Endopeptidase-24.11 in human plasma degrades atrial natriuretic factor (ANF) to ANF(99–105/106–126)

Share Embed


Descrição do Produto

Peptides.Vol. 10, pp. 891-894. ©PergamonPress plc. 1989. Printedin the U.S.A.

0196-9781/89$3.00 + .00

BRIEF COMMUNICATION

Endopeptidase-24.11 in Human Plasma Degrades Atrial Natriuretic Factor (ANF) to ANF(99-105/106-126) T I M G. Y A N D L E , S T E P H E N O. B R E N N A N , * ERIC A. E S P I N E R , M. G A R Y N I C H O L L S A N D A. M A R K R I C H A R D S

Department of Endocrinology, The Princess Margaret Hospital, Christchurch, New Zealand *Department of Pathology, Christchurch Hospital, Christchurch, New Zealand Received 23 February 1989

YANDLE, T. G., S. O. BRENNAN, E. A. ESPlNER, M. G. NICHOLLS AND A. M. RICHARDS. Endopeptidase-24.11in human plasma degrades atrial natriureticfactor (ANF) to ANF(99-105/I06--126). PEPTIDES 10(4) 891-894, 1989.--We previously demonstrated the presence of ANF(99-126), and ANF(99--126)cleaved between Cys t°S and Phe t°6 (cleaved ANF), in human coronary sinus plasma. We now report that cleaved ANF is formed when synthetic ANF(99-126) is added to human plasma. When synthetic ANF(99-126) was incubated in heparinizcd human plasma. HPLC analysis showed two degradation products. The main product was shown by amino acid and sequence analysis to be cleaved ANF. Degradation of ANF was inhibited by EDTA and phosphoramidon. These findings are consistent with the action of endopcptidase EC 3.4.24.11, which may play an important part in the biological inactivation of ANT. Atrial natriuretic factor

ANF

Cleaved ANT

ANT degradation

WE have previously identified atrial natriuretic factor [ANF(99126)] in human coronary sinus plasma (6), but also found that 20-30% of the immunoreactive ANF was ANF(99-126) cleaved between Cys l°~ and Phe 1°6 (cleaved ANF) (6). Cleaved ANF is a major product of ANF(99-126) degradation by kidney preparations due to the action of endopeptidase-24.11 (EC 3.4.24.11) (3), an enzyme which is located in a number of tissues and is present in serum (I ,3). We suspected that cleaved ANF in plasma may be derived from endopeptidase-24.11 activity in these tissues or plasma and now present evidence that ANF in serum or plasma is degraded largely by endopeptidase-24:11 to cleaved ANF, which is a primary product.

Endopeptidas¢-24.11

EC 3.4.24.11

radioimmunoassay (RIA) as previously described (7). In separate experiments, approximately 107 cpm of HPLC purified ~2SI-ANF (7) was incubated at 37°C in 1 ml of serum or citratcd plasma, or in citrated plasma containing either 4.2 mM EDTA, 100 KIU/ml Trasylol, 1 ttM phosphoramidon, or 0.1 mM phenylmcthysulfonyl fluoride (PMSF). Aliquots (20 ILl) were taken at timed intervals for HPLC. In preparative incubations, 260 p,g ANF(99-126) in 65 ixl HzO was added to 650 ~1 of hcparinized plasma and incubated at 37°C. Aliquots were extracted on Sep-Pak cartridges (7) and used for HPLC.

HPLC Conditions, Amino Acid and Sequence Analysis Labelled ANF samples were separated on a 25 cm (preparative incubations 15 cm) Zorbax ODS column using a gradient from 25% to 66.7% solvent B in solvent A over 37.5 minutes at 1 ml/minute (Solvent A: 0.1% TFA in water; Solvent B: 60% acetonitrilc + 0.1% TFA in water). UV absorbance was measured at 215 nm, Peptides were hydrolyzcd (110°(2 for 16 hr), the amino acids converted to phcnylthiocarbamyl dcriviatives, and quantified by HPLC (2). A manual Edman degradation procedure was used for sequencing (2).

METHOD

Blood Collection and Incubation Conditions Peripheral venous blood was drawn from a normal subject into chilled tubes containing either citrate anticoagulant or heparin, or EDTA/Trasylol (7). Preparative incubations used Heparin Sodium (Weddel Pharmaceuticals, Australia) 50 units/ml of blood. To assess loss of immunoreactive ANF in serum or hcparinized plasma, synthetic ANF(99-126) (Bachem, Torrance, CA) was added to the sample (final concentration 1 nmol/l) with or without EDTA/Trasylol and incubated at 37°C for intervals up to 16 hours. The samples were extracted on Sep-Pak cartridges and assayed by

RESULTS

Loss of lmmunoreactivity in Serum and Heparinized Plasma When synthetic ANF (1 nM) was incubated in human serum or

891

YANDLE ET AL

892

B 6°!o.

A O~r

i

__ f

15 1 0

i

.

