The metabolism of luteinizing hormone. plasma clearance, urinary excretion, and tissue uptake

Molecular and Cellular Endocrinology 3 (1975) 21-36. 0 North-Holland Pub]. Comp.

THE METABOLISM OF LUTEINIZING HORMONE. PLASMA CLEARANCE, URINARY EXCRETION, AND TISSUE UPTAKE* Mario Department

ASCOLI,

of Biochemistry,

Rodger

A. LIDDLE

Vanderbilt

University,

and David Nashville,

Received 15 October 1974

PUETT**

Tennessee

37232,

U.S.A.

Accepted 15 January 1975

The kinetics of plasma clearance, tissue uptake, and urinary excretion of tritiated ovine pituitary luteinizing hormone in adult male rats are reported. Most of the intravenously injected tritiated gonadotropin is cleared from circulation with a half-life of five minutes, and this is independent of the injected amount of hormone over a wide dose range. It was found that the hormone is rapidly removed from circulation by the kidneys, probably by glomerular filtration, and excreted in the urine. The radioactivity present in the urine is associated with material of the same molecular size as the native hormone and, moreover, the urinary hormone retains a significant amount of biological activity. A small amount of the hormone is catabolized by the kidney and liver, and our data suggest that this occurs in the cortex and hepatocytes, respectively. Keywords:

gonadotropin;

gonadotropin

metabolism;

luteinizing hormone.

There is little information available regarding the mechanisms by which polypeptide and glycoprotein hormones are cleared from circulation. The general problem of catabolism of extracellular proteins has been dealt with primarily using denatured or aggregated proteins (Benacerraff et al., 1957; Freeman et al., 1958; Mego and McQueen, 1965), or with proteins that are not normally found in the bloodstream (Straus, 1962, 1964; Davidson et al., 1971). These studies pointed out that the reticuloendothelial system, especially the Kupffer cells of the liver, is capable of removing and degrading intravenously injected denatured proteins (Freeman et al., 1958; Mego and McQueen, 1965). It was also found that the kidney plays an important role in removing intra*Supported by Research Grant AM-15838 and Population Center Grant HD-05797 from the National Institutes of Health. Portions of this work were presented at the Conference on Hormone Binding and Activation in Testis, Houston, Texas (February, 1974) and at the Joint Biochemistry-Biophysics 1974 Meeting, Minneapolis, Minnesota (June, 1974), Federation Proceedings (1974), 33, 1357. **Camille and Henry Dreyfus Foundation Teacher-Scholar Awardee.

22

M. Ascoli et al.

venously injected foreign proteins (Straus, 1964; Davidson et al., 1971). Various studies have shown that several plasma asialoglycoproteins are removed from circulation and degraded by hepatocytes (Gregoriadis et al., 1970; Morel1 et al., 1971). However, the physiological significance of this phenomenon is yet to be demonstrated. Early studies on the metabolism of insulin and glucagon in the rat (Elgee et al., 1954; Cox et al., 1957), showed that the kidneys were able to take up most of the injected hormone. More recently, it has been shown that the kidneys also play an important role in removing human follicle-stimulating-hormone (hFSH) (Vaitukaitis et al., 1971; Butt et al., 1974), human chorionic gonadotropin (hCG) (Braunstein et al., 1972) and human luteinizing hormone (hLH) (de Kretser et al., 1969, 1973) from the circulation of pseudopregnant and immature female rats, immature male rats, and sheep, respectively. In the present study, we have quantitatively followed the kinetics of plasma disappearance, urinary excretion, and tissue uptake of tritiated ovine pituitary luteinizing hormone (oLH) in intact and hypophysectomized mature male rats. Our findings indicate that urinary excretion, without extensive degradation, is the main mechanism by which this hormone is removed from circulation. In subsequent papers we will discuss the analysis of a compartment model for oLH metabolism and the intracellular catabolism of the hormone by various tissues.

