Estrogen-dependent trypsin-like activity in the rat uterus

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Estrogen-Dependent Localization

173, 347-354 (1976)

Trypsin-Like

of Activity

Activity

in the Rat Uterus

in the 12,000g Pellet and Nucleus’

JOSEPH KATZ, WALTER TROLL, MILTON LEVY, KAREN FILKINS, JOSEPH RUSSO, AND MORTIMER LEVITZ2 The Departments

ofobstetrics

and Gynecology and Environmental Medicine, Medicine, New York, New York 10016 Received August

New York University

School of

4, 1975

Direct evidence was obtained for the presence of hormone-stimulated trypsin-like protease activity in the rat uterus. Ovariectomized rats were either untreated (U), treated with estradiol (E), or estradiol plus progesterone (EP). The uteri were excised and subcellular fractions were prepared. Each fraction was assayed for protease activity using protamine as substrate, the cleavage products being quantitated fluorometrically following reaction with 4-phenylspiro[furan-2(3H),l’-phthalanl-3,3’dione (Fluram). Fractions from U rats yielded negative results, whereas the 12,000g pellets and nuclei from the uteri of E and EP rats exhibited appreciable activities. No significant increase in protease activity was observed in thymus and diaphragm following hormone treatment, indicating organ specificity. The enzyme (or enzymes) from the 12,000g pellet was solubilized and some characteristics were determined. The apparent K, is about 1.0 x 1OmfiM, the temperature optimum is about 44°C and maximum velocity is achieved in the alkaline range (pH - 8.5). The protease is a plasminogen activator and is inhibited by diisopropyl fluorophosphate, Antipain, and Leupeptin. These properties resemble those of trypsin.

A recent publication from this laboratory presented indirect evidence for the presence of hormone-stimulated trypsinlike proteolytic activity in rat uterine chromatin. In ovariectomized rats treated with estradiol or estradiol plus progesterone the enzyme activity was reflected in the polyacrylamide gel electrophoresis profiles of uterine histones which showed extensive breakdown of the lysine-rich band provided chromatin was incubated at 37°C prior to extraction of the histones (2). Protamine, added to the incubation mix’ This investigation was supported by Grant M74.80 from The Population Council, New York, N.Y., and Grants CA 02071 and ES 00260 from the United States Public Health Service. A preliminary report has appeared (1). 2 To whom all correspondence should be addressed: Dr. Mortimer Levitz, New York University Medical Center, 550 First Avenue, New York, N.Y. 10016. Copyright All rights

Q 1976 by Academic Press of reproduction in any form reserved.

ture prevented histone breakdown, presumably by acting as a competitive substrate (3). A new sensitive assay for trypsin-like proteases has been developed using protamine as the substrate (4). The liberated amino groups are reacted with 4-phenylspiro[furan-2(3H),l’-phthalanl-3,3’-dione (Fluram) and the adduct is quantitated fluorometrically (5). The method was applied to the analysis of subcellular fractions of rat uterus in different hormonal states. The results presented in this paper indicate that the protease activity expressed is indeed hormonally controlled 3 Common names and abbreviations used: Fluram, 4-phenylspiro [furam-2(3H), l’-phthalanl3,3’dione; Antipain, L(s)-1-carboxy-2-phenylethyl] carbamoyl-L-arginyl-L-valyl-L-argininal; Leupeptin, acetyl-L-leucyl-L-leucyl-L-argininal; U, untreated; E, estradiol-treated; EP, estradiol plus progesterone treated; PF, protamine-Fluram assay. 347

348

KATZ

and is localized in the 12,000g granules as well as in the nucleus. A second aspect of the paper concentrates on the solubilization and on some of the properties of the granule-associated enzyme.

ET AL.

