468
BIOCHEMICAL SOCIETY TRANSACTIONS
We thank the Muscular Dystrophy Group of Great Britain for
financial support, Mike Wallis for advice and help with antisera and
immunoassays,and Robin Cole for providing laboratory facilities.
Morris, G. E. & Cole, R. J. (1972) Exp. CellRes. 75, 191-199
Morris, G. E. & Cole, R. J. (1979) Deu. Biol. 69, 146-158
Morris, G. E., Piper, M. & Cole, R. J. (1976) Nature (London) 263,
Bickerstaff, G. F.& Price, N. C. (1976) FEBS Lerr. 64, 3 19-322
Davis, B. J . (1964) Ann. N.Y. Acad. Sci. 121,404427
Eppenberger, H. M., Dawson, D. M. & Kaplan, N. 0.(1967) J. Biol.
Chem. 242.204-209
Pennington,R. J. T. (1980)Br. Med. Bull. 36, 123-126
Turner, D. C., Gmur, R., Lebherz, H. G., Siegrist, M., Wallimann, T. &
Eppenberger, H. M. (1976) Deu. Biol. 48, 284-307
76-77
Golgi vesicles isolated from rat mammary tissue contain endogenous caseins and 0.1 mM free
calcium
DAVID W. WEST and ROGER A. CLEGG
Department of Biochemistry, The Hannah Research Institute,
Ayr KA6 5HL, Scotland, U.K.
Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis
and quantitative radioautography indicate that when Golgi
vesicles, isolated from lactating rat mammary glands, are
incubated at 0°C in iso-osmotic medium with [y-32PIATP,four
of the proteins present in the preparation are phosphorylated.
One of these is only transiently phosphorylated, but the amount
of phosphorus incorporated into the other three increases
linearly with time over the period studied (0-2min). These latter
proteins are the caseins of rat milk and have been identified on
the basis of identical electrophoretic behaviour with authentic
rat caseins (both native and iodinated) in two different gel
systems. Caseins purified from rat milk, when added to the
Golgi-vesicle incubation in iso-osmotic medium, incorporate no
j2P, although they do become phosphorylated when Triton
X-100 (0.05%) is added. Thus both the caseins endogenous to
the preparation and the kinase responsible for their phosphorylation are enclosed by a permeability barrier within the vesicles.
The properties of the casein kinase of rat mammary gland
have been studied in solubilized Golgi-membrane preparations,
and it is known that casein phosphorylation is Ca2+-dependent
(Bingham & Farrell, 1974; Mackinlay et al., 1977). Furthermore, the Golgi-vesicle membrane is known to contain an
ATP-dependent Ca*+-transport system (West, 1980, 1981).
Thus, although Ca2+ will traverse the membrane (from outside
to inside) when supplied with an energy source, the membrane is
normally impermeable to Ca2+. Therefore the rate of phosphorylation of the endogenous caseins that is observed on
incubation of Golgi vesicles in iso-osmotic medium without
addition of extra Ca2+ suggests that the intravesicular CaZ+
concentration is high enough to support activity of the kinase.
Although the total calcium content (free plus sequestered) of
the vesicles may be determined by atomic-absorption spectroscopy, it was expected that the Ca2+-dependenceof the casein
kinase would provide a method of estimating the intravesicular
free CaZ+concentration. This could be achieved by comparing
the rate of casein phosphorylation in the isolated vesicles against
an experimentally determined curve relating casein phosphorylation to intravesicular Ca2+concentration.
Addition of the ionophore A23 187 allows equilibration of
Ca2+ across normally Ca2+-impermeablebiological membranes.
Therefore, by including ionophore A23 187 in incubations of
Golgi vesicles with [y-32PIATP, and by manipulating CaZt
concentration by using Ca2+-EGTA buffers, it was possible to
determine the quantitative relationship between intravesicular
free CaZt concentration and endogenous casein phosphorylation. The results of such a calibration are shown in Fig.
