PRELIMINARY NOTES 2 I 5

Since dityrosine and Compound n have identical optical properties and migrate in the same manner on ion-exchange columns and in paper chromatography it can be concluded that they are identical.

During the enzymic oxidation of tyrosine, another compound was also found (present in the peak at 370 ml in Fig. I) which with respect to ultraviolet absorption and fluorescence corresponds closely to Compound I from resilin. By analogy with the results from the oxidation of p-cresol 4 this compound call be assumed to be tri- tyrosine (B, R = CH~CHNH2COOH ) although conclusive evidence for this structure is still lacking.

As far as I am aware, this is tile first demonstration of the natural occurrence of these two amino acids in a protein. In resilin their function must be to link the peptJide chains together in a stable three-dimensional network with rubber-like proper- ties. A detailed discussion of these results is ill preparation.

Zoophysiological Laboratory B, University of C@enhagen, Copenhagen (Denmark)

SVEND 0LAV ANDERSEN

1 S. O. ANDERSEN, Biochim. Biophys. Acta, 69 (1963) 249. 2 S. O. ANDERSEN AND T. WEIS-Fo~H, Advan. Insect 2Physiol., 2 (1964) I.

S. O. ANDERSEN AND B. KRISTENSEN, Acla 2Physiol. Scan&, 59, Suppl. No. 213 (1963) 15. 4 W. W. WESTERFELD AND C. LOWE, J. Biol. Chem., 145 (1942) 463 . 5 A. J. GRoss AND I. W. SIZER, J. Biol. Chem., 234 (1959) 1611.

Received Ju ly 27th, 1964 Biochim. Biophys. Acla, 93 (1964) 213-215

P N 21o47

Feedback inhibition by pregnenolone: A possible mechanism

Previous investigations in this laboratory have established the fact that pregnenolone inhibits the conversion of E7~-3Hlcholesterol to EaH]pregnenolone by acetone-dried powder of adrenal mitochondria 1. This inhibition takes place at the first step in the series of reactions between cholesterol and pregnenolone, i.e., tile conversion of chol- esterol to 2o~-hydroxycholesterol. Since the conversion of pregnenolone to steroid hormones in the adrenal cortex involves extramitochondrial enzyme systems, it is clear that pregnenolone must leave the mitochondrioI1 to undergo appropriate trans- formation to corticosterone, cortisol, etc. 2.

Cholesterol Pregnenololle ~ 17~-hydroxylation

~ "-~ 2 I -hydroxyla t ion 2oa-hydroxyeholesterol > 2oa,22~-dihydroxycholesterol 3fl-ol-dehydrogenase*

Mitochondrial Ext rami tochondr ia l

These observations suggest tha t the conversion of cholesterol to pregnenolone constitutes a discrete segment of a biosynthetic pathway, confined within a subcelhilar

* The sys temat ic name of this enzyme is 3-fl-hydroxysteroid: NAD oxidoreduetase ( E C 1 . 1 . 1 . 5 1 ) .

Biochim. Biophys. Acta, 93 (1964) 215-217

216 P R E L I M I N A R Y N O T E S

particle, with the result that inhibition by pregnenolone regulates the rate of its own production by a feedback or end-product inhibition s. The present findings suggest a possible mechanism for this inhibition.

Acetone-dried powder of bovine adrenal cortex was prepared and freshly ex-- tracted with phosphate buffer before use in the following experiments (the extract is here called adrenal extract). Adrenal extract was incubated with [7~-aH]eholesteroi, TPN, a reducing system and buffer for Io rain with constant shaking in air at 37 °, as described elsewhere ~. Following incubation the mixture was extracted in methylene chloride and applied to paper in the system ligroin-propylene glycol ~ for Io h. Radio- activity present in the form of [3H]pregneno!one was measured by liquid scintillation spectrometry. Proof of the identity and radiochemical purity of the [aH]pregnenolone has been given elsewhere ~.