MATERIALS

AND

METHODS

Supplies and hormones [ 1,2- 3H]Testosterone (43.5 Ci/mmol) and tritiated water standard (1 uCi/ml) were obtained from New England Nuclear (Boston, Massachusetts). Rabbit antiserum against testosterone was from Calbiochem (San Diego, California), collagenase (type I, 207 units/mg), lima bean trypsin inhibitor, and CZ. per. neuraminidase (0.8 units/mg) were from the Worthington Biochemical Corporation (Freehold, New Jersey), and bovine serum albumin (Cohn Fraction V) was from the Sigma Chemical Company (St. Louis, Missouri). Fetuin (99 % pure) was obtained from Grand Island Biological Company (Grand Island, New York). Crude urinary hCG (3300 IU/mg) was purchased from Organon, Inc. (West Orange, New Jersey). Protein modcjications Formaldehyde-treated albumin was prepared by the method of Mego and McQueen (1965). Asialofetuin was prepared by incubating 50 mg of protein

Gonadotropin

23

metabolism

with 0.8 units

of neuraminidase

in 2.0 ml of 0.1 M sodium

acetate

buffer,

pH 5.0, for 20 h. The incubation was performed at room temperature under a toluene atmosphere. The reaction mixture was then extensively dialyzed against water and lyophilized. Analysis of total sialic acid (Miettin and Takki-Lukkainen, 1959) showed that 90% of the sialic acid was removed. Hormone purification and labeling oLH was extracted from sheep pituitaries (Koenig and King, 1950) and purified using diethylaminoethyl-Sephadex A-25 and the starting discontinuous buffers described by Pierce et al. (1971), then carboxymethyl-cellulose and the buffer system of Ward et al. (1959, 1961), and finally gel filtration as described elsewhere (Puett et al., 1974). HCG was purified using diethylaminoethylSephadex A-25 and the buffer system of Bahl (1969), followed by gel filtration (Puett et al., 1974). The biological activity of the purified hormones, as determined by the ovarian ascorbic acid depletion assay, was as follows: oLH 1.97 x NIH-LH-S18 (95 % confidence limits = 1.27-3.53); HCG 13,588 IU/mg (95 % confidence limits = 8243-28,787). The highly purified oLH was labeled by reductive methylation using borohydride as previously described (Ascoli and Puett, 1974). The labeled product was purified by dialysis and then exclusion chromatography on Bio-Gel P-100. Gonadotropin injections and assa)’ of tissue radioactivity Intact or hypophysectomized adult male rats (Sprague-Dawley) weighing 200-300 gm were anesthetized with sodium pentobarbital (50 mg/kg body weight). The femoral veins from both sides were exposed and the radioactive gonadotropin (in 0.1 ml isotonic saline) was injected into one of the veins using a Hamilton syringe. Blood (about 0.7 ml) was collected in heparinized tubes from the femoral veins at the times indicated. The animals were sacrificed at different times, and the liver, kidneys, and testes were removed, placed on ice and washed with isotonic saline. The urine present in the bladder was also collected. For the long term experiments (424 h), the animals were kept in metabolic cages for collection of urine. The tissues were homogenized in water using a Polytron PT-10 homogenizer to give a final concentration of 0.25-0.5 g tissue/ml homogenate. Plasma was obtained from blood by centrifugation. Duplicate aliquots, e.g., 0.5 or 1.0 ml of the tissue homogenates and 0.2 ml of urine and plasma, were combusted in a Packard 306 Tri-Carb Sample Oxidizer and counted in a Packard Tri-Carb

