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Danpure, unpublished work), then the dissociation constant (K2) = [aurothiomalate] [albumin]/[gold-albumin complex][thiomalate]. In the present study, the rate of interaction of aurothiomalate and albumin was much lower than would be expected from the apparent dissociation constant. Preliminary evidence (C. J. Danpure & P. J. Sadler, unpublished work) suggests that in 0.15~-NaCl aurothiomalate polymerizes. If the interaction between aurothiomalate and albumin involves only the drug monomer, then the overall rate of reaction may depend on the rate of depolymerizationof the drug, which may be slow. If, therefore, the concentration of the unbound monomer is small compared with the concentration of the polymer, then the estimation of the free drug will be too large, resulting in an even lower true dissociation constant than found in the present study. Carnpion, D. S.,Olsen, R.,Bohan, A. &Bluestone, R. (1974)J. Rheumatol. 1, Suppl. 1,112 Danpure, C. J. (1974) Biochem. SOC.Trans. 2,899-901 Dournas, B. T., Watson, W. A. & Biggs, H. G. (1971) Clin. Chim. Actu 31,87-96 Gerber, D. A. (1964) J. Phurmacol. Exp. Ther. 143,137-140 King, T . P. (1961) J. Bid. Chem. 236, PC5 Kolthoff, I. M., Anastasi, A., Stricks, W., Tan, B. H. & Deshrnukh, G. S. (1957) J. Am. Chem. SOC.79,5102-5110

Mascarenhas, B. R., Granda, J. L. & Freyberg, R. M. (1972) Arthritis Rheum. 15, 391-402 McQueen, E. G. & Dykes, P. W. (1969) Ann. Rheum. Dis.28,437-442 Scatchard, 0. (1949) Ann. N. Y. Acad. Sci. 51,660-672

The Degradation of Porcine Neurophysin I by Alkaline Conditions DAVID EDGAR and DEREK B. HOPE University Department ofPharmacology, South Parks Road, Oxford OX1 3 QT, U.K.

In addition to the three characterized neurophysins of both bovine (Rauch et al., 1969) and porcine (Uttenthal & Hope, 1970) posterior pituitary glands, several minor components exist which are resolvable by disc-gel electrophoresis, by using the method of Pickup et al. (1973). We noted, however, that the two most significant porcine minor components had electrophoretic mobilities greater than that of the fastest-migrating major neurophysin, porcine neurophysin I. Further, the relative proportion of minor to major components was not constant, varying from preparation to preparation. Those samples with the greatest proportion of fast-migrating components had previously been extensively exposed to mildly alkaline solutions during their purification. We therefore examined, in detail, the consequences of exposure of porcine neurophysin to alkaline conditions. Neurophysin was dissolved in pH8.1 buffer [0.06% (w/v) Tris/O.29 % (w/v) glycine in 0.5mmolof HCl/litre], to give a final concentration of 1mg of porcine neurophysin I/ml. The solution was left at room temperaturefor 4 days, samples being removed for electrophoretic examination throughout this time. The relative proportions of two fast-migrating minor components were found to increase with time of exposure to the buffer. To determine if the high mobility of these minor components was a consequence either of their being smaller, or of their possessing a greater net charge than neurophysin I, the solution (after 4 days exposure to alkaline buffer) was electrophoresed on disc gels of polyacrylamide concentration ranging from 4% to 10% (w/v). The mobility data were plotted as described by Ferguson (1964), to show an exponential relationship between the relative mobility (R,) of each component and the gel concentration (Fig. 1). The slope of such a plot is a function of the apparent molecular weight of the component (Rodbard & Chrambach, 1971), hence the Ferguson plot for neurophysin I1has a steeper gradient than those of neurophysins I and 111, reflecting its greater molecular weight (Uttenthal & Hope, 1970). However, as the plots of both minor components and neurophysin I are parallel, then these must have identical apparent molecular weights.

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r

-

s

0.8

-

0.6

-

X

9

-8

- --o *-----@--a-*------0 4

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10

Acrylamide concentration (%) Fig. 1. Fergusonplot showing effect of acrylamide concentration on relative mobility (R,) of the multiple components of porcine neurophysin A, A, Fast-running minor components; 0, pig neurophysin I ; 0 , pig neurophysin 11;

pig neurophysin 111.The slope for each component was determined with the aid of a least-squares computer program. @,