Neck, N.Y.). The nuclei were suspended in 0.25 M sucrose (2 uteri/ml). Small aliquots of nuclei were assayed for DNA (10). The protamine-Fluram (PF) assay. In a typical assay 0.36 ml of 0.1 M phosphate buffer, pH 8.5, to which 100 pg protamine and 20 ~1 of the test solution or suspended pellet were added, was incubated MATERIALS AND METHODS at 37°C for 0, 2 or 4 h. Then 0.6 ml of 0.2 M borate Animals. Young adult female rats (CDcR’) weighbuffer, pH 8.0, was added. While vigorously shaking ing 200-250 g were obtained from Charles River this mixture on a vortex shaker, 0.3 ml of a 1% Laboratories, Inc. Two weeks following ovariectomy solution of Fluram in dry acetone was added. The they were divided into three groups, each receiving sample was centrifuged and the fluorescence detersubcutaneous injections at 10 AM and 4 PM daily for 4 mined in a Farrand Ratio Fluorometer equipped days. The first group (I-7) received only 0.2 ml of with Corning filter 7-51 in the exciting beam and sesame oil. The second (E) was treated with 0.3 pg of Corning tilters 3-72 and 4-70 in the secondary light estradiol in 0.2 ml of oil. The third group (EP) repath. Assays were performed in duplicate. In paralceived the same estradiol regimen but 3 mg of prolel incubations the test substance or protamine was gesterone were added to the injection medium on withheld from the medium. The data are usually Days 2,3, and 4. On the fifth day uteri were excised reported as fluorescence units/4 h/100 pg DNA, perand pooled according to hormone treatment. In one mitting normalization of protease values in extraexperiment E rats were treated as described above nuclear fractions to the approximate cell number. except that rats were sacrificed following 1, 2,3, or 4 Assay for plasminogen activation. The PF assay days of hormone treatment. was adapted to the measurement of plasminogen Materials. Protamine chloride was obtained from activation as outlined by Kessner et al. (11). These authors kindly provided the details of the assay Sigma and succinylated (6). Fluram was bought from Fisher and diisopropyl fluorophosphate from prior to publication. The assay was carried out in two stages. First, a mixture of 0.07 CTA units of Aldrich. Human plasminogen, plasmin, and urokinase were generously donated by Dr. Alan Johnson. plasminogen, the test substance (usually 25% of that used for the direct PF assay) and 0.3 ml of 0.1 M Antipain and Leupeptin were kindly donated by Dr. phosphate buffer, pH 8.5, was incubated for 1 h at T. Sugimura, Tokyo, Japan. 37°C. In the second stage, designed to measure the Preparation of subcellular fractions. Extranuclear released plasmin, 100 pg of succinylated protamine fractions. With all operations at 0-4”C, rat uterine in 0.06 ml of the same buffer were added and the samples (either pooled or separate) were homogeincubation was continued for 0,2, or 4 h. The sample nized by hand (25 strokes in a Dounce homogenizer) was then submitted to the standard PF assay as The resultin 0.25 M sucrose solution (3 ml/uterus). ing homogenates were centrifuged for 20 min at SOOg described in the preceding section. Several controls were run in which the test substance was either and the resulting pellets were resuspended in 0.25 M omitted or replaced by 0.05 units of urokinase or 0.01 sucrose (0.4 ml/uterus) and saved for use in the casein units of plasmin. protamine-Fluram (PF) assay. In some studies the Solubilization of the protease in the 12,000g granwashes were tested for enzyme activities. In other ule. The PF assay showed the protease activity to be studies the supernatant fractions from the 12,000g concentrated in the 12,000g extranuclear granules centrifugation were centrifuged at 100,OOOg. The reand the nuclei. In attempts to solubilize the granulesulting microsomal and soluble fractions were sublocalized enzyme, aliquots of the pellet were susmitted to the PF assay. pended in 1 ml of 0.25 M sucrose and subjected to the Nuclei. Three types of nuclear preparations, A, following conditions: (1) freeze-thaw in dry ice (three B, and C, were tested for protease activity in the PF times) in the presence of 0.1% Triton X-100; (2) soniassay. A was a crude nuclear fraction prepared by cation for 1 min at 0°C using the sonifier cell disrupwashing the 600g pellet described above twice and ter Model W 140 (Branson Sonic Power Co.) at conresuspending it in 0.25 M sucrose. B and C were trol setting 3; (3) shearing in a Polytron, PT 10 on purified nuclear fractions prepared as described by setting 3 for intervals totaling 1 min at 0°C; (4) Sepherd et al. (7) and Szego et al. (8), respectively. incubation at 37°C for 0.5, 1, or 2 h. After each Both nuclear preparations were purified by high procedure the mixture was centrifuged at 12,000g for speed centrifugation in 2.4 M sucrose. The major 20 min at 0°C and the resulting supernate and pellet difference was in the mode of homogenization. In C were assayed by the PF method. In one experiment the uteri were homogenized by hand in the Dounce the 12,OOOgpellet from E rats was lysed for 2 h at apparatus (9), whereas in B homogenization was 37°C and the mixture was centrifuged at 50,OOOg. achieved in a Polytron PT 10 (Brinkmann, Great