24

M. Ascoli et al.

Scintillation Spectrometer at 60% gain. The efficiencies obtained by internal standardization were as follows: testes 24.7 %, liver 23.1x, kidneys 21.9 %, urine and plasma 28.5 %. Polyacrylamide gel electrophoresis Urine samples collected at different times after injection of [3H]oLH were lyophilized and redissolved in a suitable volume of 10 mM Tris-phosphate buffer with 1% sodium dodecylsulfate (SDS) and 0.2% dithiothreitol, pH 7.2. Electrophoresis was carried out by the method of Weber et al. (1972) except that we used Tris-phosphate instead of sodium phosphate buffer, and N,N’diallyl tartardiamide instead of bisacrylamide in order that the gel slices could be easily solubilized (Anker, 1970). At the end of the run the gels were frozen and sliced. Each slice was placed in a scintillation vial and solubilized by incubation with 1.0 ml of 2% periodic acid for 2 h at room temperature. The samples were then counted in 10 ml of Aquasol. In vitro biological activity of [3H]oLH Leydig cells were prepared from testes of mature male rats by collagenase digestion using essentially the method of Moyle and Ramachandran (1973) except that 1.0 mg of collagenase/testis was used (2 ml of a 0.5 mg/ml solution in Krebs-Ringer bicarbonate buffer). This procedure normally yields 5-7 x lo7 cells per testis. For measurements of testosterone production, the cells were diluted in Krebs-Ringer bicarbonate buffer with 2 mg/ml bovine serum albumin and 0.1 mg/ml lima bean trypsin inhibitor to obtain 10’ cells/ml and incubated at room temperature for 15 min. Then, 1 ml of the cell suspension was incubated with increasing amounts of hormone in 0.1 ml of 0.1 M sodium phosphate buffer (pH 7.5) with 0.15 M NaCl and 1 mg/ml bovine serum albumin at 37 “C with constant shaking under a 95 % O,-5 % CO, atmosphere. At the end of 4 h the tubes were placed on ice, sonicated for 30 s, and centrifuged at 12,000 g for 20 min. The supernatant was diluted 10 fold with isotonic saline for testosterone radioimmunoassay (Dufau et al., 1972). The data from the radioimmunoassays were analyzed as described elsewhere (Ascoli and Puett, 1974) and are expressed as ng testosterone plus dihydrotestosterone per lo7 cells per 4 h. The relative potency values for the urinary [3H]oLH were calculated using the measured radioactivity and the known specific radioactivity as a measure of the dose of hormone added to the cells in order to obtain the dose-response curve.

Gonadotropin metabffIisn2

25

RESULTS Hormone characterization

[“H]oLH is homogeneous and is indistinguishable from the unlabeled hormone on both gel filtration and SDS-polyacrylamide gel electrophoresis. The specificity of this reaction for oLH lysyl residues has been demonstrated and, moreover, under the conditions employed, approximately 85% of the oLH lysylresidues are converted to monomethyllysine and dimethyllysine with the latter representing the major product (Ascoli and Puett, 1974). The specific radioactivity and potency of the two 13H]oLH preparations used in the present experiments are given in table 1. The increased potency of the methylated derivatives has been documented before (Ascoli and Puett, 1974; De la Llosa et al., 1974a, b). No significant differences (e.g., specific activity, biological activity, and circulatory half-life) were detected in the two preparations. Circulatory behavior

Fig, 1 shows the plasma disappearance curves of several doses of 13H]oLH. We have previously reported that up to 12 h after injection these curves can be resolved into at least two components (Ascoli and Puett, 1974). One, which accounts for most of the labeled material, has an apparent circulatory half-life of 5 min. The other component has an apparent circulatory half-life of about 29 h and represents only 2-3 % of the injected [ 3H]oLH. The similarity of the curves suggests that the different tissues responsible for removing the hormone from circulation are not easily saturated. Gel filtration of plasma samples following injection of [3H]oLH indicates that much of the radioactivity is associated with a compound of similar size to that of LH. The biological activity of the plasma material is not yet known.

Table 1

Characteristicsof t3H]oLH. Two batches of oLH were tritiated by reductive methylation using the procedure described elsewhere (Ascoli and Puett, 1974). Biological activity was measured itt vitro as described in the text. The standard was purified, unlabeled oLH (i.e., 1.97 x NIH-LH-S18); the values in parentheses represent 95% confidence limits.

Preparation

Specific radioactivity (Ci/mmol)

-I._~ _-._.._ ___1

2

-20.8 18.1

Biological activity

___-_______

_____--~.“-_~

2.50 (1.91-3.60) 1.94 (1 M-2.30)

26

M. Ax&

DPMt

f-)x

et al.

100

DPMt min

0

20

40

60 80 t, minutes

100

120

Fig. 1. Plasma disappearance patterns of different amounts of r3H]oLH given intravenously to intact male rats at time zero. The values have been normalized to the amount of radioactivity present in plasma 1 min after injection to correct for differences in the amount of radioactivity recovered.