.

.

..

.

.

.

.

OI ~,5

hi m

I"-1t 50

,o

r )

20-

10 ; i

I

I

o

Io

20

/

b

)o

10 20 30 40 @O aO 110 80 liME (mln)

FIG. 1. (A) HPLC of ANF(99-126) in heparinized plasma after incubation at 37°C for 0 hr (top panel), 2.5 hr (middle) and 4.25 hr (bottom panel). Aliquots were extracted on Sep-Pak carlxidges and analysed by HPLC with UV absorbance detected at 215 rim. Peak c is ANF(99-126). Peaks b (cleaved AND")and y (unknown) are new products. Other peaks at time 0 are plasma or anticoagulant components. (B) HPLC of [:2~I]ANF(99-126) incubated in serum. Samples were incubated at 37°C for the times indicated, mixed with TFA-acetonitrile and injected. Radioactivity was counted in the 0.5 ml fractions collected, and expressed as a percentage of the total activity eluted. Recovery of radioactivity was close to 100%. Peak c is labelled ANT, peak b is labelled cleaved ANT and peak x is an unidentified product.

heparinized plasma there was a progressive loss of ANF immunoreactivity. The disappearance rate (TI/2) of IR-ANF in serum and hepatinized plasma was almost identical (62 and 68 rain, respectively), but was greatly reduced in EDTA/Trayslol plasma (TI/2 913 min).

Preparative Incubation of ANF(99--126 ) in Heparinized Plasma and Identification of Cleaved ANF When synthetic ANF(99-126) (364 p.g/ml final concentration)

was added to heparinized plasma at 0°C, extracted and separated by HPLC, a large peak of ANF(99-126) (peak c, Fig. IA) was detected. This peak was resolved from other UV absorbing material present in plasma. After incubation at 37*(2 for 2.5 hr, most of the ANF(99-126) had disappeared and two new UV absorbing peaks (peaks b and y, Fig. IA) could be identified. At 4.25 hr, only traces of ANF remained whereas peaks b and y were still clearly present. The larger of these two new peaks (peak b) was subjected to amino acid analysis, and was sequenced. The amino acid composition was essentially the same as that of full

ANF DEGRADATION IN HUMAN PLASMA

893

B

100

80 - ~



80

...'" 60

c~

...'"'"

40

c~

""

40

.'"'""'"'"'"'""" . ..R

N

0 0

20

~,0

60

INCUBATION lINE

80

180

120

0

( minutes )

20

kO

60

80

100

120

INCUBATION TIME ( minutes )

=

C

D

80

80

60

40

40

20 ¸ ..... ,."" ...... , . . . . . " ° " ' " " =

"'""

0

20

40

D

i

60

80

v

10(]

0

120

INCUBATION TIME ( minutes )

0

2*0

40 I~ATION

60

80

100

200

TIME ( minutes )

FIG. 2. Incubation of [~2~I]ANF(99-126) in serum (A) or into the HPLC citrated plasma (B) or with EDTA (C) or phosphoranaidon (D). Samples were incubated at 37°C, mixed with TFA-acetonitrile and injected. Radioactivity was counted in the 0.5-ml fractions collected, and expressed as a percentage of the total activity eluted. Continuous line, filled circles: labelled ANF; continuous line, open circles: labelled cleaved ANF; interrupted line: unidentified product.

length ANF(99-126), containing the following amino acids (mole ratio): Asp (1.0); Glu (0.9); Ser (4.6); Gly (5.3); Arg (4.1); Ala (1.1); Tyr (1.0); Met (1.0); lie (1.0); Leu (2.0); Phe (2.1).

However, sequence analysis over three cycles gave molar yields from two free N-termini commencing at the expected Ser 99 and also at Phe t°6. These data established that peak b represented

894

YANDLE ET AL.

ANF(99-126) cleaved after Cys 1°5 and held together by the disulfide bridge to Cys 121. Incubation of Labelled ANF With Serum or Citrated Plasma On incubation of [ ~ I ] A N F in serum, the amount of labelled ANF detected after HPLC rapidly decreased (Fig. 1B), as was observed with unlabelled ANF (Fig. 1A). At the same time, two new radioactive peaks appeared; one (peak b) eluted just before ANF(99-126) in the labelled cleaved ANF position, and the other (peak x, unidentified material) eluted early from the HPLC. The pattern of these radioactive products was identical in serum and citrated plasma (not shown), suggesting that clotting factors such as thrombin are not involved. Labelled ANF degradation in citrated plasma (TV2 = 46 min) was considerably slower than that in serum (T~/: = 23 rain) (Fig. 2A,B) presumably due to the mild chelating activity of citrate. Addition of 4.2 mM EDTA, or 1 ~m phosphoramidon, a specific endopeptidase-24.11 inhibitor, to citrated plasma (Fig. 2C, D) further inhibited ANF degradation by 70 and 74%, respectively (TV2= 156 and 175 rain). Similarly, addition of 1 IxM phosphoramidon to serum inhibited ANF degradation by 67% (TI/2 serum = 27.9, T~/2 serum + phosphoramidon = 85.1 min). Trasylol and PMSF had no effect on ANF degradation in citrated plasma (data not shown). As shown in Fig. 2A, while labelled ANF progressively decreased, peak b (corresponding to cleaved ANF) behaved like an intermediate product, increasing in the first 30 min and later decreasing. In citrated plasma (Fig. 2B) both the increase and decrease in the cleaved ANF peak were slower, when compared to serum (Fig. 2A). The content of peak x progressively accumulated, but its formation was also retarded in citrated plasma. All these reactions were further inhibited in the presence of EDTA or phosphoramidon (Fig. 2C, D),