ESTROGEN-DEPENDENT The resulting supernate and pellet were assayed for protease activity. The relative efficiencies of solubilization were assessed. Enzyme properties. Several properties of the enzyme from E and EP rats were studied. The enzyme was solubilized by autolysis at 37°C for 1 h. In each experiment the assay was run in the usual way except that one of the conditions was varied. To study substrate dependence, the protamine concentration was varied from 5 to 100 pg/incubation. In other studies the temperature was varied from 25 to 60°C. In a third series of experiments the pH was varied between 4 and 10. Inhibition studies. The influence of Antipain and Leupeptin on protease derived from estradioltreated rata was examined. The assay was run in the customary way except that the final volume was 0.38 ml and the enzyme was exposed to 5-160 pg of inhibitor prior to the addition of the protamine substrate. The incubations were run for 4 h. The data are given as percent inhibition. In another inhibition study the influence of diisopropyl fluorophosphate on the protease activity was determined in the following way. The solubilized enzyme was preincubated in the absence or presence of 1 x lo-* M diisopropyl fluorophosphate at pH 8.0 for 1 hat 37°C. Then the sample was submitted to the PF assay and the results with and without inhibitor were compared. Suspensions of nuclei were also submitted to the diisopropyl fluorophosphate inhibition test. RESULTS

Protease Activity in Rat Uteri. Comparison with Other Tissues Subcellular fractions prepared from uterine homogenates of hormone-treated ovariectomized rats were assayed for protease activity by the protamine-Fluram (PF) method. Hormone-dependent activity was demonstrable in the 12,000g pellet. As shown in Table I, there is virtually no activity in the U rats. There is considerable activity in the E animals and slightly less in the EP rats. In two studies a fourth group of rats were treated with progesterone without estradiol. The results were similar to those obtained with the U rats. No activities were detectable in the 100,OOOg pellets or supernatant fractions in any rats. In two experiments crude and purified nuclei were submitted to the PF assay. The results in Table II indicate appreciable activities in specimens derived from the hormone-treated rats but virtually

RAT UTERINE

349

PROTEASE

none was detected in uterine nuclei obtained from the untreated animals. To determine the course of onset of protease activity, rats were treated with estradiol for shorter times than usual and uterine extracts were assayed. The data in Table III show that the response is not acute. More than 1 day of treatment is required for the expression of enzyme activity in the 12,000g pellet and an additional day is required for appearance of the activity in the nucleus. In one experiment the enzyme activity in the uterus was compared to those in diaphragm and thymus. The results in Table IV indicate that only the uterus exhibited a significant response to hormone treatment. The hormone induction thus, is organ specific. Curiously, this is the only experiment in which the value for the uterine extract from EP rat exceeded that of the E rat. Whereas the difference in the protease concentration in the extranuclear fraction between the E and EP rat uterus appears to be statistically significant (Table I), the physiological importance of this observation is obscure. Solubilization

of Protease Activity

The 12,000g uterine pellets from U, E, and EP rats were submitted to several conditions designed to solubilize the enzyme TABLE

I

PROTEA~E ACTIVITY IN THE 12,000~ PELLETS OF HOMOGENATES OF UTERI FROM HORMONE-TREATED OVARIECTOMIZED RATS” Hormone

Experiment Untreated 1 2 3 4 5 6

3.00 2.5 5.0 2.5 0.3 1.6

treatment

Estradiol 33 19 30 30 18 22

Estradiol + progesterone 16 13 20 19 10 9

a In each experiment the 12,000g pellets from three to five uteri were pooled and suspended in 0.25 M sucrose (0.4 ml/uterus). b Values are in fluorescence units per 4 h normalized to 100 pg of tissue DNA. Each assay was done in duplicate and the average deviations were invariably less than 10% for the hormone-treated rata.

