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Effect of growth hormone on enzyme activities in the hypophysectomized rat
ARCHIVES
OF
BIOCHEMISTRY
AND
Effect of Growth
BIOPHYSICS
113, 718-724 (1966)
Hormone
on Enzyme
Hypophysectomized JOSEPH P. LIBERTI:
Activities
Rat’
JOHN C. COLLA,3 JOHN F. VAN PILSUM, UNGAR
Department of Biochemistry, University of Minnesota, Received
in the
AND
FRANK
College of Medical Sciences, Minneapolis, 1Vinnesota
November
2, 1965
The effects of hypophysectomy and growth hormone administration on the activities of various enzyme systems in rat kidney and liver extracts have been studied. Removal of the pituitary gland resulted in either essentially no changes in the enzyme activity compared to the sham-operated animals, e.g., glucose B-phosphate dehydrogenase, isocitric dehydrogenase, aconitase, and hepatic glucose 6-phosphatase,or in a change in the catalytic activity of the enzymes, e.g., lactic dehydrogenase (437,), glutamic dehydrogenase (-2470), 5’-nucleotidase (-207G), kidney glucose 6-phospha(-6Oa/,). In most of tase (-31%), tryptophane pyrrolase (-150/c), a nd transamidinase the reactions studied, the administration of growth hormone to hypophysectomized rats resulted in little alteration in activities compared with the hypophysectomized rats. The activity of kidney transamidinase, which was decreased to a marked degree by hypophysectomy, was increased by the administration of three daily doses of 20 pg of growth hormone to the hypophysectomized rats. These alterations in transamidinase activity by hypophysectomy and growth hormone treatment are unique when compared to the response of a number of other enzyme systems in the liver and kidney.
Kidneys from hypophysectomized rats have only a fraction of the transamidinase activities, in vitro, of kidneys from intact or sham-hypophysectomized rats. Hypophysectomized rats that had been given injections of growth hormone had higher kidney transamidinase activities than hypophysectomized rats (1). The response of the kidney enzyme under these circum-
stances was specific for growth hormone. The loss of body protein from hypophysectomized rats and the retention of nitrogen in hypophysectomized rats treated with growth hormone are well documented (2). Recently a low rate of nL-C14-leucine incorporation into protein was demonstrated in a cell-free system prepared from livers (3, 4) obtained from hypophysectomized rat,s. The rate of incorporation in the in vitro system was partially restored to normal in livers from hypophysectomized rats treated with growth hormone. It is reasonable to expect that an effect on protein synthesis would also be reflected in observable changes in enzyme activity. The activities of certain other enzyme systems were examined to see if any were altered as markedly by hypophysectomy as rat kidney transamidinase. The choice of enzymes for study was
1 This research was supported in part by grants from the American Cancer Society, North Dakota Division, No. P-375, and from the National Institutes of Health, U.S. Public Health Service, Nos. A-2731 and AM-02583. 2 Postdoctoral trainee in the Training Program for Steroid Biochemistry, University of Minnesota. Present address: Sloan Kettering Institute, New York. 3 Postdoctoral trainee in the Training Program for Steroid Biochemistry, University of Minnesota. 718
ENZYME
ACTIVITIES
AND
determined by their possible significance in terms of the known physiological effects of growth hormone, the faci1it.y of determination, and the possibilit,y of purification if further studies were warranted. A few of the enzymes previously had been investigated to some degree and a comparison with the older studies as reviewed by Knox et al. (5) could be of value. Tissues were obtained from hypophysectomized rats, sham-hypophysectomized rats, and hypophysectomized rats treated with growth hormone. The enzyme activities of liver and kidney preparations were measured and compared with the changes in kidney transamidinase activities in the same animals. In no instance could changes be described in any of the enzymes investigated which were comparable to the effects seen for kidney transamidinase. This enzyme at present is unique in the degree of response to hypophysectomy and to subsequent treatment with growth hormone. EXPERIMENTAL
PROCEDURE
Materials Reagent grade solvents and inorganic compounds were used. The following were employed in the enzyme assays in the form as purchased: reduced and oxidized pyridine mlcleotides (Sigma, St. Louis, Missouri), disodium-glucose B-phosphate (California Corporation Biochemical Research, Los Angeles), sodium pyruvate, nL-isocitric acid, sodium succinate, ATP and AMP (Nutritional Biochemicals Company, Cleveland, Ohio), and bovine albumin (Pentex Incorporated, Kankakee, Illinois). Ovine growth hormone (GH) was supplied by the Endocrine Study Section of the National Institutes of Health (NIH-GH-S6) and had an activity of 1.6 units per milligram. The reaction involving the production of cisaconitate, and the reduction of NAD or oxidation of NADP were monitored continuously in a recording double-beam spectrophotometer (Bausch and Lomb, model 505). Male Sprague-Dawley rats weighing 100 gm were hypophysectomized or sham-operated via the parapharyngeal route at the Hormone Assay Laboratory, Chicago, Illinois, and shipped to our laboratory 3 days after the operation. The animals were housed individually with access to a complete synthetic diet (6) and water ad libitum. The hypophysectomized animals were given daily intraperitoneal injections of 20 pg of GH in 0.9% saline on days 7 to 10 after hypophysectomy (1).
GROWTH
HORMONE
719
Control animals received injections of saline on the same days. The animals were weighed when the injections were started and at t,he time of sacrifice. The effectiveness of the GH preparation was verified by t,he weight gains of the hypophysectomized animals that received GH and by the activities of the kidney enzyme, transamidinase.
Tissue Preparation The suspending medium and the enzyme concentrations of tissue preparations varied with the different enzymes assayed. The general procedure was as follows: the animals were killed by decapitation, after which the livers or kidneys were removed and placed on ice or in ice-cold 0.25 M sucrose or 0.14 &Z KCl. The tissues were then blotted, weighed, minced, and homogenized in a Potter-Elvehjem homogenizer fitted with a Teflon pestle. FlIrther steps in the tissue preparations are noted in the text.
Enzyme A says Preliminary test)s were done to determine the enzyme concentrations with which zero-order kinetics were obtained. The linearity of the enzyme catalyzed reactions m-as determined as a function of both the incubation t,ime and tissue concentration. The rate of srtbstrate utilized or product formed was directly proportional to the tissue concentrations and incubation periods. The enzyme assays were performed in duplicate, except for transamidinase and xanthine oxidase, and also at two enzyme concentrations in the presence of excess substrate. The tissues from all of the animals were handled in as much the same manner as possible in order to minimize changes in enzymic activit,y due to procedural manipulations. The activity of each enzyme preparation was also monitored at varions times after homogenization to check its stability. Protein concentration was determined by the spectrophotometric method of Lowry (7) or by the biuret reaction (8). Crystalline bovine serum albumin was used as the standard. All results are listed in Table I as the average values f the standard deviations. Transamidinase. The method of Van Pilsum et al. (9) was used in the measurement of kidney hotransamidinase activity. A 5% rat kidney mogenate was used as the source of enzyme. The enzyme activity was expressed as milligrams guanidinoacetic acid formed per gram kidney per hour. Kidneys from hypophysectomized rats had 19% of the transamidinase activities of kidneys from the sham-operated rats. The kidney transamidinase activites of the hypophysectomized rats that had been given daily injections of 20
720
LIBERTI
ET AL.