Tissue distribution

Fig. 2 shows the time course of the tissue distribution of 13H]oLH after a single intravenous injection. Most of the labeled hormone is taken up by the kidney and excreted in the urine. The liver also takes up a significant amount of radioactivity. When the results are expressed per g of tissue, the specific activity of the liver is about one order of magnitude lower than that of the kidney. However, due to the larger size of the liver, both tissues have a similar capacity to remove [3H]oLH from the circulation. The testicular uptake of t3H]oLH is somewhat lower than that of the other tissues. It reaches a maximum 60-90 min after injection (about 1.2 rig/g tissue) and remains essentially constant for 12 h. It is noteworthy that the hormone undergoes degradation in these tissues. The amount of trichloroacetic acid soluble radioactivity in the non-target tissues, liver and kidneys (Puett et al., 1974), and in testes (unpublished observations) increases with time after injection of i3H]oLH. Two important differences were observed when hypophy-

27

~~nadotrQ~~~metabolism

IO3 0

2

4

6

8

10

12

t , hours Fig, 2. Tissue distribution of [%I]oLH in intact male rats following a single intravenous injection of 1.6 pg (0.5 uCi) at time zero. Each point represents a separate experiment. The radioactivity recovered was always 90-100’~ of the amount injected for the early time points (O-2 h). For the late time points (e.g., 4-12 h), the amount recovered was 3%100% of that injected. Testes (II), Liver (V), Kidneys (A), Plasma (0). Urine (0).

were used, The testicular uptake was both more rapid (e.g., the maximum occurred at about 30 min) and greater (2-3-fold increase). The relative uptake of 13H]oLH by the other tissues remained unchanged. Fig. 3 shows the relationship between the amount of [3H]oLH injected and the amount taken up by the different tissues. It can be seen that the liver and kidneys have a large capacity to remove E3H]oLH from the bloodstream. Under these conditions (i.e., 15 min after injection) the amount of [3H]oLH taken up by the testes increases from about 1.5 to 30 rig/g tissue when the amount of hormone injected was increased from 0.05 to 28 ng. Data (not reported) similar to those shown for intact animals were also obtained with sectomized

male

rats

28

M. Ascoli et al. 9c I 2

80

70

[

I

60 c

DPM x IO+ gm tissue

c

50 c c

40

30

20

IO

L 0.1

1.0

pg L3~] oLH

Fig. 3. Dose-uptake relationship of Testes (m), Liver (D), Plasma (@), average of two different experiments. expanded scale. The bars extend

IO

injected

[3H]oLH in intact male rats 15 min after injection. Kidneys (A), Urine (a). Each point represents the The insert shows the lower part of the curves in an to the individual values of duplicate experiments.

hypophysectomized animals at 15 and 30 min after injection of the hormone. The specificity of [3H]oLH uptake by the different tissues was tested by injecting the labeled gonadotropin with a large excess of hCG, which has the same gonadotropic action as LH and binds to the same testicular receptor (Catt et al., 1972). The testis appeared to be the only specific tissue for [ 3H]oLH (table 2). Table 3 shows the results of several experiments devised to determine the cellular location of [3H]oLH in the liver. Blockage of the reticuloendothelial system with charcoal (Freeman et al., 1958) prior to injection of [3H]oLH did

Gonadotropin

metabolism

29

Table 2 Effect of the simultaneous injection of hCG on the tissue uptake of [3H]oLH. Intact mature male rats were intravenously injected with [3H]oLH (1 x lo6 dpm) alone or with [3H]oLH and hCG. The rats were sacrificed 15 min after injection and the tissues assayed for radioactivity as described under Materials and Methods.