1. Control experiments (results not shown) have demonstrated
that endogenous casein phosphorylation, under these conditions,
proceeds at a linear rate. Thus rates of phosphorylation are
represented by the single time-point values in Fig. 1. The rate of
~
9
~~
8
7
6
5
4
3
-log [concn. of CaZ+(M)I
Fig. 1. Calibration curve: casein phosphorylation as a function
of Ca2+concentration within rat mammary Golgi vesicles
32P incorporation into the 42000-mol.wt. rat casein was
measured after incubation of Golgi vesicles (42pg of protein) for
40s in a medium containing 250m~-lactose, 5 0 m ~ - 2 -12(
hydroxy - 1 , l - bis(hydroxymethy1)ethyllamino Iethanesulphonic
acid (Tes), pH 7.0, ~ P M [ ~ - ~ ’ P I A (3.5
T P Ci/mmol), 9pM-iOnOphore A23187 and Ca2+ as indicated. Arrows denote extent of
casein phosphorylation under other conditions: 1, as above but
without Ca2+ or ionophore A23 187: 2, as 1 but with ionophore
A23 187.
casein phosphorylation measured in the absence of both
ionophore A23187 and added Ca2+ (arrow 1, Fig. 1) corresponds to a free Ca2+ concentration of about O . l m ~ .We
conclude that this is the Ca2+ concentration within the Golgi
vesicles: the value is much greater than that in the extravesicular aqueous phase. This conclusion is supported by the
observed response of casein phosphorylation on addition of
ionophore A23 187 to vesicles incubated without added Ca2+
(arrow 2, Fig. l), i.e. phosphorylation diminished to a value
which indicated that ionophore A23187 had lowered the intravesicular CaZt concentration to around 3 0 ~ ~ .
Thus, although these Golgi vesicles take up ‘ T a 2 + when
incubated in micromolar concentrations of this cation, results of
such uptake experiments must be interpreted with caution, since
measurement of the true initial rate of Ca2+ translocation is
precluded by the pre-existence of a Ca2+concentration gradient
across the limiting membrane of the vesicles.
1981
595th MEETING, MANCHESTER
We thank Mrs. Anne Shields for skilled technical assistance.
469
Mackinlay, A. G., West, D. W. & Manson, W. (1977) Eur. J. Biochem.
76,233-243
Bingham, E. W. & Farrell, H. M., Jr. (1974) J. B i d . Chem. 249,
3647-365 1
West, D. W. (1980)Biochem. SOC.Trans. 8,337-338
West, D. W. (1981) Biochim. Biophys. Acta 673,374-386
Insulin binding to rat mammary-gland Golgi membranes
DAVID J. FLINT* and DAVID W. WEST?
Departments of *Physiology and t Biochemistry, The Hannah
Research Institute, Ayr KA6 5HL, Scotland, U.K.
In recent years binding sites for insulin have been detected in a
number of subcellular organelles, including microsomal fractions
(Kahn et al., 1973: Horvat et al., 1975), nuclei (Goldfine &
Smith, 1976) and Golgi fractions (Posner et a[., 1978). The
Golgi fractions obtained from rat liver have been the subject of a
more extensive investigation, and it has been demonstrated that
insulin injected into the portal vein of rats concentrates in an
intact form in highly purified Golgi fractions (Posner, et al.,
1980). These observations raise the question as to whether these
insulin-binding sites are intracellular precursors for plasmalemma1 receptors, arising from the Golgi membranes during
secretion of proteins by exocytosis, or whether they represent
points within the cell at which hormonal action can be effected.
Insulin is one of the major hormones controlling mammary
cell function, and as part of a continuing study of the properties
of Golgi membranes of the lactating rat mammary gland (West,
198 1: West & Clegg, 1980), we have investigated the binding of
insulin to these membranes. Specific insulin binding was
determined by using '251-labelled insulin after subtraction of
non-specific binding, which was measured in the presence of
unlabelled insulin (30pg/ml). Maximum specific binding was
obtained after 18h at 4OC, and exhibited a sharp pH optimum
at pH 7.8-8.0. Somatotropin (growth hormone), prolactin,
thyrotropin and glucagon failed to compete with '251-labelled
insulin for binding to the Golgi membranes. Scatchard analysis
revealed curvilinear plots which indicate either negative cooperativity or multiple classes of binding sites. Insulin binding
was unaffected by incubation in a hypo-osmotic medium or by
freeze-thawing (four times) in iso-osmotic medium, whereas
EGTA ( 2 m ~ )and CaCI, ( 1 . 5 m ~ )plus EGTA ( 2 m ~ )decreased specific insulin binding by 15% and 42% respectively.