TABLE ENZYME
ACTIVITY HORMONE
I
CHANGES IN THE R.LT AFTER SHAM OPERATION, HYPOPH~SECTDMY, .IND GILOWTH ADYINISTL~TION (20 PG/D.IU FOIX 4 D:LYS) TO THE H~PDPHYSECTDMIZED ROT
Enzyme assay
Tissue IIypophysectomized
Sham-operated
~
1. Transamidinase 2. Lactic dehydrogenase 3. (+lucose B-PO1 dehydrogenase 4. Isocitric dehydrogenase 5. Sllccinoxidase 6. Aconitase 7. Glutamic dehydrogenase 8. 5’.Nucleotidase 9. Arginase 10. Glucose G-phosphatase 1Oa. Glucose O-phosphatase 11. Xanthine oxidase 12. Tryptophane pyrrolase
Kidnel Liver ” Liver
3.2 f 0.2 19,600 * 1900 311 & 10
Kidney 1 Kidney Kidney Liver
21.9 + 1.5 /
87f4 1589 Z!Z 153 5900 + 212
~
5% Change from sham .~
Hyvwbsectomized with GH Wh4day)
0.G + 0.2 11,000 * 9io 304&14
-8lh ~ -43h -2
1.5 It 0.1 9800 f 1100 281 f 11
23.1 rt 1.9
~ +5
29.8 zt 1.3
+3
89 f 3 1448 + 140 4655 xt 142
90 i 1592 * 4585 f
3 185 180
- 24h
1.2 14 0.16
-20” -45O -5
Liver Liver Liver
39.6 =I= 1.6 461 & 83 2.00 It 0.26
31.6 * 253 f 1.90 f
Kidney
2.15 + 0.15
1.48 + 0.21
Liver Liver
7.3 f 0.7 11.7 + 3.4
10.8 f 9.5 f
0.9 4.1
0
35.2 f 1 251 f 2.22 f
r,fC&w (Iwpophyectomized
1.2 20 0.19
-3lb
1.48 f
0.20 I
1 +48” , -19
10.2 * 7.9 *
1.0 1.9
-1 +li” 0 -G
-17
ClUnits of activity: (1) transamidinase, mg guanidinoacetic acid/gm kidney/hour; (2) lactic dehydrogenase, rmoles pyruvate/mg liver/hour; (3) glucose H-phosphate dehydrogenase, Mmoles G-fi-P/gm liver/hour; (4) isocitric dehydrogenase, units/mg kidney where 1 unit = 01) increase of O.Ol/min at room temperatrlre; (5) succinoxidase, ~1 OJmg kidney/30 min; (6) aconitase, units/my kidney where 1 unit = OD increase of O.Ol/min at 25°C; (7) gltttamic dehydrogenase, rmoles cr.ketoglutarate/mg liver/ hour; (8) 5’.nrlcleotidase, rmoles Pi/mg liver/hour; (9) arginase, pmoles urea/gm liver/min; (10) glucose 6.phosphatase, ,umoles P;/mg liver/hour; (lOa) glucose 6-phosphatase, rmoles Pi/mg kidney/hour; (11) xanthine oxidase, pmoles uric acid/gm liver/hour; (12) tryptophane pyrrolase, pmoles kynurenine/mg liver (dry weight)/hour. * Significant difference (p < 0.01). fig of GH for 3 days were 2507; of the activities of the hypophysectomized rats (Table I, Assay 1). Lactic dehydrogenase. The assay method and liver tissue preparation were essentially those described by Weber (10). The oxidation of NADH was measured continuously for 5 minutes at 340 rnp at room temperatllre. The control cuvette contained all the reagents except the liver homogenate. Enzyme activity was expressed as pmoles pyruvate reduced per milligram liver per hour. As seen in Table I (Assay 2)) the livers from hypophysectomized rats had lower enzyme activities than livers from the intact controls. The injections of GH rrere without any effect on the enzyme. Glucose 6.phosphate dehydrogenase. The spec-
trophotometric assay of Kornberg and Horecker (11) for glucose G-phosphate (G-6-P) dehydrogenase in a 5yc liver homogenate was employed. Enzyme activity was expressed as micromoles G-6-P oxidized per gram liver per hoL[r. The results are shown in Table I, Assay 3. All three groups of rats had similar liver G-6-P dehydrogenase activities. Isocitric dehydrogenase. Isocitric dehydrogenase activity was measured spectrophotometrically at 340 rnr in a lye kidney homogenate according to the method of Ochoa (12). i\ctivity was expressed as units per milligram kidney. One unit, represent,ed an increase in optical density of 0.01 per minute. As shown in Table I, Assay 4, the removal of the pituitary gland had little or no effect on the enzyme-catalyzed conversion of isocitrate to
ENZYME
ACTIVITIES
AND
a-ketoglutarat,e. The hypophysectomized rats that received GH had greater kidney isocitric dehydrogenase activities than the intact controls or the untreated hypophysectomized rats. Succinoridase. It was shown by Melhuish and Greenbaum (13) that, the administration of GH to the rat resulted in a low P/O ratio when p-hydroxybutyrate was used as the oxidizable substrate, and that this decrease was due to increased oxidation of the substrate. A 10% rat kidney homogenate in 0.25 M sucrose was prepared. The oxidation of succinic acid was measured manometrically at 37” with a shaking rate of 120 cycles per minute. Readings were t,aken every 10 minutes for 30 minutes. Activity was expressed as microliters oxygen utilized per milligram kidney per 30 minutes. The data are presented in Table I, ilssay 5. All rats had similar kidney succinoxidase act,ivities. Bconitase. A 2% kidney homogenate prepared in 0.14 J4 KC1 was used as the source of aconitase. The enzyme activity was measured according to the method of Racker (14) by the appearance of eis-aconitate observed as 240 rnr after 4 minutes of incubation at 25°C. Activity was expressed in units per milligram kidney where one unit represented an increase in optical density of 0.01 per minute. From Table I, Assay 6 it can be seen that hypophysectomy or treatment of hypophysectomized rats with GH did not alter the kidney aconitase activities. Glutamic dch@ogenase. The procedure employed for the assay of glutamic dehydrogenase was a modification of the methods of Olson (15) and Endahl (16) using a 1% liver homogenate in distilled water. The reaction vessels contained, in a final volume of 3.0 ml, 12 pmoles of a-ketoglutarate adjusted to pH 6.8 with NaOH, 190 pmoles of ammonium acetate, 0.11 pmole of EDTA, 100 pmoles of tris buffer, pH 7.8, and the homogenate. The reaction was started by the addition of 0.33 pmole of NADH, and the oxidation of NADH R-as measured continuously against a control cuvette containing all the components except NADH. Activity was expressed as pmoles a-ketoglutarate reduced per milligram liver per hour. Livers from hypophysectomized rats had slightly less glutamic dehydrogenase activity than livers from the sham-operated rats, but a single dose of 20 pg of GH daily for 4 days did not alter this effect. 6’.Nucleotidase. The source of 5’.nucleotidase (17, 18) was a 10yo liver homogenate in 0.14 al KC1 which was diluted 1: 10 with isotonic KC1 and then strained through several layers of cheesecloth before use. The incubation reaction was stopped with 107; TCA and samples of the centrifuged supernatant fluid were u-ithdrawn and
GROWTH
HORMONE
721