The high mobility of the minor components must therefore be a consequence of their possessing a higher charge than neurophysin I under the conditions of the electrophoresis. Alkali-catalysed deamidation of glutamine or asparagine residues of human growth hormone and bovine prolactin has been described (Cheever & Lewis, 1969). Species of higher electrophoretic mobility, but withno apparent decrease in molecular weight, were produced. It is probable that such a deamidation (with consequent increase in charge) occurs with porcine neurophysin I, to give rise to the fast-migrating minor components. No degradation of porcine neurophysins I1 or I11was detected after treatment under the same incubation conditions. It is noteworthy that porcine neurophysin I is also susceptible to enzymic degradation, resulting in the loss of the C-terminal leucine residue (Wuu et al., 1971). The cause of the relative instability of neurophysin I in alkaline solution, together with any connexion between this and the molecule’s susceptibility to enzymic degradation, is not known. Minor components of bovine neurophysin have been described by Vilhardt & Robinson (1975). Although the porcine and bovine minor components are similar in possessing the ability to bind to an affinity-chromatography column of agarossvasopressin, they differ in that the bovine minor components are present in neurosecretory granules prepared from fresh tissue. It i s therefore unlikely that an alkali-catalysed degradation process, which results in the production of rapidly migrating porcine minor components in uitro, is responsible for the production of other minor components of neurophysin in viuo. This work was supported by a Medical Research Council grant. D. H. E. is a Christopher Welch Scholar. Cheever, E. V. & Lewis, U. J. (1969) Endocrinology 85,465-473 Ferguson, K. A. (1964) Metab. Clin. Exp. 13, 985-1002 Pickup, J. C., Johnston, C. I., Nakamura, S.,Uttenthal, L. 0. &Hope, D. B. (1973) Biochern.J. 132,361-371

Rauch, R., Hollenberg, M. D. & Hope, D. B. (1969) Biochern. J. 115,473479 Rodbard, D. & Chrambach, A. (1971) Anal. Biochern. 40,95-134 1976

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Uttenthal, L. 0. & Hope, D. B. (1970) Biochem. J. 116,899-909 Vilhardt, H. & Robinson, I. C. A. F. (1975) J. Neurochem. 24, 1275-1276 Wuu, T. C., Crumm, S. & Saffran, M. (1971) J. Biol. Chem. 246, 6043-6063

Solubilization and Peptide ‘Mapping’ of a Large External Glycoprotein Fraction Labelled by Lactoperoxidase-CatalysedIodination of Cultured Fibroblasts RODERICK NAIRN and R. COLIN HUGHES

National Institute for Medical Research, Mill Hill, London NW7 IAA, U.K. A glycoprotein(s) of apparent molecular weight 220000-250000 can be labelled by lactoperoxidase-catalysed iodination of fibroblasts from a variety of species (Hynes, 1973;Hogg, 1974; Pearlstein & Waterfield, 1974; Yamada & Weston, 1974). The glycoprotein of hamster fibroblasts appears at the cell surface during interphase and is lost from the surface at mitosis (Hynes &Bye, 1974). Further, the glycoprotein is absent from the surface of virally transformed derivatives of hamster, chick, human and mouse fibroblasts (Hynes, 1974) and has been termed LETS (large external transformationsensitive) glycoprotein (Hynes & Bye, 1974). There are exceptions to this correlation, however (Meager et al., 1975). At present no function has been defined for the LETS glycoprotein, although its external location, apparently in a fraction related to the cell coat or glycocalyx (Graham et al., 1975), and relativity to growth state of the cells suggest a possible involvement in cellular processes mediated via the cell surface. To study further the structure and function of the LETS glycoprotein it is necessary to isolate the material in pure form. In this report we describe the solubilization characteristics of LETS glycoprotein and also show that extensive differences exist in the primary structures of the LETS glycoproteins obtained from chick or hamster fibroblasts. Baby-hamster kidney BHK21 C13 cells, hamster NIL 8 cells, primary chick-embryo fibroblasts and primary human skin fibroblasts were grown at 37°C in Glasgow-modified minimal essential medium supplemented with 10% (v/v) foetal bovine serum, 10% (v/v) tryptose phosphate broth,0.2 %(w/v) NaHC03, penicillin(0.1 M-i.u./l) andstreptomycin (0.1 g/l). Confluent monolayers of cells were labelled on 6Omm Falcon plastic culture dishes with a mixture of lactoperoxidase (Sigma Chemical Co., London S.W.6, U.K.) glucose oxidase (type V, Sigma) and carrier-free NalZSI(The Radiochemical Centre, Amersham, Bucks., U.K.) as described by Meager et al. (1975). After labelling, the cell sheets were washed with phosphate-buffered saline, pH7.4, scraped off with arubber policeman and finally dissolved in 1%sodium dodecyl sulphate/ 1 % 2-mercaptoethanol by heating at 90°C for 5 min. Samples were analysed on sodium dodecyl sulphate/polyacrylamide gels by the discontinuous gel system described by Maize1 (1971). After resolution the gels were sliced into 2mm slices and counted directly for radioactive iodine associated with protein bands. As a routine the separations show a slow-moving major iodinated band (LETS glycoprotein) which is well separated from other faster-moving labelled bands. The LETS glycoprotein of BHK cells is not extracted into solution by either 0.5 % Nonidet (NP-40) (Fig. 1) or 1% sodium deoxycholate. Extractions were carried out for up to 45min at 4°C. Notably, the other iodinated bands were readily solubilized under these conditions. These findings strongly suggest that the LETS glycoprotein is differently organized within the cell surface structure and is not released, as are the integral components, after extensive membrane disassembly with detergents. Similar behaviour has been described for non-integral membrane components such as spectrin (Steck, 1974). However, the LETS glycoprotein, unlike extrinsic components of the erythrocyte membrane, is also resistant to extraction with 0.2 % EDTA (results not shown). Under these conditions essentially no iodinated components are made soluble. The LETS

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