Proteins injected

DPM/g of tissue

__Testes

Liver

Kidneys

Plasma

1972

12558

95145

9668

950

12518

94538

14342

[3H]oLH (0.8 pg) [3H]oLH (0.8 pg) with 10 mg hCG

not produce a significant decrease in the amount of radioactivity taken up by the liver. Also, HCHO-treated albumin, which seems to be taken up by the Kupffer cells (Mego and McQueen, 1967), did not prevent the hepatic uptake of [3H]oLH. Therefore, it seems that the hormone is removed by the hepatocytes rather than by the Kupffer cells. The finding that asialofetuin did not inhibit this hepatic uptake indicates that it occurs in a different manner than

Table 3 Cellular localization of [3H]oLH in the liver. ‘A Recovered radioactivity present in the liver 15 minutes after injection of [3H]oLH

Injected materials

17.8

VHloLH (1 ug) [3H]oLH (1 ug) in charcoal-loaded [3H]oLH (1 pg) with 10 mg HCHO-treated albumin* * [3H]oLH (1 ug) with 10 mg asialofetuin** _

rats*

15.0

17.4

19.8

*Mature male rats were injected intravenously with 75 mg of charcoal in isotonic saline for 4) days prior to injection of the labeled hormone. **The proteins were injected simultaneously.

30

M. Ascoli et al. Table 4

Renal distribution of [3H]oLH. The cortex and medulla were prepared by gross dissection. 200-mg samples were combusted and assayed for radioactivity as described under Materials and Methods. DPM x 1O-5 Time after injection (min)

1 15 30 120

Cortex

Medulla

1.02 3.55 3.76 2.08

0.27 0.25 0.52 0.15

that reported for asialoglycoproteins (Pricer and Ashwell, 1971; Van Lenten and Ashwell, 1972). In table 4, results are presented which show that the radioactivity present in the kidney at various times after injection is localized mainly in the cortex. These results suggest that some [3H]oLH is either secreted into or reabsorbed from the tubules. Urinary excretion Figs. 4 and 5 show that most of the radioactivity excreted in the urine for up to 24 h after injection is associated with a compound of similar size to that of [3H]oLH. Thus, it appears that most of the hormone is excreted in essentially the intact form, or at least has not been extensively degraded. It can also be concluded that there is no dissociation of the hormone into subunits prior to excretion. Fig. 6 shows the in vitro biological activity of urine obtained from a hypophysectomized mature male rat 2 h after injection of [3H]oLH. A potency value cannot always be calculated using [3H]oLH as standard because nonparallelism of the dose-response curves is often observed. However, when the assay for [3H]oLH is run in the presence of 0.1 ml aliquots of urine from a control hypophysectomized rat diluted in the same way as that of the rat injected with [3H]oLH (l-20 fold), parallel curves are obtained. Using these two dose-response curves and defining the specific biological activity of [3H]oLH in the presence of control urine as 1.0, a specific biological activity of 0.57, with 95% confidence limits of 0.46-0.72, was obtained. In an independent experiment, parallelism was observed between standard [3H]oLH without added urine and urinary [3H]oLH obtained from a hypophysectomized rat 2 h

31 5-

4-

CPM x lO-5

I

c3H]oLH

3-

URINE

2 hr

URtNE

24

CPM x IO+

8

hr

CPM x lO-3 4

0~ 1.2

2.4

3.6

4.8

Ve/Vo

Fig. 4. Gel filtration patterns of urine samples collected several times injection of 1.6 ug [3H]oLH. About 1 ml of urine was chromatographed Bio-Rad P-30 column equilibrated and developed with 0.154 M NaCl in pH 7.45. Fractions (2 ml) were collected and a 0.1-0.5 ml ahquot was activity using 10 ml Aquasol as scintillation fluid.

after intravenous on a 1.5 x 8.5 cm 10 mM Tris-HC1, assayed for radio-

after injection. Defining the specific biological activity of control L3H]oLH as 1.O,a specific biological activity of 0.47 with 95 % confidence limits of 0.41-0.55 was found for urinary E3H]oLH. A 0.1 ml aliquot of undiluted urine from the control rat gave a response of 14.6 f 0.4 (n = 3) ng testosterone/lO’ cells/4 h. The control value, with no addition of urine or hormone, was 13.5 rt 0.63 (n = 3) ng testosterone/lO’ cells/4 h. Therefore, it seems that although urine does not have effect on the basal level of testosterone production, it does inhibit to some extent the stimulation produced by oLH.

32

M. Ax&

et al.

3

2 CPM x I o-5 I

0

3 URINE

15 min

URINE

6Omin

CPM x 10-3

:i I CPMx

10-3 .5

Rm

Fig. 5. SDS-polyacrylamide gel electrophoresis of urine samples coilected at different times after intravenous injection of I.6 pg [3H]oLH. Rm = relative migration to that of the tracking dye.

