Post-translational Processing in Xenopus Oocytes Includes Carboxyl-terminal Amidation”

Xenopus oocytes are versatile cells capable of car-rying out many post-translational processes. Although previously reported not to be capable of C-terminal amidation, this report demonstrates that Xenopus OO- cytes do indeed have an amidating enzyme. The amidating activity from Xenopus ovaries is compared to the known amidating activity found in porcine pituitaries. The demonstration of C-terminal amidation by Xenopus oocytes extends their usefulness in studying post-translational events. as just described. The HPLC-purified product yielded a protonated fast atom bombardment mass ion at m/z 513 as expected. Using the pure dansyl-D-Tyr-Val-Gly and dansyl-D-Tyr-Val-CONH,, methods for separating the product and substrate of the amidating reaction could be evaluated. Rapid and complete separation were obtained using the reverse phase thin layer chromatography plates described above.

Many small peptide hormones are amidated at their carboxyl termini. The C-terminal amide groups appear to be important both for biological activity and for protection from degradation. Amidation occurs as a post-translational modification, most probably in the Golgi apparatus or in secretory vesicles of the cells. An amidating enzyme from porcine pituitaries has been partially purified and characterized. The mechanism of action is oxidative with the amide nitrogen derived from a C-terminal glycine (see reviews in Refs 1 and 2).
Xenopus oocytes, injected with the relevant macromolecules, are a useful test system for studying many biological processes including translation and post-translational processing. Injected foreign mRNAs are translated efficiently. Most interestingly, the primary translation products can often be correctly modified in post-translational processes such as precursor cleavage, acetylation, glycosylatioin, hydroxylation, and phosphorylation (see review in Ref. 3). Although Xenopus oocytes have been an excellent system for studying most posttranslational events, they have been reported to be unable to synthesize terminal amides (4, 5).
Venom glands of young queen bees synthesize the toxic peptide melittin as their main product. Melittin is synthesized as a preprohormone. Several enzymatic reactions are required to convert the primary translation product to active melittin, one of which is conversion of a C-terminal glutaminylglycine to a glutamine amide (6). When Xenopus oocytes were injected with honeybee venom gland RNA, stable polypeptides similar to insect promelittin were found. The oocyte products, however, were not amidated at the C terminus. The authors suggested, therefore, that Xenopus oocytes can not synthesize terminal amides (2, 5).
In preliminary control experiments with uninjected oocytes, clear amidating enzyme activity was observed in homogenized * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. oocytes. Given the published reports discussed previously, this was an unexpected result. Further work was undertaken to confirm this result and to compare the amidating enzyme present in Xenopus oocytes to that present in porcine pituitaries.

MATERIALS AND METHODS
Live, female Xenopus laeuis were obtained from Xenopus Ltd., South Nutfield, Surrey, United Kingdom. Ovarian tissue was removed and used directly (3). Frozen porcine pituitaries were obtained from Walls Meat Co., Hyde, Cheshire, United Kingdom. The Xenopus and porcine tissues were processed identically.
The starting tissue was weighed and then homogenized at 1 g/ml in ice-cold buffer (100 mM NaC1,25 mM PIPES,' pH 6.8,0.1% Triton X-100, 100 units/ml catalase, 1 mM phenylmethylsulfonyl fluoride). The mixture was centrifuged at 13,000 X g at 4 "C for 20 min. Supernatant was saved at 4 "C and the pellet was rehomogenized in a volume of buffer equal to that used in the first homogenization step. The mixture was centrifuged and the supernatants were pooled. The supernatants were assayed immediately for amidating activity. Each amidating reaction contained 35 p1 of tissue homogenate, 5 pl of dansylated D-Tyr-Val-Gly (61 pglml), 5 pl of catalase (1.7 X lo6 units/ml), 5 p1 of 10 mM ascorbate, 5 pl of 10 mM cu(II), and 1 pl of 650 mM KI. The reaction was incubated overnight at 37°C. Volumes of 10 pl were spotted onto a thin layer chromatography plate (HPTLC, RP-18 Fz=S, Merck 13724) and dried, and the chromatography was done at 37 "C in a solvent of methanol/water/dichloromethane (6:3:1). The plates were dried and photographed under longwave UV light.
The assay for amidating activity is a modification of a published procedure (7,8). A fluorescent label has been substituted for the radioactive label. To synthesize the fluorescent substrate, a solution of D-Tyr-Val-Gly (1 mg, 2.9 pmol) in sodium bicarbonate (100 pl, 0.2 M) was lyophilized and the residue was dissolved in acetone (500 pl) containing dansyl chloride (4.0 mg, 14.7 pmol). Distilled water was added (500 pl), and the reaction was incubated at 45°C for 1 h and the products were separated by HPLC (Synchropak RP-P 25 X 1-cm column, 0.1% HCl). One principal product peak was observed and when analyzed by fast atom bombardment mass spectroscopy it yielded an ion of m/z 359 corresponding to sodiated dansyl-D-Tyr-Val-Gly. As a control for monitoring the amidating reaction, fluorescent product was also chemically synthesized. A solution of D-Tyr-Val-CONH,.HCl(l mg, 3.2 pmol) in sodium bicarbonate (100 pl, 0.2 M) was dansylated as just described. The HPLC-purified product yielded a protonated fast atom bombardment mass ion at m/z 513 as expected. Using the pure dansyl-D-Tyr-Val-Gly and dansyl-D-Tyr-Val-CONH,, methods for separating the product and substrate of the amidating reaction could be evaluated. Rapid and complete separation were obtained using the reverse phase thin layer chromatography plates described above.