analyzed for inorganic phosphate by the method of Fiske and SubbaRow (19). The activity was expressed as micromoles inorganic phosphate liberated per milligram liver per hollr. A small but significant inhibitory effect of hypophysectomy on the activity of 5’.nucleotidase is shown in Table I, Assay 8. Hypophysectomieed rats that received 20 rg GH per day for 4 days had a small increase in liver 5’.nucleotidase activity compared with the untreated hypophysectomized rat. Panda and Gael (18) showed that the administration of large quantities of growth hormone to normal rats resulted in a greatly increased activity of this enzyme. drginase. The a,ctivity of arginase was determined by the method of Folley and Greenbaum (20). A lyc liver homogenate in isotonic saline served as the enzyme source. A unit activity was expressed as the quantity of enzyme that liberated 1 pmole of urea irl 1 minute at 37”C, pH 9.45, from 0.023 iU L-arginine solution. Arginsse activities of the livers from the hypophysectomized rats were 5576 of the activit,ies of livers from the shamoperated control rats (Table I, Assay 9). The enzyme activity remained at 557, of the controls in the hypophysectomized rats that received 10 fig of GH daily for 7 days. In this same experiment it was found that kidneys from the hypophysectomized rats had 377, of the transamidinase activities from the sham-operated controls. Kidneys from hypophysectomized rats that had received seven daily injections of 10 pg of GH had 70yo of the activities of kidneys from the sham-operated rats. Tryptophane pyrrolase. Tryptophane pyrrolase was assayed according to the method of Knox (21). A 12.5yo liver homogenate was used as the enzyme source. The enzyme activity was expressed as micromoles kynurenine formed per milligram liver (dry weight) per hour. The enzyme activity in the liver was lower in the hypophysectomized rats and in hypophysectomized rats treated with GH as compared with the intact controls (Table I, Assay 12). These differences, however, were not significant. In another group of rats, administrat’ion of tryptophane resulted in an eighfold increase in tryptophane pyrrolase and cortisol administration caused an activity, increase of 255yo over the control vallles. Xanthine oxidase. The livers were assayed for xanthine oxidase activity by the method of Van Pilsum (22) with a 10% homogenate in 0.066 11f phosphate buffer, pH, 7.4. The activities were expressed as micromoles uric acid formed per gram liver per hour (Table I, Assay 11). Livers from hypophysectomized rats had 48% greater xanthine oxidase activities than livers from the sham-
722
LIBERTI
ET AL.
jetted with a small dose of GH, 20 pg, for only 3 days. It is quite possible that by increasing the periods of time after the operat,ion, the dose of GH, and the period of administrat,ion, different results for certain of the enzymes may have been obtained. The unique changes in t,ransamidinase activity under the conditions stated are interpreted to indicat’e that a factor(s) is operating which does not affect the activity this treatment (Table I, Assay lOa), i.e., hypophy- of t*he ot,her enzymes. It is possible that this sectomy decreased glucose 6-phosphatase ac- difference is due, in part, to inanition and had no tivity by 317~, and CH administration to the init’ial increase in creatine level (1) effect on this low enzyme activity. which occurs immediately following operation and which will decrease transamidinase DISCUSSION activity (24). The administ#ration of GH Twelve enzyme activities, in addition t.o could affect’ all enzyme systems, as a result the kidney enzyme transamidinase, were of increased protein synhheses, but in admeasured in the livers or kidneys obtained dition, it may have a specific stimulatory from hypophysectomized rats, hypophyUnder effect on transamidinase activity. sectomized rat)s t.hat had been given in- chronic conditions in terms of days after jections of GH, or from sham-operated rats. hypophysectomy and the length of time and Hypophysectomized rats had low kidney dosage of GH administration, the effect of glucose 6-phosphatase and transamidinase GH on prot,ein (enzyme) syntheses would activities, and in the livers the activities be manifest. In the acute situation existing were low for the following: lactic dehydroin the first few days of these experiments, a genase, glutamic dehydrogenase, arginase, decrease of enzyme activit’y by a creatine 5’-nucleotidase, and tryptophane pyrrolase. feedback mechanism could be a prominent In the case of liver xanthine oxidase an factor (1). actual increase was observed as a result of The general problem of t’he role of INhypophysectomy. Ko alteration in enzyme trition on enzyme activity has been disactivities after hypophysectomy was ob- cussed in more detail previously (1) and served for t,wo liver enzymes, glucose 6- will be considered only briefly here. Some phosphate dehydrogenase and glucose 6- of t,he enzymes st,udied, such as lactic phosphatase, and for three kidney enzymes, dehydrogenase and glucose 6-phosphatase, isocitric dehydrogenase, succinoxidase, and have been shown to be affected by diet (10) aconitase. In comrast to t#he marked re- while others are relatively insensitivo. sponse of transamidinase activities in Changes in dietary int,ake, particularly in kidneys of hypophysectomized rats t#reated the first few days after operation, could with GH, there was no stimulation of this account, at least in part, for the differences order of magnitude for any of the other in enzyme activities noted in the hypophyenzyme systems studied. sectomized rat. In the first 3 days after A number of factors should be considered hypophysectomy there is a period of inaniwhen comparing the large changes in ac- tion with a rapid decrease in transamidinase tivity observed for rat kidney transamidiactivity (1). The enzyme changes are nase with the changes observed for t,he similar to those observed with inanition in other enzyme systems. The length of time the sham-operated rat. These early changes after hypophysectomy was relat,ively short. are not reversed by GH (1). Stimulat.ion by In most cases, rats were sacrificed only ten GH occurs approximately seven days and days after hypophysectomy or sham opera- thereafter following hypophysectomy. Studtion. Furthermore, t,he animals were in- ies with force-feeding or pair-feeding over hypophysectomized rats. Growth hormone injections, however, did not alter the xanthine oxidase activities. Glucose 6.phosphatase. The assay for glucose 6.phosphatase was carried out by the method of Cori and Cori (23) with a 2.5(r, homogenate of rat, kidney or liver in isotonic KCl. Enzyme activity was expressed as micromoles inorganic phosphate released per milligram protein per hour. Hepatic glttcose 6.phosphatase was unaffected by the removal of t,he pituitary (Table I, Assay 10). The kidney responded in a totally different manner to
ENZYME
ACTIVITIES
AND