It is noteworthy that the biological activity of the [jH]oLH excreted in the urine does not change appreciably with increasing time after injection. As early as 15 min after injection, the specific biological activity of the urinary [ 3H]oLH was found to be about 50% of that of the injected hormone, i.e., about the same as that found 2 h after injection. Although the molecular size of the urinary E3H]oLH is similar to that of control [3H]oLH, we have recently noted that the two compounds chromatograph differently on a cation exchanger.

DISCUSSION In contrast to other glycoproteins of similar molecular size but with different carbohydrate composition (Morel1 et al., 1971), oLH exhibits biphasic circulatory behavior (Ascoli and Puett, 19’74; Puett et al., 1974) with most of the material exhibiting a relatively short half-life. The rapidly disappearing compo-

Gonadotropin metabolism

33

a,-

IO3

ng [3H]

IO4

OLH

105

DPM

Fig. 6. One hypophysectomized male rat was injected with 5 pg t3H]oLH (16 h post-hypophysectomy) and sacrificed 2 h after the injection. The urine present in the bladder was collected, assayed for radioactivity and diluted l-20 fold to obtain solutions with enough t3H]oLH to stimulate testosterone production in collagenase-dispersed Leydig cells. [‘H]oLH (0); urinary [3H]oLH (A); [3H]oLH in the presence of 0.1 ml aliquots of l-20 fold dilutions of control urine (m). Each point represents the average of two different samples. The bars extend to the individual values of these samples.

with a half-life of 5 min appears to be due to the rapid hepatic and renal uptake. A small amount of hormone binding to plasma proteins, degradation products and, perhaps, the presence of sialic acid (Walborg and Ward, 1963), if distributed unevenly among the LH molecules, could account for the component with the apparent longer half-life.

nent

With respect to other gonadotropins of comparable size but containing significant amounts of sialic acid, e.g., hCG (Morel1 et al., 1971; Braunstein et al., 1972) and hFSH (Morel1 et al., 1971; Vaitukaitis et al., 1971), oLH has a much shorter circulatory half-life. Thus, the carbohydrate moiety of oLH, which contains mannose, galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and small amounts of sialic acid (Papkoff, 1973), does not lead to a long circulatory half-life. We have also found (unpublished results) that the plasma disappearance of human LH is more similar to oLH than to

34

M. Ascoli et al.

hCG, although human LH contains a higher content of sialic acid, e.g., 2% (Hartree et al., 1971), than does oLH. The plasma disappearance pattern of oLH is independent of the amount of hormone injected and the transfer of hormone from the bloodstream to the urine is rather efficient even at very low concentrations of hormone corresponding to physiological levels. In fact, most of the hormone (80-90x) has been excreted in the urine 2 h after injection and the kidneys and urine account for most of the radioactivity even at very low concentrations of hormone (e.g., 50 ng injected). This urinary excretion seems to be faster than that of insulin and glucagon (Elgee et al., 1954; Cox et al., 1957). Also, unlike these two hormones, most of the radioactivity excreted in the urine is associated with a protein of molecular size very similar to that of [3H]oLH. In contrast to our findings, de Kretser et al. (1973) have reported that although the kidneys are quite capable of removing [ I2 ‘I]hLH from the circulation of sheep, only a very small amount of the radioactivity present in the urine seems to be associated with protein. We have observed, however, that hLH labeled with tritium either in the protein or the carbohydrate moiety behaves very similarly to oLH in mature male rats, and that most of the radioactivity present in the urine 1 h after injection behaves like the injected hormone on gel filtration (unpublished results). We have found that the excreted [ 3H]oLH retains about 50 % of the specific biological activity of control [3H]oLH and that most of the urinary hormone fraction has a different apparent charge relative to [3H]oLH. These observations indicate that the hormone undergoes some type of modification (e.g., limited proteolytic cleavage or binding of an ionic inhibitor) prior to or during excretion. The nature of this modification is currently under investigation. The radioactivity that remains in the kidney is found primarily in the cortex, suggesting that some of the hormone is reabsorbed from the urine. This is supported by the studies of de Kretser et al. (1969) who reported that the [lz51]hLH found in the kidneys of immature male rats after intravenous injection of the hormone is associated with the cells of the proximal convoluted tubules. A similar phenomenon has been observed with insulin and glucagon (Narahara et al., 1958). Therefore, from our data we conclude that the main mechanism by which [3H]oLH is removed from circulation is glomerular filtration, with much of the labeled hormone excreted without extensive degradation. The relatively small proportion of [ 3H]oLH that is not excreted is retained in the kidney cortex and hepatocytes where it is degraded. This phenomenon will be discussed in a subsequent paper.