350

KATZ

ET AL.

TABLE

II

PROTEASE ACTIVITY IN UTERINE NUCLEIC FROM HORMONE-TREATED OVARIECTOMIZED RATS Experiment

Hormone Untreated

Estradiol Nuclear

1 2

treatment Estradiol

+ progesterone

preparations

A

B

C

A

B

C

A

B

C

l.Ob 1.0

1.1 1.0

0.8 1.3

7.9 13.9

9.0 10.8

7.2 9.1

14.6 11.8

18.4 13.4

12.3 9.8

’ Nuclei preparation A (crude) and B (purified according to Shepherd et al., 7) were from the same animal, one horn being used for each preparation. Nuclei C (purified, according to Szego et al., 8) were obtained from different animals. In each experiment three to four uteri or horns were pooled. ’ Values are in fluorescence units per 4 h (not normalized as in Table I). The suspended nuclei were assayed for DNA and each incubation tube contained about 15 pg DNA. Assays were done in duplicate, the average deviations being about 10%. About 1-2 ng of trypsin afford the values shown for the hormonetreated animals. TABLE III TIME COURSE OF ONSET OF PROTEASE ACTIVITY IN ESTRADIOL-TREATED OVARIECTOMIZED RAT@ E treatment (days)

Uterine weight (mg)

Protease Nuclei

Ob 1 2 3 4

80 172 195 260 238

0.0’ 0.0 1.9 9.8 19.2

activity 12,000g pellet 1.1 0.5 10.5 13.0 21.2

a Following treatment with estradiol (El for 1, 2, 3, or 4 days, uteri were excised and homogenized. The 12,OOOg pellets and nuclei were analyzed for protease activities. b Treated with sesame oil only. c Values are in fluorescence units per 4 h normalized to 100 pg DNA and are averages of three uteri. The standard errors were less than 10%. Values less than 4-5 are indistinguishable from zero.

(or enzymes). These included mechanical destruction, the combined application of thermal shock and detergent action, and autolysis. The results are shown in Table V. Shearing released only 5% of the protease from the granules. Greater solubilization was achieved by sonication, but the results were variable. Freeze-thaw cycles in the presence of Triton X-100 caused the solubilization of about 40 and 25%, respectively, of the protease activity contained in the pellets from E and EP rats. Incubation of the 12,OOOgpellet at 37°C for 1 h consistently solubilized about 50% of the activity.

This procedure was adopted for the routine solubilization of the enzyme except that in some instances the incubation time was extended to 2 h, resulting in slightly higher yield. All the procedures described in Table V were also applied to uterine extracts from U rats. In no case was protease detected in either the soluble fraction or the pellet. Properties

of the Protease

Some of the properties of the crude solubilized enzyme are shown in Fig. 1. It is apparent that under the conditions of the assay nearly maximum reaction velocity is achieved at a concentration of 50 pg of protamine/incubation, one-half the concentration routinely used in the assay. The TABLE

IV

PROTEOLYTIC ACTIVITY IN 12,000G PELLETS OF TISSUE HOMOGENATES FROM HORMONE-TREATED OVARIECTOMIZED RATS Hormone

Tissue Untreated

treatment

Estradiol

Estradiol

+

progesterone Uterus Thymus Diaphragm

0.0” 0.4 3.3

33.6 0.7 8.0

41.5 1.0 4.4

D Values are in fluorescence units (per 100 pg of tissue DNA per 4 h) as determined by the protamine-Fluram method.

