form higher aggregates. Furthermore the size of the monomer appears to closely resemble that of the corresponding molecules hitherto identified (Peters, 1975) from a number of other species, including avian albumins (Peters et al., 1958). During equilibrium centrifugation the PEGalbumin fraction (at three different concentrations), gave rise to linear plots of log A280vs r 2 over the entire radial distance along the cell axis, thus lending support to the intrinsic homogeneity of the preparation. All centrifugation data are summarized in Table 1, including the determination of s20,w and D20,w. It is evident from Table 1 that s20,w and M, show little if any concentration dependence. The average values obtained were: s20,w= 4.03 x 10-13 s, M r--- 71,666 and D20.w=5.07x 10-7cm 2s-l. (s20,w=4.6 and 4.5 × 10-13s and D20.w=6.1 and 5.9 x 10-Tcm2s -l for HSA and BSA respectively (Peters, 1975)). The amino acid composition of ostrich PEGalbumin fraction compares favorably with those of the mammalian species and the fowl with respect to several amino acids including Asx, Thr, Glx, half Cys, Val, Tyr and Lys (Table 2). The two avian types are also quite similar with respect to Ser, Thr, Ala and Tyr content and they both appear to exceed the

833

mammalian types with respect to Ser, Pro, Gly, Met and lie content. OsSA, however is remarkably higher in Pro, Gly, lie and His content than all the other types, including the fowl. At the same time it appears considerably lower in half Cys, Phe and Arg content than the fowl type. These findings would seem to indicate a considerable deviation of the ostrich derived molecule even from its avian counterpart. N-terminal analysis of the PEG-albumin fraction indicated a single aspartic acid residue which is in agreement with all mammalian (Peters, 1975) and avian (Peters et al., 1958) species so far investigated. From the above physical and chemical data, we conclude that OsSA is obtainable in good yields and in a high state of purity by means of the relatively simple heat fractionation-PEG precipitation procedure of Schneider et al. (1975). In general the physical and chemical properties of the purified product appeared to closely resemble those described previously for serum albumins isolated from a variety of sources including avian species. However, certain striking differences in primary structure would seem to be implicated as well, and their full elucidation will have to await the determination of the complete amino acid sequence. We have also used our PEG-albumin fraction in an in vitro incubation system for the study of the lipolytic responsiveness of isolated chicken adipocytes to added glucagon. The results were compared with those obtained with BSA which is commonly used in such media (Fig. 5). The adipocytes appeared to be remarkably less responsive to added glucagon in the presence of 4% OsSA in the medium as compared to 4% BSA, all other factors being equal. Nonlinear regression analysis revealed a K,, value of 6.359 nM for the OsSA system as compared to 1.135nM for the BSA system, a highly significant difference (P < 0.001). It is possible that this apparent increase in the level of glucagon needed for half-maximal

Table 2. Amino acid composition (molar proportions) of serum albumin from ostrich, fowl~, human b, bovine b and rat b Amino acid residue Asp + A s n Thr Ser Glu + Gin Pro Gly Ala Half Cys Val Met lle Leu Tyr Phe His Lys Trp Arg Total number of residues

Ostrichc

Fowl e

Human

Bovine

Rat

52.1 (52) 31.3 (31) 31.3 (31) 83.3 (83) 41.7 (42) 41.7 (42) 41.7 (42) 31.3 (31) 31.3 (31) 10.4 (10) 31.3 (31) 52.1 (52) 20.8 (21) 20.8 (21 ) 20.8 (21) 52.1 (52) 1d 20.8 (21)

60 27 34 82 34 32 44 38 34 14 23 45 19 27 11 47 ND 29

54 30 22 83 25 12 63 35 39 6 8 61 18 30 16 58 I 23

54 34 28 79 28 15 46 35 36 4 14 61 19 27 17 59 2 23

51 32 24 81 30 18 62 35 33 6 14 55 21 24 14 51 1 23

600

584

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575

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aSchjeide (1963). bpeters (1975). CAverage of three determinations. aAssumed value. eExpressed as #moles/600 ,umole of amino acids. ND, not determined.

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Dose (nM) Fig. 5. Effect of two different serum albumins on the in vitro lipolytic response of chicken adipocytes to glucagon. The washed cells (approx. 7 # g adipocyte DNA/tube) were incubated in the presence of different concentrations of glucagon at 40°C for 1 hr in KRB-buffered medium (without added glucose) in the presence of 4 g/I00 ml of either BSA ([[] I-1) or OsSA (C)---O). The glycerol released was measured in the infranate and was expressed as nmol/hr//~g adipocyte DNA.