a period of 7-10 days demonstrated that with regard to transamidinase (1) enzyme levels were low in the hypophysectomized rat compared with the sham control that was fed the same amount of diet. Only GH stimulated the enzyme under these conditions. In the studies reported here for certain other enzyme systems, the small differences seen in the hypophysect)omized and intact’ rats could have been due to dietary fact’ors. With few exceptions, changes in enzyme activity were not large. It should be noted that liver xanthine oxidase, a most sensit,ive indicator of dietary protein intake, was higher in hypophysectomized rats than in sham-operated rats, but. administration of GH to hypophysectomized rats did not affect the enzyme activity. Special comment is warranted for the results observed for glucose 6-phospha0ase in which a decrease was seen in the kidney enzyme activity whereas the activity in the liver showed no change. When the values are expressed per milligram protein nit,rogen the total enzyme activity per animal is much lower than indicat,ed, since the kidney weight of the hypophysectomized rats is also low. The fact, that certain enzymes in the kidney will respond to hypophysectomy and possibly to GH suggests that the kidney, in this respect, may be considered to be a target organ for this pituitary factor. An effect seen with kidney glutaminase but not liver glutaminase previously had been shown by others (25). Tryptophane pyrrolase (26) and arginase (25) are two examples of liver enzymes in the rat which have been shown to be stimulated by cort,isol administration. These enzymes showed a decrease of activity after hypophysectomy, but did not respond to the treatment of GH administration as employed here. Isocitric dehydrogenase is of some interest with regard to GH since both citrate and isocitrate are metabolized in the kidney. These substances are linked wit,h calcium metabolism by virtue of the formation of a soluble c$alcium complex, and calcium excretion is well document,ed as a consequence of GH administration. In this study no changes in activit,ies from the control
GROWTH
HORMONE
723
levels were seen in kidneys from hypophysect,omized rats. However, kidneys from rats injected wit,h GH had higher activities than those from t’he control rats. This effect cannot, be explained at present but the possible relationship of this enzyme with GH artivit’y merits further investigation. Another kidney enzyme related to citrate metabolism, aconitase, did not show any changes in act)ivity with the various treatments. Further st,udies wit,h growth hormone on some of these enzyme systems would be of value particularly under chronic conditions. It is obvious that the aherations of enzyme activities observed under the experimental circumstances used in this report, require more detailed study since these enzyme changes were not nearly as marked as those seen for kidney transamidinase. Considering the possible importance of transamidinase in terms of maintaining creatine levels in the body, there may be a pronounced sensitivity and specificity of GH for this one enzyme alone. However, a more likely premise at the present time is that the changes in transamidinase activit,y arc the result of a secondary effect, namely, a lowered enzyme activity due to inanition followed by increasing levels of creatine. The levels of creatine have been shown to reach a maximum in the blood, kidney, and urine in the third to fourth day after hyljophysect,omy (1) just as t’hey do in starvation. The changes observed for kidney transamidinase may involve a feedback mcchanism or repression (24) by t.he increased levels of creatine. In addition, there would be a lack of protein (enzyme) synthesis during periods of prot.ein depletion, starvation, or after hypophysectomy. These combined fa(%ors would result in a more profound decrease in transamidinase activity as comI)ared t,o other enzymes. The apparent, specificity of GH action would then bc a result, of its normal anabolic activit,y under conditions in which the transamidinase activity is at a very low level. The over-all effect, of GH would appear to be cnhanccd due to t)hc conditions prevailing after hypophysectomy which have an exaggerated cffec&t on transamidinase.
724
LIBERTI
The response of kansamidinase in the hypophysectomized rat to GH administration could be used as a sensitive and useful model system for t,he study of the actions of GH on enzyme act,ivation or enzyme synthesis, or both. REFERENCES 1. UNGAR, F., AND VAN PILSUM, J. F., Endocrinology (submitted). 2. SMITH, R. W., JR., GAEBLER, O., ASD LONG, C. N. H., “The Hypophysial Growth Hormone, Nature and Actiotts.” McGraw Hill, New York (1955). 3. KORNER, A., Riochem. J. 73, 61 (1959). 4. KORNER, A., Rec. Prog. Horm. Res. 21, 205 (1965). 5. KNOX, W. E., AI-ERBAcH, V. H., AND LIN, E. C. C., Physiol. Rev. 36, 164 (1956). 6. VAN PILSUM, J. F., WICKENG, E. Z., AND FILONOM-ICH, L. K., I’oxicology and Applied Pharmacology 3, 431 (1961). 7. LOWRY, 0. H., FARR, A. I,., ROSEBROUGH, N. J., AND RANDALL, I<. J., J. Viol. Chem. 193, 265 (1951). 8. LAYNE, E., in “Methods in Enzymology” S. P. Coiourick and N. 0. Kaplan, eds.), Vol. III, p. 450. Academic Press, New York (1957). 9. v.4~ PILSUM, J. F., BERMAN, I). A., AND vl’oLIN, E. A., Proc. Sac. E’xptl. Biol. Med. 96, 96 (1957). 10. WEBER, G., AND CANTERO, A., Cancer Res. 19, 763 (1959). 11. KORNBERQ, A., AXI) HORECKER, B. L., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 323. Academic Press, New York (1955).
ET AL. 12. o('HO.%. s., in “Methods in Enzymology” (8. P. Colowick and N. 0. Kaplan, eds.), \‘ol. I, p. 739. Academic Press, New York (1955). 13. MELH~-ISH, A. H., APZI) (~REENBAC-M, A. L., Hiochem. J. 78, 392 (1961). 14. RACKER, I?., Eiochem. Biophys. Ada 4, 211 (1950). 15. OLSOX, J. A., AND AIVFINSEX, C. B., J. Biol. Chem. 197, 67 (1952). 1G. ENDAHL, P., Proc. Enptl. Biol. Med. 24, 192 (1957). 17. HEPPEL, L. A., ASD HILMOE, R. J.,in “Methods itt Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 565. Academic Press, New York (1955). 18. PAND.4, N. c., (;OEL, B. K., M~NsOOR, &I., AXD TAI,I\.AR, (;. P., Biochem. J. 82, 176 (1962). 19. FISKE, C. H., AND S~BBAROX, Y., J. Hiol. Chem. 66, 375 (1925). 20. FOLLEI., S. J., AND C;REEKBAUM, A. L., BioChem. .3. 43, 537 (1948). 21. Ksos, W. E., in “Methods in Enzymology” i&S. P. Colon-ick and N. 0. Kaplan, eds.), Vol. II, p. 242. Academic Press, New York (1957). 22. VAN PILRCM, J. F., J. Biol. Chem. 204, (i13 (lQ53). 23. &RI, C. F., .4x11 &RI, (>. T., J. Biol. Chem. 199, 661 (1952). 24. WALKER, J. B., AND WALKER, M. S., J. Biol. Chem. 237, 473 (1962). 25. SCHMKE, R. T., J. Biol. Chem. 239, 3808 (1964). 26. FEIGELSOE;, P., FEIGELSOX, M., AND GREENGARD, O., Rec. Prog. Harm. Res. 18, 491 (1962).