RESULTS AND DISCUSSION
Crude preparations of homogenized Xenopus ovaries and porcine pituitaries were prepared as outlined under "Materials and Methods." In both preparations the concentration (grams of tissue/volume of homogenization buffer) was kept the same. The frog and porcine extracts were assayed for amidating activity immediately after preparation. The assay employs a synthetic substrate, D-Tyr-Val-Gly, that was originally designed from the known amino acid sequences of a-melanotropin, melittin, and their precursors. This synthetic substrate was also designed to be relatively stable and reasonably resistant to the wide variety of proteolytic enzymes found in The abbreviations used are: PIPES, 1,4-piperazinediethanesulfonic acid; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; HPLC, high pressure liquid chromatography.

A.
B. tissue homogenates. In the published assay for amidating activity, '2sI-D-Tyr-Val-Gly is converted to '251-D-Tyr-Val-CONH, (see review in Ref. 1). In our study, dansylated D-Tyr-Val-Gly is the substrate. It is converted by amidating enzyme to dansylated D-Tyr-Val-CONH2. The substrate and product can then be separated on a reverse phase TLC plate and visualized by long-wave UV light (see "Materials and Methods").
The Xenopus ovary and porcine pituitary preparations were assayed for amidating activity using concentrated and diluted preparations. The results are presented in Fig. 1. In both panels, lune 1 is a positive control using partially purified amidating enzyme from porcine pituitaries. Lane 2 in both panels is a negative control where no enzyme was added to the reaction. Arrows on the side point out the positions of the substrate (5') and product ( P ) . Panel A, lanes 3 to 6, presents the results analyzing the Xenopus preparation at IO", IO-', and lo-" dilutions, respectively. Using the concentrated Xenopus preparation, a substantial proportion of the substrate was converted to product (Fig. lA, lane 3). When the preparation was diluted 10-fold, a small amount of product I was still formed (Fig. lA, lune 4 ) . Clearly, there is an amidating activity in Xenopus ovaries. respectively. Using the concentrated porcine preparation, virtually all of the substrate was converted to product (Fig. 1B,   lune 3 ) . Even a 100-fold dilution of the porcine preparation could give a significant amount of product formation (Fig. 1B, lune 5 ) . As expected, the porcine pituitary preparation contained high levels of amidating activity. Thus, both Xenopus ovaries and porcine pituitaries contain amidating enzyme. Quantitative comparisons are difficult from such crude preparations, but the original tissues were homogenized in equal volumes of buffer with respect to the tissue wet weight. By comparing the amount of amidating activity per gram of original tissue, porcine pituitaries are estimated to contain 20 to 50 times more amidating activity then Xenopus ovaries (see Fig. 1).
Concentrated preparations from both Xenopus ovaries and porcine pituitaries were tested for thermolability of the amidating enzymes. Amidating enzyme from porcine pituitary is relatively stable at 60 "C.' Both preparations were heated to 60 "C for 10 min. Concentrated preparations were then assayed before and after heat treatment. The heat treatment had no detectable effect on amidating activity from porcine pituitaries (Fig. 2, lane 3 and 4 ) . With the preparation from Xenopus ovaries, however, the heat treatment destroyed most, if not all, of the amidating activity. The reason for this difference in thermolability between amidating enzyme from Xenopus ovaries and porcine pituitaries is not known. It does suggest that the amidating activities observed in the two tissues are not due to identical enzymes.
The tissue distribution of amidating enzyme is not well known (2). It is assumed that amidating enzymes will be found in a variety of tissues involved in the amidation of peptide hormones (8). These peptides are widely distributed in neural and endocrine tissues. The presence of amidating enzyme has been reported in pituitary, thyroid, pancreas, hypothalamus, submandibular glands, cerebrospinal fluid, and serum (1,9,10). Xenopus ovarian tissue, as well as individual mature oocytes (data not presented), have now been demonstrated to have amidating enzyme. Besides extending the range of tissues demonstrated to have amidating enzyme, knowing that Xenopus oocytes are capable of Cterminal amidation broadens their potential usefulness in studying post-translational events. D. Low, personal communication.