stimulation of lipolysis in the presence of OsSA as compared to BSA can be related to a subtle difference in the binding characteristics and subsequent interactions of these two proteins with the chicken adipocyte surface. In view of the recently described specificity and saturability in the binding properties of BSA for rat adipocytes and its potential functional implications (Brandes et al., 1982) such a possibility cannot be discounted. It is also possible that differences in the binding affinity of BSA as compared to OsSA for glucagon itself may affect the apparent Km values. F r o m Fig. 5 it furthermore became evident that the maximal rate of glycerol release (Vm~x) in the presence of OsSA (36.70nmol/hr//~g adipocyte D N A ) was about 22% lower than in the BSA system (46.72 n m o l / h r / p g adipocyte DNA). This difference once again was high significant (P < 0.001) and might indicate a lowered trapping efficiency of OsSA for released free fatty acids in the in vitro system which would be a result comparable to the lowering of Vmax observed in rat adipocytes when the BSA concentration was lowered from 4 to 1% in the medium (Oelofsen and Ramachandran, 1983). Further studies on the nature of the interaction between OsSA and adipocytes and the binding properties of OsSA for hormonal agents are needed to elucidate the basis of the present observations. Acknowledgements--This work has been supported by grants from the South African Council for Scienific and Industrial Research (C.S,I.R.), Pretoria and from the University of Port Elizabeth. The kind co-operation of the Klein Karoo Agricultural Co-operation at Oudtshoorn, in supplying ostrich blood, is gratefully acknowledged. REFERENCES

Behrens P. Q., Spiekerman A. M. and Brown J. R. (1975) Structure of human serum albumin. Fedn Proc. Fedn Am. Socs exp. Biol. 34, 591. Braatz J. A. and Mclntire K. R. (1977) A rapid and efficient method for the isolation of proteins from polyacrylamide gels. Prep. Biochem. 7, 495-509.

Brandes R., Ockner R. K., Weisiger R. A. and Lysenko N. (1982) Specific and saturable binding of albumin to rat adipocytes: Modulation by epinephrine and possible role in free fatty acid transfer. Biochem. biophys. Res. Commun. 105, 821-827. Brown J. R. (1974) Structure of serum albumin: Disulfide bridges. Fedn Proc. Fedn Am. Socs exp. Biol. 33, 1389. Bruce Edwards F., Rombauer R. B. and Campbell B. J. (1969) Thiol disulfide interchange reactions between serum albumin and disulfides. Biochem. Biophys. Acta 194, 234-245. Ceriotti G. (1952) A microchemical determination of deoxyribonucleic acid. J. biol. Chem. 198, 297 303. Chervenka C. H. (1970) in A Manual o f Methods" ['or Analytical Ultracentrifuge (edited by Chervenka C. H.), pp. 44-50. Spinco Division, Beckman Instruments, Palo Alto, CA. Gray W. R. (1967) Dansyl chloride procedure. Meth. Enzymol. 11, 139-151. Houston L. L. (1971) Amino acid analysis of stained bands from polyacrylamide gels. Anal. Biochem. 44, 81-88. Korn E. D. (1955) Clearing factor, a heparin-activated lipoprotein lipase. J. biol. Chem. 215, 1 14. Langslow D. R. and Lewis R. J. (1972) The compositional development of adipose tissue in Gallus domesticus. Comp. Biochem. Physiol. 43B, 681-688. Li C. H., Dixon J. S., Lo T. B., Schmidt K. D. and Pankov Y. A. (1970) Studies on pituitary lactogenic hormone XXX. The primary structure of the sheep hormone. Archs Biochem. Biophys. 141, 705 737. Naud6 R. J. and Oelofsen W. (1977) The isolation and characterization of corticotropin from the pituitary gland of the ostrich Struthio camelus. Biochem. J. 165, 519 523. Oelofsen W. and Ramachandran J. (1983) Studies of corticotropin receptors on rat adipocytes. Archs Biochem. Biophys. 225, 414-421. Peters T., Jr. (1975) The Plasma Proteins (edited by Putnam F. W.), Vol. 1, pp. 133 181. Academic Press, New York. Peters T., Logan A. C. and Sanford C. A. (1958) Terminal amino acid residues of chicken, duck and turkey serum albumins. Biochim. Biophys. Acta 30, 88-92. Ramachandran J., Lee V. and Li C. H. (1972) Stimulation of lipolysis and cyclic AMP accumulation in rabbit fat cells by human growth hormone. Biochem. biophys. Res. Commun. 48, 274-279.

Ostrich serum albumin Rodbell M. (I 964) Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239, 375-380. Saifer A., Robin M. and Ventrice M. (1961) Starch-gel electrophoresis of purified albumins. Archs Biochem Biophys. 92, 409-419. Schjeide O. A. (1963) Lipoproteins of the fowl-serum, egg and intracellular. Prog. Lipid Res. 6, 253-289. Schneider W., Lef6vre H., Fiedler H. and McCarty L. J. (1975) An alternative method of large scale plasma fractionation for the isolation of serum albumin. Blut 30, 121-134. Short E. C., Warner H. R. and Koerner J. F. (1968) The

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effect of cupric ions on the indole reaction for the determination of deoxyribonucleic acid. J. biol. Chem. 243, 3342-3344. Spackman D. H., Stein W. H. and Moore S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. Swank R. T. and Munkres K. D. (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal. Biochem. 39, 462-477. Tamura H. and Ui N. (1972) A new buffer system for disc electrophoresis. Suitable for slightly basic proteins. J. Biochem. 71, 543-545.