OF
BIOCHEMISTRY
AND
Effect of Growth
BIOPHYSICS
113, 718-724 (1966)
Hormone
on Enzyme
Hypophysectomized JOSEPH P. LIBERTI:
Activities
Rat’
JOHN C. COLLA,3 JOHN F. VAN PILSUM, UNGAR
Department of Biochemistry, University of Minnesota, Received
in the
AND
FRANK
College of Medical Sciences, Minneapolis, 1Vinnesota
November
2, 1965
The effects of hypophysectomy and growth hormone administration on the activities of various enzyme systems in rat kidney and liver extracts have been studied. Removal of the pituitary gland resulted in either essentially no changes in the enzyme activity compared to the sham-operated animals, e.g., glucose B-phosphate dehydrogenase, isocitric dehydrogenase, aconitase, and hepatic glucose 6-phosphatase,or in a change in the catalytic activity of the enzymes, e.g., lactic dehydrogenase (437,), glutamic dehydrogenase (-2470), 5’-nucleotidase (-207G), kidney glucose 6-phospha(-6Oa/,). In most of tase (-31%), tryptophane pyrrolase (-150/c), a nd transamidinase the reactions studied, the administration of growth hormone to hypophysectomized rats resulted in little alteration in activities compared with the hypophysectomized rats. The activity of kidney transamidinase, which was decreased to a marked degree by hypophysectomy, was increased by the administration of three daily doses of 20 pg of growth hormone to the hypophysectomized rats. These alterations in transamidinase activity by hypophysectomy and growth hormone treatment are unique when compared to the response of a number of other enzyme systems in the liver and kidney.
Kidneys from hypophysectomized rats have only a fraction of the transamidinase activities, in vitro, of kidneys from intact or sham-hypophysectomized rats. Hypophysectomized rats that had been given injections of growth hormone had higher kidney transamidinase activities than hypophysectomized rats (1). The response of the kidney enzyme under these circum-
stances was specific for growth hormone. The loss of body protein from hypophysectomized rats and the retention of nitrogen in hypophysectomized rats treated with growth hormone are well documented (2). Recently a low rate of nL-C14-leucine incorporation into protein was demonstrated in a cell-free system prepared from livers (3, 4) obtained from hypophysectomized rat,s. The rate of incorporation in the in vitro system was partially restored to normal in livers from hypophysectomized rats treated with growth hormone. It is reasonable to expect that an effect on protein synthesis would also be reflected in observable changes in enzyme activity. The activities of certain other enzyme systems were examined to see if any were altered as markedly by hypophysectomy as rat kidney transamidinase. The choice of enzymes for study was
1 This research was supported in part by grants from the American Cancer Society, North Dakota Division, No. P-375, and from the National Institutes of Health, U.S. Public Health Service, Nos. A-2731 and AM-02583. 2 Postdoctoral trainee in the Training Program for Steroid Biochemistry, University of Minnesota. Present address: Sloan Kettering Institute, New York. 3 Postdoctoral trainee in the Training Program for Steroid Biochemistry, University of Minnesota. 718
ENZYME
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determined by their possible significance in terms of the known physiological effects of growth hormone, the faci1it.y of determination, and the possibilit,y of purification if further studies were warranted. A few of the enzymes previously had been investigated to some degree and a comparison with the older studies as reviewed by Knox et al. (5) could be of value. Tissues were obtained from hypophysectomized rats, sham-hypophysectomized rats, and hypophysectomized rats treated with growth hormone. The enzyme activities of liver and kidney preparations were measured and compared with the changes in kidney transamidinase activities in the same animals. In no instance could changes be described in any of the enzymes investigated which were comparable to the effects seen for kidney transamidinase. This enzyme at present is unique in the degree of response to hypophysectomy and to subsequent treatment with growth hormone. EXPERIMENTAL
PROCEDURE
Materials Reagent grade solvents and inorganic compounds were used. The following were employed in the enzyme assays in the form as purchased: reduced and oxidized pyridine mlcleotides (Sigma, St. Louis, Missouri), disodium-glucose B-phosphate (California Corporation Biochemical Research, Los Angeles), sodium pyruvate, nL-isocitric acid, sodium succinate, ATP and AMP (Nutritional Biochemicals Company, Cleveland, Ohio), and bovine albumin (Pentex Incorporated, Kankakee, Illinois). Ovine growth hormone (GH) was supplied by the Endocrine Study Section of the National Institutes of Health (NIH-GH-S6) and had an activity of 1.6 units per milligram. The reaction involving the production of cisaconitate, and the reduction of NAD or oxidation of NADP were monitored continuously in a recording double-beam spectrophotometer (Bausch and Lomb, model 505). Male Sprague-Dawley rats weighing 100 gm were hypophysectomized or sham-operated via the parapharyngeal route at the Hormone Assay Laboratory, Chicago, Illinois, and shipped to our laboratory 3 days after the operation. The animals were housed individually with access to a complete synthetic diet (6) and water ad libitum. The hypophysectomized animals were given daily intraperitoneal injections of 20 pg of GH in 0.9% saline on days 7 to 10 after hypophysectomy (1).
GROWTH
HORMONE
719
Control animals received injections of saline on the same days. The animals were weighed when the injections were started and at t,he time of sacrifice. The effectiveness of the GH preparation was verified by t,he weight gains of the hypophysectomized animals that received GH and by the activities of the kidney enzyme, transamidinase.
Tissue Preparation The suspending medium and the enzyme concentrations of tissue preparations varied with the different enzymes assayed. The general procedure was as follows: the animals were killed by decapitation, after which the livers or kidneys were removed and placed on ice or in ice-cold 0.25 M sucrose or 0.14 &Z KCl. The tissues were then blotted, weighed, minced, and homogenized in a Potter-Elvehjem homogenizer fitted with a Teflon pestle. FlIrther steps in the tissue preparations are noted in the text.