ESTROGEN-DEPENDENT

RAT UTERINE TABLE

351

PRGTEASE

V

COMPARISON OF METHODS FOR SOLUBILIZING PROTEASE IN THE 12,000~ EXTRANUCLEAR PELLETS FROM RAT UTERI IN DIFFERENT HORMONAL STATES’ Method

Hormonal

state

Estradiol

Estradiol

Pellet None Shearing Sonication Freeze-thaw in Triton Incubate 1 h at 37°C

X-100

100 45 26 36 37

k f * t

Soluble

lib 7 13 I

+ progesterone

Pellet 100 39 37 69 60

525 34 + 14 38 + 8 51 ? 3

+ + c 2

Soluble 13 20 30 20

52-5 15 + 13 21 t 9 47 k 19

n Details of the procedures for solubilization are in the text. b Values are in percent of activity in the nontreated pellet which is taken as 100. All assays are by the protamine-Fluram assay. Averages are of two to four determinations.

pH Optimum

7.-‘“, 1: : /’ _,’ 14 6 8 10 PH FIG. 1. Properties of the rat uterine protease. Ovariectomized rats were treated with estradiol or estradiol plus progesterone. The uteri were excised and subcellular fractions prepared. The 12,000g pellet was lysed for 2 h at 37°C and the soluble supernatant submitted to the protamine-Fluram (PF) assay as described in the text except that in each study one condition was varied. The influences of substrate concentration, temperature, and pH on the fluorescence yields are shown. The arrows in the third panel indicate the pH at the start and end of the incubation. The PF assay is constrained to buffers containing no primary amines or borate in the incubation medium. Rather than change buffers, phosphate, a poor buffer in the high pH ranges was used throughout.

apparent K, calculated from this and similar studies is 1.0 x lop6 M and 1.1 x lo-” M for the proteases, respectively, from E and EP rat uteri. Figure 1 also shows that maximum velocity is achieved by carrying out the incubation at about 44°C. Finally, the third panel in Fig. 1 indicates that optimum rates are obtained at alkaline PI-I.

The data in Table VI indicate that the protease is an effective plasminogen activator. Preincubation of the protease in the presence of plasminogen resulted in approximately fivefold amplification of enzyme activity. Plasminogen activation is also hormone-dependent as indicated by lack of activity in the uterine extracts from untreated rats. TABLE

VI

PLASMINOGEN ACTIVATION BY PROTEASE FROM 12,000~ PELLETS OF RAT UTERINE HOMOGENATES~ Experimentb

Hormone treatment

Protease activity - Plasminogen

1

2

3

None E EP None E EP None E EP

0.0’ 5.0 2.2 0.0 5.3 1.6 0.1 2.7 0.3

.-

+ Plasminogen 0.3 13.8 10.2 1.6 15.5 12.6 0.0 8.8 9.2

0 Ovariectomized rats were either untreated or treated with estradiol (E) or with estradiol + progesterone (EP) as described in the text. The extranuclear 12,000g pellets were lysed at 37°C for 1 h and the soluble supernates were submitted to the PF assay with or without prior incubation with plasminogen as described in the text. b In each experiment analyses were done on extracts of two to three rat uteri, pooled according to hormonal treatment. c The values are in fluorescence units/4 h.

352

KATZ

ET AL.

Inhibition Studies Antipain and Leupeptin were tested for their ability to inhibit the enzyme extracted from E rat uterus. The data shown in Table VII indicate that they are both effective inhibitors. Diisopropyl fluorophosphate inhibited the hormonally induced rat uterine protease of the 12,OOOg pellet (Table VIII) and nucleus (results not shown) with equal effectiveness.

TABLE

Experiment

TABLE

0

5 10 20 40 160

treatment!’