T A B L E I

THE INFLUENCE OF ITgg-HYDROXYPREGNENCLONE UPON THE INHIBITION" BY PREGNENOLONE OF THE CONVERSION OF [7C~-3H]CHOLESTEROL TO [3H]PREGNENOLONE BY ADRENAL EXTRACT

A ddi~io~¢

PregnenoIone z7x-Hydroxypregmnolom (~g/beake~ (#g/be~keO

[~HJPregnenolone (disintegm~ions/mi~)

o o 4 5 6 o o o o o 472 o o o 5 o 322 o o o 5 o 3o8 o o o

i o o 318 o o o i o o 298 ooo

5 5 418 o o o

5 5 4o7 o o o 5 I o 4~8 o o o 5 i o 4 IO ooo

i o i o 334 ooo IO s o 377 ooo

The data presented in Table I show that pregnenolone produced an inhibition of the conversion of [7~-aH]cholesterolto [3H]pregnenolone but that the same concen- tration of iT~-hydroxypregneno!one was without demonstrable effect. On the other hand, when I7~-hydroxypregnenolone (in doses which were without effect when added alone) was added with pregnenolone, the inhibition produced by the latter was reversed. In six other experiments identical With that shown in Table t, using three separate preparations of acetone powder, this reversal of pregnenolone inhibition by i7e-hydroxypregnenolone was observed. Although such reversal was seen m each experiment, maximal reversal in various experiments was produced by a ratio of I7c~-hydroxypregnenolone to pregnenotone in/,g/beaker of 5:5 or of io:5. This vari- ation was not related to the use of different powders and at present remains un- explained.

When pregnenolone inhibition and maximal reversal of inhibition by i7c~-hy- droxypregnenolone were expressed as percentages of the conversion of [7c~-~H]chol - esterol to [aH]pregnenolone in the absence of either steroid, the mean inhibition for 7 experiments (duplicate determinations for each condition tested) was 59 % with a standard error of ± 6.! yo for pregnenolone at 5/,g/beaker and for maximal reversal

Bioch~:m. B i o p h y s . A c t a 93 (z964) z I 5 2x7

PRELIMINARY NOTES 2 I 7

was 92 % with a standard error of ~ 3-4 %- In each experiment it was shown that I7e-hydroxypregnenolone (at concentrations of 5 and IO/,g/beaker) was without significant effect upon the conversion of [7~-~H~cholesterol to [3H~pregnenolone in the absence of pregnenolone.

In a number of cases of feedback inhibition, evidence has been presented to show, tha t the inhibitor acts by combining with the enzyme at some site other than that at which the substrate attaches 3. The inhibitor is then capable of producing a change in the conformation of the enzyme such as to inhibit the action of the enzyme upon its substrate 3. One line of evidence for such so-called allosteric effects is the obser- vation tha t substances which resemble the inhibitor in chemical structure, are able to bind to the allosteric site but are unable to produce the conformational changes necessary for inhibition. Such analogues do not therefore inhibit the reaction con- cerned but by binding the allosteric site can reverse the action of the inhibitor (ref. 3 for review and further references).

In the experiments reported here, it appears that I7e-hydroxypregnenolone re- versed the inhibition caused by pregnenolone while at the same concentration i7~- hydroxypregnenolone did not itself inhibit the conversion of [7e-all,cholesterol to [3H]pregnenolone. These observations suggest that inhibition by pregnenolone may involve an allosteric mechanism. I t is realized that the present findings offer only one line of evidence to support this view. However, the surprising reversal of inhibition b y I7~-hydroxypregnenolone encourages a search for further evidence. Such evidence will, however, require a highly purified enzyme system.

This investigation was supported by grants AM-o3 676-04 END and AM-o6 459-02 END from the National Insti tutes of Health, U.S. Public Health Service.

Departments of Biochemistry and Physiology, University of Pittsburgh School of Medicine,

Pittsburgh, Pa. (U.S.A.)

SEYMOUR B. KORITZ

PETER 1 ~. HALL

1 S. B. I{ORITZ AND P. F. HALL, Biochemistry, in t he press. 2 X. T. SAMUELS, in D. M. GREENBERG, Metabolic Pathways, Academic Press, New York, 196o. 3 j . MONOD, J. CHANGBUX AND F . JACOB, or. Biol. Chem., 6 (1963) 306. 4 R. O. BRADY, jr. Biol. Chem., 193 (1951) 145.

Received June 29th, 1964

Biochim. Biophys. Acta, 93 (1964) 215-217

P~ 21045

The isolation of human urinary follicle-stimulating hormone

Interest in the extraction of gonadotropic material of pi tui tary origin from human non-pregnant urine is reflected in numerous papers. Most of these are concerned with concentrating the total gonadotropic activity; tha t is, the activity of both FSH and of LH. There are very few papers 1-a in which a t tempts have been made to separate

Abbreviations: FSII, follicle-stimulating hormone; LH, luteinizing hormone; I-ICG, human chorionic gonadotropin.

Biochim. Biophys. dcta, 93 (1964) 217-22o