Enzyme A says Preliminary test)s were done to determine the enzyme concentrations with which zero-order kinetics were obtained. The linearity of the enzyme catalyzed reactions m-as determined as a function of both the incubation t,ime and tissue concentration. The rate of srtbstrate utilized or product formed was directly proportional to the tissue concentrations and incubation periods. The enzyme assays were performed in duplicate, except for transamidinase and xanthine oxidase, and also at two enzyme concentrations in the presence of excess substrate. The tissues from all of the animals were handled in as much the same manner as possible in order to minimize changes in enzymic activit,y due to procedural manipulations. The activity of each enzyme preparation was also monitored at varions times after homogenization to check its stability. Protein concentration was determined by the spectrophotometric method of Lowry (7) or by the biuret reaction (8). Crystalline bovine serum albumin was used as the standard. All results are listed in Table I as the average values f the standard deviations. Transamidinase. The method of Van Pilsum et al. (9) was used in the measurement of kidney hotransamidinase activity. A 5% rat kidney mogenate was used as the source of enzyme. The enzyme activity was expressed as milligrams guanidinoacetic acid formed per gram kidney per hour. Kidneys from hypophysectomized rats had 19% of the transamidinase activities of kidneys from the sham-operated rats. The kidney transamidinase activites of the hypophysectomized rats that had been given daily injections of 20
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TABLE ENZYME
ACTIVITY HORMONE
I
CHANGES IN THE R.LT AFTER SHAM OPERATION, HYPOPH~SECTDMY, .IND GILOWTH ADYINISTL~TION (20 PG/D.IU FOIX 4 D:LYS) TO THE H~PDPHYSECTDMIZED ROT
Enzyme assay
Tissue IIypophysectomized
Sham-operated
~
1. Transamidinase 2. Lactic dehydrogenase 3. (+lucose B-PO1 dehydrogenase 4. Isocitric dehydrogenase 5. Sllccinoxidase 6. Aconitase 7. Glutamic dehydrogenase 8. 5’.Nucleotidase 9. Arginase 10. Glucose G-phosphatase 1Oa. Glucose O-phosphatase 11. Xanthine oxidase 12. Tryptophane pyrrolase
Kidnel Liver ” Liver
3.2 f 0.2 19,600 * 1900 311 & 10
Kidney 1 Kidney Kidney Liver
21.9 + 1.5 /
87f4 1589 Z!Z 153 5900 + 212
~
5% Change from sham .~
Hyvwbsectomized with GH Wh4day)
0.G + 0.2 11,000 * 9io 304&14
-8lh ~ -43h -2
1.5 It 0.1 9800 f 1100 281 f 11
23.1 rt 1.9
~ +5
29.8 zt 1.3
+3
89 f 3 1448 + 140 4655 xt 142
90 i 1592 * 4585 f
3 185 180
- 24h
1.2 14 0.16
-20” -45O -5
Liver Liver Liver
39.6 =I= 1.6 461 & 83 2.00 It 0.26
31.6 * 253 f 1.90 f
Kidney
2.15 + 0.15
1.48 + 0.21
Liver Liver
7.3 f 0.7 11.7 + 3.4
10.8 f 9.5 f
0.9 4.1
0
35.2 f 1 251 f 2.22 f
r,fC&w (Iwpophyectomized
1.2 20 0.19
-3lb
1.48 f
0.20 I
1 +48” , -19
10.2 * 7.9 *
1.0 1.9
-1 +li” 0 -G
-17
ClUnits of activity: (1) transamidinase, mg guanidinoacetic acid/gm kidney/hour; (2) lactic dehydrogenase, rmoles pyruvate/mg liver/hour; (3) glucose H-phosphate dehydrogenase, Mmoles G-fi-P/gm liver/hour; (4) isocitric dehydrogenase, units/mg kidney where 1 unit = 01) increase of O.Ol/min at room temperatrlre; (5) succinoxidase, ~1 OJmg kidney/30 min; (6) aconitase, units/my kidney where 1 unit = OD increase of O.Ol/min at 25°C; (7) gltttamic dehydrogenase, rmoles cr.ketoglutarate/mg liver/ hour; (8) 5’.nrlcleotidase, rmoles Pi/mg liver/hour; (9) arginase, pmoles urea/gm liver/min; (10) glucose 6.phosphatase, ,umoles P;/mg liver/hour; (lOa) glucose 6-phosphatase, rmoles Pi/mg kidney/hour; (11) xanthine oxidase, pmoles uric acid/gm liver/hour; (12) tryptophane pyrrolase, pmoles kynurenine/mg liver (dry weight)/hour. * Significant difference (p < 0.01). fig of GH for 3 days were 2507; of the activities of the hypophysectomized rats (Table I, Assay 1). Lactic dehydrogenase. The assay method and liver tissue preparation were essentially those described by Weber (10). The oxidation of NADH was measured continuously for 5 minutes at 340 rnp at room temperatllre. The control cuvette contained all the reagents except the liver homogenate. Enzyme activity was expressed as pmoles pyruvate reduced per milligram liver per hour. As seen in Table I (Assay 2)) the livers from hypophysectomized rats had lower enzyme activities than livers from the intact controls. The injections of GH rrere without any effect on the enzyme. Glucose 6.phosphate dehydrogenase. The spec-
trophotometric assay of Kornberg and Horecker (11) for glucose G-phosphate (G-6-P) dehydrogenase in a 5yc liver homogenate was employed. Enzyme activity was expressed as micromoles G-6-P oxidized per gram liver per hoL[r. The results are shown in Table I, Assay 3. All three groups of rats had similar liver G-6-P dehydrogenase activities. Isocitric dehydrogenase. Isocitric dehydrogenase activity was measured spectrophotometrically at 340 rnr in a lye kidney homogenate according to the method of Ochoa (12). i\ctivity was expressed as units per milligram kidney. One unit, represent,ed an increase in optical density of 0.01 per minute. As shown in Table I, Assay 4, the removal of the pituitary gland had little or no effect on the enzyme-catalyzed conversion of isocitrate to
ENZYME
ACTIVITIES
AND
a-ketoglutarat,e. The hypophysectomized rats that received GH had greater kidney isocitric dehydrogenase activities than the intact controls or the untreated hypophysectomized rats. Succinoridase. It was shown by Melhuish and Greenbaum (13) that, the administration of GH to the rat resulted in a low P/O ratio when p-hydroxybutyrate was used as the oxidizable substrate, and that this decrease was due to increased oxidation of the substrate. A 10% rat kidney homogenate in 0.25 M sucrose was prepared. The oxidation of succinic acid was measured manometrically at 37” with a shaking rate of 120 cycles per minute. Readings were t,aken every 10 minutes for 30 minutes. Activity was expressed as microliters oxygen utilized per milligram kidney per 30 minutes. The data are presented in Table I, ilssay 5. All rats had similar kidney succinoxidase act,ivities. Bconitase. A 2% kidney homogenate prepared in 0.14 J4 KC1 was used as the source of aconitase. The enzyme activity was measured according to the method of Racker (14) by the appearance of eis-aconitate observed as 240 rnr after 4 minutes of incubation at 25°C. Activity was expressed in units per milligram kidney where one unit represented an increase in optical density of 0.01 per minute. From Table I, Assay 6 it can be seen that hypophysectomy or treatment of hypophysectomized rats with GH did not alter the kidney aconitase activities. Glutamic dch@ogenase. The procedure employed for the assay of glutamic dehydrogenase was a modification of the methods of Olson (15) and Endahl (16) using a 1% liver homogenate in distilled water. The reaction vessels contained, in a final volume of 3.0 ml, 12 pmoles of a-ketoglutarate adjusted to pH 6.8 with NaOH, 190 pmoles of ammonium acetate, 0.11 pmole of EDTA, 100 pmoles of tris buffer, pH 7.8, and the homogenate. The reaction was started by the addition of 0.33 pmole of NADH, and the oxidation of NADH R-as measured continuously against a control cuvette containing all the components except NADH. Activity was expressed as pmoles a-ketoglutarate reduced per milligram liver per hour. Livers from hypophysectomized rats had slightly less glutamic dehydrogenase activity than livers from the sham-operated rats, but a single dose of 20 pg of GH daily for 4 days did not alter this effect. 6’.Nucleotidase. The source of 5’.nucleotidase (17, 18) was a 10yo liver homogenate in 0.14 al KC1 which was diluted 1: 10 with isotonic KC1 and then strained through several layers of cheesecloth before use. The incubation reaction was stopped with 107; TCA and samples of the centrifuged supernatant fluid were u-ithdrawn and