35

ACKNOWLEDGMENTS It is a pleasure to thank Betty Kay Wasserman, Gudrun Moustafa, and John D. Ford for expert technical assistance in hormone purification. We also thank Wendeil E. Nicholson for performing the hypop~ysectomies and Dr. Leslie A. HolIaday for his assistance with the ele~trophoresis and for many helpful discussions.

REFERENCES Anker, H. S. (1970) FEBS Lett. 7, 293. Ascoli, M. and Puett, D. (1974) Biochim. Biophys. Acta 371, 203. Bahl, 0. P. (1969) J. Biol. Chem. 244, 567. Benacerraff, B., Halpern, B. N., Biozzi, G., Stiffel, C. and Mouton, D. (1957) Br. J. Exp. Pathol. 38, 35. Braunstein, G. D., Vaituka~tis, J. L. and Ross, G. T. (1972) Endocrinology 91, 1030. Butt, W. R., Ryle, M. and Shirley, A. (1973) J. Endocrinol. 58, 275. Cart, K. J., Dufau, M. L. and Tsuruhara, T. (1972) J. Clin. Endocrinol. Metab. 34, 123. Cox, R. W., Henley, E. D., Narahara, H. T., Vanarsdel, Jr., P. P. and Williams, R. H. (1957) Endocrinology 60, 277. Davidson, S. J., Hughes, W. L. and Barnwell, A. (1971) Exp. Cell Res. 67, 171. De Kretser, D. M., Catt, K. J., Burger, H. G. and Smith, G. C. (1969) J. Endocrinol. 43, 105. De Kretser, D. M., Atkins, R. C. and Paulsen, C. A. (1973) J. Endocrinol. 58,425. De la Llosa, P., Durosay, M., Tertrin-Clary, C. and Jutisz, M. (1974a) Biochim. Biophys. Acta 342, 97. De la Llosa, P., Marche, P., Morgat, J. L. and De la Llosa-Hermier, M. P. (1974b) FEBS Lett. 45, 162. Elgee, N. J., Williams, R. H. and Lee, N. D. (1954) J. Clin. Invest. 33, 1252. Freeman, T., Gordon, A. H. and Humphrey, J. H. (1958) Br. J. Exp. Pathol. 39,459. Gregoriadis, G., Morel], A. G., Sternlieb, I. and Scheinberg, H. I. (1970) 3. Biol. Chem. 245, 5833. Hartree, A. S., Thomas, M., Braikevitch, M., Bell, E. T., Christie, D. W., Spaul, G. V. Taylor, R. and Pierce, J. G. (1971) J. Endocrinol. 51, 169. Koenig, V. L. and King, E. (1950) Arch. Biochem. Biophys. 26, 219. Mego, J. L. and McQueen, D. J. (1965) Biochim. Biophys. Acta 100, 136. Mego, J. L. and McQueen, D. J. (1967) J. Cell Physiol. 70, 115. Miettin, T. and Takki-Lukkainen, L. T. (1959) Acta Chem. Stand. 13, 856. Morel], A. G., Gregoriadis, G., Scheinberg, H. I., Hickman, J. and Ashwell, G. (1971) J. Biol. Chem. 246, 1461. Moyle, W. R. and Ramachandran, J. (3973) Endocrinology 93, 127. Narahara, H. T., Everett, N. B., Simmons, B. S. and Williams, R. H. (1958) Am. J. Physiol. 192, 227. Papkoff, H. (1973) In: Hormonal Proteins and Peptides, Vol. 1, Ed.: Li, C. H. (Academic Press, New York) p. 59.

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