Protease

activity’

-DFP

+DFP

+ progester-

12.0 9.5

0.4 0.1

+ progester-

20.0 8.5

0.2 0.3

a In each experiment the 12,OOOg pellets from three pooled uteri were lysed for 1 h at 37”C, centrifuged, and an aliquot of the supernate was analyzed for protease activity. The test sample was preincubated for 1 h at pH 8.5 in the absence or presence of DFP (1 x 1O-2 M) at 37°C. Then the solutions were submitted to the protamine-Fluram assay as described in the text. b Ovariectomized rats were treated with estradiol or estradiol plus progesterone as described in the text. r The values are in fluorescence units/4 h. 14

VII

EFFECT OF ANTIPAIN AND LEIJPEPTIN ON THE PROTEA~E EXTRACTED FROM UTERI OF ESTRADIOLTREATED OVARIECTOMIZED RATS Inhibitor added” (CLB)

Hormone

Estradiol E&radio1 one E&radio1 E&radio1 one

DISCUSSION

The sensitive method of Brown et al. (4) was adapted for the assay of trypsin-like protease activity in subcellular fractions of uterine homogenates. The method depends on the hydrolysis of protamine and the assay of the newly formed amino groups with Fluram. Under the conditions described in this paper, 1 ng of trypsin is readily detected, the precision is good, the blanks are negligible, and the fluorescence yield increases in reproducibly regular fashion for at least 4 h (Fig. 2). The interexperiment variance is quite small for a biological study of this kind in which precise control was exercised only over the injection regimen and the time, postoopherectomy, of utilization of animals. Furthermore, some 50 assays can be performed on extracts of a single rat uterus and they can be handled routinely by one technician. Exploiting this novel method, a dramatic increase in estradiol-mediated

VIII

INHIBITION OF HORMONALLY-INDUCED RAT UTERINE PROTEASE ACTIVITY BY DIISSOPROPYL FLIJOROPHOSPHATE (DFPY

Percent Antipain 100 32* 71 56 48 23

of initial

activity Leupeptin 100 63 58 35 30 16

p The protamine-Fluram assay was run as described in the text with the addition of the inhibitors in amounts indicated. The molecular weights are: Antipain, 696; Leupeptin (acetyl form), 481. b The estimatedKi is about 1 x 10e5 M.

0

05

10

1.5

L 2.0 25 Time (hr)

1 30

35

40

FIG. 2. Typical assay of rat uterine protease by the protamine-Fluram method. The protease was obtained by lysing the 12,000g extranuclear pellet obtained from estradiol-treated rats. The curve shows the averages of assays done in duplicate.

proteolytic activity was demonstrated. Moreover, the activity is specific to the uterus. Although the application of gentle techniques does not preclude the translocation of macromolecules during homogenization; it is a reasonable supposition that in the hormone-treated rat the uterine pro-

ESTROGEN-DEPENDENT

tease is concentrated in the nucleus. This view is reinforced by the time-course study (Table III) in which after 2 days of estradiol treatment, protease was found in the 12 ,OOOgextranuclear granule, but not in the nucleus. After another day of hormone administration, protease activity was detected in both subcellular fractions. The protease activity in the nuclei was independent of the purity of the preparations (Table II), suggesting that the enzyme is intimately associated with the nuclear matrix rather than being loosely bound to the membrane. The extranuclear granule harboring the protease activity merits elucidation. Mitochondria, lysosomes, plasma membrane fragments, and less-defined particles sediment at 12,000g. Ordinarily, mitochondria are not associated with trypsin-like enzymes , whereas lysosomes concentrate acid proteases (12) and to a lesser extent neutral proteases (13). A resolution of the problem may result from the fractionation of the 12,000g sediment followed by comparisons of concentrations of well-established marker enzymes for each of the subcellular fractions with those of the protease. Studies are underway to purify and characterize the enzyme more completely. Nevertheless, the properties exhibited by the crude preparations are stongly suggestive of the enzyme type. The high pH optimum (8.591, the preference for basic amino acid peptides (arginine comprises more than 50% of the amino acid residues in protamine), and the effective inhibition by diisopropyl fluorophosphate indicate the presence of a serine trypsin-like enzyme (14). Plasminogen activation is also associated with cleavage of arginine peptides by serine protease (15). The protease is present during the estrus cycle of the rat and mouse (unpublished results) and in the normal human uterus (16), but its biological role has not been clarified. The enzyme possesses the capability of degrading histones (2) and of enhancing RNA synthesis (9). These phenomena may be related but our studies do not permit quantifications relative to protease activity. Uterine nuclei from the EP