GROWTH
HORMONE
721
analyzed for inorganic phosphate by the method of Fiske and SubbaRow (19). The activity was expressed as micromoles inorganic phosphate liberated per milligram liver per hollr. A small but significant inhibitory effect of hypophysectomy on the activity of 5’.nucleotidase is shown in Table I, Assay 8. Hypophysectomieed rats that received 20 rg GH per day for 4 days had a small increase in liver 5’.nucleotidase activity compared with the untreated hypophysectomized rat. Panda and Gael (18) showed that the administration of large quantities of growth hormone to normal rats resulted in a greatly increased activity of this enzyme. drginase. The a,ctivity of arginase was determined by the method of Folley and Greenbaum (20). A lyc liver homogenate in isotonic saline served as the enzyme source. A unit activity was expressed as the quantity of enzyme that liberated 1 pmole of urea irl 1 minute at 37”C, pH 9.45, from 0.023 iU L-arginine solution. Arginsse activities of the livers from the hypophysectomized rats were 5576 of the activit,ies of livers from the shamoperated control rats (Table I, Assay 9). The enzyme activity remained at 557, of the controls in the hypophysectomized rats that received 10 fig of GH daily for 7 days. In this same experiment it was found that kidneys from the hypophysectomized rats had 377, of the transamidinase activities from the sham-operated controls. Kidneys from hypophysectomized rats that had received seven daily injections of 10 pg of GH had 70yo of the activities of kidneys from the sham-operated rats. Tryptophane pyrrolase. Tryptophane pyrrolase was assayed according to the method of Knox (21). A 12.5yo liver homogenate was used as the enzyme source. The enzyme activity was expressed as micromoles kynurenine formed per milligram liver (dry weight) per hour. The enzyme activity in the liver was lower in the hypophysectomized rats and in hypophysectomized rats treated with GH as compared with the intact controls (Table I, Assay 12). These differences, however, were not significant. In another group of rats, administrat’ion of tryptophane resulted in an eighfold increase in tryptophane pyrrolase and cortisol administration caused an activity, increase of 255yo over the control vallles. Xanthine oxidase. The livers were assayed for xanthine oxidase activity by the method of Van Pilsum (22) with a 10% homogenate in 0.066 11f phosphate buffer, pH, 7.4. The activities were expressed as micromoles uric acid formed per gram liver per hour (Table I, Assay 11). Livers from hypophysectomized rats had 48% greater xanthine oxidase activities than livers from the sham-
722
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ET AL.
jetted with a small dose of GH, 20 pg, for only 3 days. It is quite possible that by increasing the periods of time after the operat,ion, the dose of GH, and the period of administrat,ion, different results for certain of the enzymes may have been obtained. The unique changes in t,ransamidinase activity under the conditions stated are interpreted to indicat’e that a factor(s) is operating which does not affect the activity this treatment (Table I, Assay lOa), i.e., hypophy- of t*he ot,her enzymes. It is possible that this sectomy decreased glucose 6-phosphatase ac- difference is due, in part, to inanition and had no tivity by 317~, and CH administration to the init’ial increase in creatine level (1) effect on this low enzyme activity. which occurs immediately following operation and which will decrease transamidinase DISCUSSION activity (24). The administ#ration of GH Twelve enzyme activities, in addition t.o could affect’ all enzyme systems, as a result the kidney enzyme transamidinase, were of increased protein synhheses, but in admeasured in the livers or kidneys obtained dition, it may have a specific stimulatory from hypophysectomized rats, hypophyUnder effect on transamidinase activity. sectomized rat)s t.hat had been given in- chronic conditions in terms of days after jections of GH, or from sham-operated rats. hypophysectomy and the length of time and Hypophysectomized rats had low kidney dosage of GH administration, the effect of glucose 6-phosphatase and transamidinase GH on prot,ein (enzyme) syntheses would activities, and in the livers the activities be manifest. In the acute situation existing were low for the following: lactic dehydroin the first few days of these experiments, a genase, glutamic dehydrogenase, arginase, decrease of enzyme activit’y by a creatine 5’-nucleotidase, and tryptophane pyrrolase. feedback mechanism could be a prominent In the case of liver xanthine oxidase an factor (1). actual increase was observed as a result of The general problem of t’he role of INhypophysectomy. Ko alteration in enzyme trition on enzyme activity has been disactivities after hypophysectomy was ob- cussed in more detail previously (1) and served for t,wo liver enzymes, glucose 6- will be considered only briefly here. Some phosphate dehydrogenase and glucose 6- of t,he enzymes st,udied, such as lactic phosphatase, and for three kidney enzymes, dehydrogenase and glucose 6-phosphatase, isocitric dehydrogenase, succinoxidase, and have been shown to be affected by diet (10) aconitase. In comrast to t#he marked re- while others are relatively insensitivo. sponse of transamidinase activities in Changes in dietary int,ake, particularly in kidneys of hypophysectomized rats t#reated the first few days after operation, could with GH, there was no stimulation of this account, at least in part, for the differences order of magnitude for any of the other in enzyme activities noted in the hypophyenzyme systems studied. sectomized rat. In the first 3 days after A number of factors should be considered hypophysectomy there is a period of inaniwhen comparing the large changes in ac- tion with a rapid decrease in transamidinase tivity observed for rat kidney transamidiactivity (1). The enzyme changes are nase with the changes observed for t,he similar to those observed with inanition in other enzyme systems. The length of time the sham-operated rat. These early changes after hypophysectomy was relat,ively short. are not reversed by GH (1). Stimulat.ion by In most cases, rats were sacrificed only ten GH occurs approximately seven days and days after hypophysectomy or sham opera- thereafter following hypophysectomy. Studtion. Furthermore, t,he animals were in- ies with force-feeding or pair-feeding over hypophysectomized rats. Growth hormone injections, however, did not alter the xanthine oxidase activities. Glucose 6.phosphatase. The assay for glucose 6.phosphatase was carried out by the method of Cori and Cori (23) with a 2.5(r, homogenate of rat, kidney or liver in isotonic KCl. Enzyme activity was expressed as micromoles inorganic phosphate released per milligram protein per hour. Hepatic glttcose 6.phosphatase was unaffected by the removal of t,he pituitary (Table I, Assay 10). The kidney responded in a totally different manner to
