MONTAJE,INSTALACIÓN,PRUEBAS Y RESULTADOS.

Western blotting of glucose transporters in mammary homogenate, shown in Section 4.2, had indicated abundant expression of GLUTl and low level expression of GLUT4 at mid-lactation. The sub-cellular distribution of these two glucose transporter isoforms in mid-lactating ra t mammary gland was next investigated by using differential centrifugation to prepare mammary sub-cellular membrane fractions and Western blotting to detect the glucose transporters, coupled to analysis of membrane purity and cross-contamination of membranes by using marker enzyme activities.

Mammary fractions enriched in plasma membranes, Golgi membranes or milk fat globule membranes were prepared by sub-cellular fractionation of mammary tissue or milk according to the methods in Sections 2.3.1, 2.3.2, and 2.3.3 respectively.

4.3.1 Analysis of mammary sub-cellular membrane fractions using marker enzymes The enrichments of the plasma membrane and the Golgi membranes in the sub- cellular fractions in comparison to the homogenate were assessed using assays for the marker enzymes 5'-nucleotidase (plasma membrane marker) and galactosyltransf erase (Golgi membrane marker). Similarly the degree of contamination of plasma membranes with Golgi membranes, and vice versa, could be determined. The milk fat globule membranes were prepared directly from rat milk, and therefore were not contaminated with plasma membranes or Golgi membranes from the epithelial cells.

Plasma membranes prepared as described in Section 2.3.1 were routinely enriched in 5’-nucleotidase activity by approximately 24-fold compared to the activity of the

original homogenate. However, plasma membranes also contained on average 7-fold higher galactosyltransf erase activity per mg protein than the homogenate, which indicated there was some contamination of the plasma membrane preparation with the Golgi membrane marker enzyme.

Golgi membranes prepared as described in Section 2.3.2 were routinely found to be enriched 13-fold with galactosyltransf erase activity compared to the original homogenate, but also contained an approximately 7-fold enrichment over homogenate with the plasma membrane marker. There was therefore a substantial contamination of the Golgi membranes with plasma membranes. However, the activity of 5’-nucleotidase in the Golgi membrane fraction («650 pmol adenosine/min/mg protein) was substantially less than the activity of 5'- nucleotidase in the plasma membrane preparation («2500 pmol adenosine/min/mg protein), therefore the Golgi membrane fraction contained less than 30% of the 5'nucleotidase activity of the plasma membrane fraction, a fact which becomes relevant in Section 4.3.2, in relation to the sub-cellular distribution of glucose transporters.

4.3.2 Sub-cellular distribution of GLUTl and GLUT4 in mammary tissue

The glucose transporter contents of mammary plasma membranes (lOpg), Golgi membranes (lOpg) and milk fat globule membranes (20pg), were investigated by Western blotting using the methods described in Sections 2.5 and 2.6. Following electrophoresis on 12% SDS/polyacrylamide gels, the proteins were transferred to nitrocellulose and GLUTl or GLUT4 detected using antibodies raised against their respective C-terminal peptides, an iodinated second antibody and autoradiography.

membranes and Golgi membranes using GLUTl, but only a very faint band of labelling in milk fat globule membranes, shown in Fig. 4.2(a). In each membrane the detected protein had an apparent M^ave 50,000. GLUT4 could not be detected in lOpg of mammary plasma membranes or Golgi membranes, shown in Fig. 4.2(b). The GLUT4 content of milk fat globule membranes was not investigated. Both the mammary plasma membranes and Golgi membranes showed substantially increased levels of GLUTl/mg protein compared to the level of GLUTl in the homogenate detected by Western blotting (shown in Fig. 4.1(a)). This result indicated th at both sub-cellular membrane preparation procedures resulted in co-purification from other cellular proteins of both GLUTl and the relevant marker enzymes.

The intensity of GLUTl seen in Western blots of Golgi membrane-enriched fractions was very similar to the intensity of GLUTl seen in the plasma-membrane- enriched fractions. This finding indicated that GLUTl in the Golgi membrane could not be derived entirely from contamination of the Golgi membranes with the plasma membranes, since, although there is contamination of Golgi membranes with the plasma membrane marker enzyme, the total enzyme activity was less than 30% of the activity of the plasma membrane itself (Section 4.3.1). The absence of GLUTl in the milk fat globule membranes suggested th at either GLUTl was not present in the apical plasma membrane, or th at the milk fat globule membrane proteins were selectively sequestered from the cellular membranes, a possibility discussed in Section 4.9.

4.3.3 SDS/polyacrylamide gel electrophoresis of mammary tissue sub-cellular fractions

Fig. 4.2 Sub-cellular distribution of GLUTl and GLUT4 in mammary membranes

(a) GLUTl (b) GLUT4

1 2 3 4 Mr 5 6 7 8 9 10

42-7

310

Mammary plasma membranes (2,(0), Golgi membranes (^,4) (lOpg each) or milk fat globule membranes ( I ) (20pg) were electrophoresed on 12% SDS/polyacrylamide gels and glucose transporters detected by Western blotting as describèd in Section 2.6, using antibodies raised against the C-terminal peptides of GLUTl (a) or GLUT4 (b). Human erythrocyte membranes ( 4- : 50pg) and rat adipocyte sub-cellular fractions (lOpg each) were used as positive controls for GLUTl and GLUT4 respectively. Adipocyte membranes were a gift from Mr. L. Fryer and consisted of low density microsomal membranes ( 5 ) and plasma membranes ( 7 ) from basal adipocytes, and low density microsomal membranes ( 6 ) and plasma membranes ( 8 ) from insulin-stimulated adipocytes, prepared by the method of Cushman and Wardzala (1980).

fractions used in the experiments described in Section 4.3.2, mammary plasma membranes (20pg), Golgi membranes (20pg) and milk fat globule membranes (20pg), were made up as gel samples and electrophoresed on 10% SDS/polyacrylamide gels then stained with Coomassie blue as described in Section 2.5.

A typical example of such an SDS/polyacrylamide gel is shown in Fig. 4.3, which illustrates that each membrane fraction exhibited a characteristic and distinct set of protein bands. There were clearly some proteins of an apparently similar present in all three mammary membrane fractions, while other proteins appeared to be confined to one, or two, of the fractions only. This result lends support to the assumption that each membrane type is not severely contaminated with the other membrane types, but that some cross-contamination is occurring. The milk fat globule membranes appeared to contain fewer proteins than the plasma membrane or Golgi membrane fractions, a few proteins being very heavily expressed in the milk fat globule membranes.

4.4 Distribution of GLUTl in the fractions of a standard mammary plasma