RAT UTERINE

PROTEASE

353

rat exhibited far greater histone breakdown and enhancement of RNA synthesis than those seen in the E rat, yet the protease activity exhibited by the EP rat nuclei was only slightly greater than in E rats (Table II). Perhaps progesterone administration to the estradiol-primed rat produces changes in the physical structure of chromatin, rendering it susceptible to derepression by proteases or other agents. Investigations along these lines are being pursued. The ability of the protease to act as a plasminogen activator affords a mechanism for the amplification of activity. High concentrations of plasminogen activators have been found in endocrine organs (17, 18) and are associated with experimental tumors (19). Like trypsin (20), the protease described in this paper is inhibited by Antipain and Leupeptin. These low molecular weight inhibitors are excreted, in part, unmetabolized after ingestion by experimental animals, and have been shown to delay the onset of chemically-induced tumors (21, 22). If the trypsin-like protease activity described in this paper is essential for the expression of hormone action in the uterus, Antipain and Leupeptin may be useful agents to alter the normal or abnormal physiological functions of that important target organ. REFERENCES 1. KATZ, J., TROLL, W., LEVY, M., Russo, J., FILKINS, K., AND LEVITZ, M. (1974) Fed. Proc. 33, 1511. 2. LEVITZ, M., KATZ, J., KRONE, P., PROCHOROFF, N. N., ANDTROLL, W. (1974)EndocrinoZogy 94, 633-640. 3. KATZ, J., TROLL, W., Russo, J., FILKINS, K., AND LEVITZ, M. (1974) Endocrine Res. Commun. 1, 331-337. 4. BROWN, F., FREEDMAN, M. L., AND TROLL, W. (1973) Biochem. Biophys. Res. Commun. 53, 75-81. 5. UDENFRIEND, S., STEIN, S., BOHLEN, P., AND DAIRMAN, W. (1972) Science 178, 871-872. 6. KLOTZ, I. M. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. XI (Hirs, H. W., ed.), pp. 576-580, Academic Press, New York. 7. SHEPHERD, R. E., HUFF, K., AND MCGUIRE, W. L. (1974) Endocrine Res. Commun. 1, 73-85. 8. SZEGO, C. M., RAKICH, D. R., SEELER, B. J., AND

354

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GROSS,R. S. (1974) Endocrinology 95, 863-874. 9. KATZ, J., LEVITZ, M., GOR~TEIN, F., AND TROLL, W. (1970) Endocrinology 87, 294-301. 10. BURTON, K. (1956) Biochem. J. 62, 315-323. 11. KESSNER, A., ONG, E. B., AND TROLL, W. (1975) Fed. Proc. 34, 532. 12. DE DWE, C., PRESSMAN, B. C., GIANETTO, R., WATTIAX, R., AND APPELMANS, F. (1955) Bio&em. J. 60, 604-617. 13. DAVIES, P., RITA, G. A., KRAKAUER, K., AND WEISSMANN, G. (1971) Biochem. J. 123, 559569. 14. FAHRNEY, D. E., AND GOLD, A. M. (1963) J. Amer. Chem. Sot. 85, 997-1000. 15. SUMMARIA, L., HSIEH, B., AND ROBBINS, K. C. (1967) J. Biol. Chem. 242, 4279-4283.

ET AL. 16. NOTIDES, A. C., HAMILTON, D. E., AND RUDOLPH, J. H. (1973) Endocrinology 93, 210-216. 17. ALBRECHTSEN, 0. K. (1956) Actu Endocrinol. 23, 207-218. 18. ASTRUP, T. (1966) Fed. Proc. 25, 42-51. 19. UNKELESS, J., DAN@, K., KELLERMAN, G. M., AND REICH, E. (1974) J. Biol. Chem. 249,42954305. 20. SUDA, H., AOYAGI, T., HAMADA, M., TAKEUCHI, T., AND UMEZAWA, H. (1972) J. Antibiotics 25, 263-266. 21. TROLL, W., KLASSEN, K. A., AND JANOFF, A. (1970) Science 169, 1211-1213. 22. UMEZAWA, H. (1972) Enzyme Inhibitors of Microbial Origin, pp. 15-52, University Park Press, Baltimore.