ENZYME
ACTIVITIES
AND
a period of 7-10 days demonstrated that with regard to transamidinase (1) enzyme levels were low in the hypophysectomized rat compared with the sham control that was fed the same amount of diet. Only GH stimulated the enzyme under these conditions. In the studies reported here for certain other enzyme systems, the small differences seen in the hypophysect)omized and intact’ rats could have been due to dietary fact’ors. With few exceptions, changes in enzyme activity were not large. It should be noted that liver xanthine oxidase, a most sensit,ive indicator of dietary protein intake, was higher in hypophysectomized rats than in sham-operated rats, but. administration of GH to hypophysectomized rats did not affect the enzyme activity. Special comment is warranted for the results observed for glucose 6-phospha0ase in which a decrease was seen in the kidney enzyme activity whereas the activity in the liver showed no change. When the values are expressed per milligram protein nit,rogen the total enzyme activity per animal is much lower than indicat,ed, since the kidney weight of the hypophysectomized rats is also low. The fact, that certain enzymes in the kidney will respond to hypophysectomy and possibly to GH suggests that the kidney, in this respect, may be considered to be a target organ for this pituitary factor. An effect seen with kidney glutaminase but not liver glutaminase previously had been shown by others (25). Tryptophane pyrrolase (26) and arginase (25) are two examples of liver enzymes in the rat which have been shown to be stimulated by cort,isol administration. These enzymes showed a decrease of activity after hypophysectomy, but did not respond to the treatment of GH administration as employed here. Isocitric dehydrogenase is of some interest with regard to GH since both citrate and isocitrate are metabolized in the kidney. These substances are linked wit,h calcium metabolism by virtue of the formation of a soluble c$alcium complex, and calcium excretion is well document,ed as a consequence of GH administration. In this study no changes in activit,ies from the control
GROWTH
HORMONE
723
levels were seen in kidneys from hypophysect,omized rats. However, kidneys from rats injected wit,h GH had higher activities than those from t’he control rats. This effect cannot, be explained at present but the possible relationship of this enzyme with GH artivit’y merits further investigation. Another kidney enzyme related to citrate metabolism, aconitase, did not show any changes in act)ivity with the various treatments. Further st,udies wit,h growth hormone on some of these enzyme systems would be of value particularly under chronic conditions. It is obvious that the aherations of enzyme activities observed under the experimental circumstances used in this report, require more detailed study since these enzyme changes were not nearly as marked as those seen for kidney transamidinase. Considering the possible importance of transamidinase in terms of maintaining creatine levels in the body, there may be a pronounced sensitivity and specificity of GH for this one enzyme alone. However, a more likely premise at the present time is that the changes in transamidinase activit,y arc the result of a secondary effect, namely, a lowered enzyme activity due to inanition followed by increasing levels of creatine. The levels of creatine have been shown to reach a maximum in the blood, kidney, and urine in the third to fourth day after hyljophysect,omy (1) just as t’hey do in starvation. The changes observed for kidney transamidinase may involve a feedback mcchanism or repression (24) by t.he increased levels of creatine. In addition, there would be a lack of protein (enzyme) synthesis during periods of prot.ein depletion, starvation, or after hypophysectomy. These combined fa(%ors would result in a more profound decrease in transamidinase activity as comI)ared t,o other enzymes. The apparent, specificity of GH action would then bc a result, of its normal anabolic activit,y under conditions in which the transamidinase activity is at a very low level. The over-all effect, of GH would appear to be cnhanccd due to t)hc conditions prevailing after hypophysectomy which have an exaggerated cffec&t on transamidinase.
724
LIBERTI
The response of kansamidinase in the hypophysectomized rat to GH administration could be used as a sensitive and useful model system for t,he study of the actions of GH on enzyme act,ivation or enzyme synthesis, or both. REFERENCES 1. UNGAR, F., AND VAN PILSUM, J. F., Endocrinology (submitted). 2. SMITH, R. W., JR., GAEBLER, O., ASD LONG, C. N. H., “The Hypophysial Growth Hormone, Nature and Actiotts.” McGraw Hill, New York (1955). 3. KORNER, A., Riochem. J. 73, 61 (1959). 4. KORNER, A., Rec. Prog. Horm. Res. 21, 205 (1965). 5. KNOX, W. E., AI-ERBAcH, V. H., AND LIN, E. C. C., Physiol. Rev. 36, 164 (1956). 6. VAN PILSUM, J. F., WICKENG, E. Z., AND FILONOM-ICH, L. K., I’oxicology and Applied Pharmacology 3, 431 (1961). 7. LOWRY, 0. H., FARR, A. I,., ROSEBROUGH, N. J., AND RANDALL, I<. J., J. Viol. Chem. 193, 265 (1951). 8. LAYNE, E., in “Methods in Enzymology” S. P. Coiourick and N. 0. Kaplan, eds.), Vol. III, p. 450. Academic Press, New York (1957). 9. v.4~ PILSUM, J. F., BERMAN, I). A., AND vl’oLIN, E. A., Proc. Sac. E’xptl. Biol. Med. 96, 96 (1957). 10. WEBER, G., AND CANTERO, A., Cancer Res. 19, 763 (1959). 11. KORNBERQ, A., AXI) HORECKER, B. L., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 323. Academic Press, New York (1955).
ET AL. 12. o('HO.%. s., in “Methods in Enzymology” (8. P. Colowick and N. 0. Kaplan, eds.), \‘ol. I, p. 739. Academic Press, New York (1955). 13. MELH~-ISH, A. H., APZI) (~REENBAC-M, A. L., Hiochem. J. 78, 392 (1961). 14. RACKER, I?., Eiochem. Biophys. Ada 4, 211 (1950). 15. OLSOX, J. A., AND AIVFINSEX, C. B., J. Biol. Chem. 197, 67 (1952). 1G. ENDAHL, P., Proc. Enptl. Biol. Med. 24, 192 (1957). 17. HEPPEL, L. A., ASD HILMOE, R. J.,in “Methods itt Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 565. Academic Press, New York (1955). 18. PAND.4, N. c., (;OEL, B. K., M~NsOOR, &I., AXD TAI,I\.AR, (;. P., Biochem. J. 82, 176 (1962). 19. FISKE, C. H., AND S~BBAROX, Y., J. Hiol. Chem. 66, 375 (1925). 20. FOLLEI., S. J., AND C;REEKBAUM, A. L., BioChem. .3. 43, 537 (1948). 21. Ksos, W. E., in “Methods in Enzymology” i&S. P. Colon-ick and N. 0. Kaplan, eds.), Vol. II, p. 242. Academic Press, New York (1957). 22. VAN PILRCM, J. F., J. Biol. Chem. 204, (i13 (lQ53). 23. &RI, C. F., .4x11 &RI, (>. T., J. Biol. Chem. 199, 661 (1952). 24. WALKER, J. B., AND WALKER, M. S., J. Biol. Chem. 237, 473 (1962). 25. SCHMKE, R. T., J. Biol. Chem. 239, 3808 (1964). 26. FEIGELSOE;, P., FEIGELSOX, M., AND GREENGARD, O., Rec. Prog. Harm. Res. 18, 491 (1962).