One interesting feature of the binding experiments was the
observation that the binding determined in iso-osmotic medium
was not increased when hypo-osmotic medium was employed. If
binding sites on Golgi vesicles are precursors of plasmalemmal
receptors, it would be expected that the binding sites would be
situated on the internal membrane surface. Thus, lysing the
vesicles should have markedly increased the number of acessible
binding sites. The observed result suggests that the receptors are
in fact on the exterior (i.e. cytosolic) membrane surface and act
as intracellular sites of hormone action rather than as part of the
biogenesis of plasmalemmal proteins.
No physiological function can as yet be ascribed to these
subcellular insulin receptors. The Golgi vesicles contain the
lactose-synthesizing system of the mammary-gland secretory
cell (Kuhn & White, 1975). As part of this process, glucose is
transported across the membrane, where it reacts with UDPgalactose inside the vesicle to form lactose. A study of the
formation of lactose in the isolated Golgi vesicles has failed to
demonstrate any detectable influence of insulin in this process.
Further experimentation to determine a physiological function
for the insulin receptors on the Golgi membranes is required.
Goldfine, I. D. & Smith, G. J. (1976) Proc. Nut/. Acad. Sci. U.S.A. 73,
1427-1431
Horvat, A., Li, E. & Katsoyannis, P. G. (1975) Biochim. Biophys. Acia
382,609-620
Kahn, C . R., Neville, D. M., Jr. & Roth. J. (1973)J. B i d . Chem. 248.
244-250
Khun, N. J. & White, A. (1975) Biochem. J . 148,77-84
Posner, B. I., Josefsberg, Z. & Bergeron, J. J. M. ( I 978) J. B i d . Chem.
253,40674073
Posner. B. I., Patel, B., Verma, A. K. & Bergeron. J. J. M. (1980) J .
Biol. Chem. 255,735-74 1
West, D. W. (1981) Biochim. Biophys. Acta 673,374-386
West, D. W. & Clegg, R. A. (1980) Biochem. Soc. Trans. 8.525-526
Stimulation by 1,25-dihydroxycholecalciferolof the synthesis of a membrane-bound protein in
the chick small intestine
Furthermore, CaBP is still present in the cell in high concentrations long after Ca2+ transport has declined to basal values
(Spencer et al., 1976). In the present paper we describe
experiments which identify another protein whose synthesis is
also stimulated by 1,25(OH),D,, is membrane-bound and which
is produced coincidentally with the stimulation of Ca2+
The steroid hormone 1,25(OH),D,* stimulates the absorption of transport.
dietary CaZ+ by the small intestine of animals. However, the
Mitochondria from a wide variety of tissues can accumulate
mechanism by which this is achieved is far from clear. It has large amounts of Ca2+ (Lehninger et al., 1967) and have been
long been known that a soluble calcium binding protein (CaBP) suspected for many years of afi involvement in CaZ+transport
is produced in large amounts in intestinal cells after adminis- across intestinal cells (Omdahl & DeLuca, 1973). We have
tration of 1,25(OH),D, in vivo (Wasserman et al., 1974). CaBP looked for any changes in the protein composition of intestinal
does not, however, appear to have a direct effect on Ca2+ mitochondria after administration of 1,25(OH),D, in viuo. At
transport, since it is not synthesized until after Ca2+transport various times after administration of 125ng of 1,25(OH),D, to
has been stimulated by 1,25(OH),D, (Spencer et al., 1976). rachitic chicks, their everted jejuna were sliced and slices
* Abbreviations: I .25(OH),D,. I .25-DihydroxycholecaIciferol: incubated in Krebs Improved Ringer 1 medium (Dawson, 1969)
CaBP, calcium-binding protein.
with 2 5 p C i of fU-'4Clleucine. Mitochondria were subsequently
ADRIAN N. HOBDEN, MARILYN HARDING and D.
ERIC M. LAWSON
Dunn Nutritional Laboratory, University of Cambridge and
Medical Research Council, Milton Road, Cambridge CB4 IXJ,
U.K.
VOl. 9