keeping with our previous findings (7). Preparative incubation with exogenous ANF followed by HPLC analysis confirmed that loss of IR-ANF in heparinized plasma resulted from degradation of ANF(99-126) to other peptides, including cleaved ANF. the structure of which was confirmed by amino acid sequencing. In both serum and citrated plasma, the pattern of labelled ANF degradation products was virtually identical (not shown). In both media two distinct peptides were formed, one of which (peak b. Fig. IB) corresponded to cleaved ANF, which behaved like an intermediate in the degradation process. The other earlier eluting labelled peptide (peak x) was not identified. Inhibition of both ANF degradation and the release of degradation products, by EDTA, and to a lesser extent by citrate, indicated a metallo enzyme was responsible for ANF degradation. Further, the marked inhibition of all these reactions by the endopeptidase-24. I I specific inhibitor, phosphoramidon, demonstrated that endopeptidase24.11 was largely responsible for ANF degradation in serum and plasma. Taken together, the results suggest that endopeptidase-24. I 1 in human plasma may be responsible, at least in part, for the presence of cleaved ANF in coronary sinus plasma (6), but the physiological role of ANF degradation in serum or plasma remains to be clarified. In view of the large difference in half lives [3.1 min in vivo (8) versus 23-68 min in vitro] our findings make it unlikely that this process in plasma or serum affects the in vivo metabolic clearance of ANF. However, much of the rapid loss of ANF from blood in vivo could be associated with endopeptidase activity in tissues, or with the hormone's rapid uptake by specific tissue receptors where ANF may still be subject to endopeptidase action. Since the biological activity of cleaved ANF is minimal (4), and infusion of endopeptidase-24.11 inhibitors potentiates the biological actions of ANF (5), it is possible that endopeptidase activity in plasma, and/or within specific tissues, has a physiological role by modulating some of the regional effects on ANF.

DISCUSSION

ACKNOWLEDGEMENTS

In initial studies of the metabolism of ANF in human serum or heparinized plasma, we noted a progressive loss of IR-ANF, which was largely prevented by the presence of EDTA/Trasylol, in

We thank Stephen Fisher for his valuable technical assistance. This work was funded in part by a grant from the National Heart Foundation of New Zealand.

REFERENCES I. Almenoff, J.; Teirstein, A. S. Clinical significance of serum thermolysin-like metzlloendopeptidase and its relationship to serum angiotensin converting enzyme in sarcoidosis. Am. J. Meal. 82:33-38; 1987. 2. Brennan, S. 04 Carrell, R. W. cd-Antitrypsin Christchurch, 363 Glu Lys: mutation at the Pa position does not affect inhibitory activity. Biochim. Biophys. Acta 873:13-19: 1986. 3. Kenny, A. J.; Stephenson, S. L. Role of endopeptidase-24. I 1 in the inactivation of atrial natriuretic peptide. FEBS Lett. 232: I-8', 1988. 4. Seymour, A. A.; Swerdel, J. N.; 1)elaney, N. G.; Rom, M.; Cushman, D. W.; De Forrest, J. M. Effects of ring-opened atrial natriuretic peptides (ANP) on mean arterial pressure (MAP) and renal excretion in SliP,. Fed. Proc. 46:1296: 1987. 5. Sybertz, E. J.; Chiu, P. J. S.; Vemulapalli, S.; Watldns, R.; Foster, C.; Pitts, B. J. R.; Haslanger, M. F. SCH 39370, an inhibitor of neutral metallo endopepfidase potentiates the biological actions and plasma

levels of immunoreactive ANF and lowers blood pressure in DOC NA rat. Twelfth scientific meeting of the International Society of Hypertension, Kyoto, May 1988 (absu" 1079). 6. Yandle, T. G.; Crozier, I.; Nicholls, G.; Espiner, E.; Came, A.; Brennan, S. Amino acid sequence of atrial natriuretic peptides in human coronary sinus plasma. Biochem. Biophys. Res. Commun. 146:832-839; 1987. 7. Yandle, T. G.; Espiner, E. A.; Nicholls, M. G.; Duff, H. Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma. I. Clin. Endocrinol. Metab. 63:72-79; 1986. 8. Yandle, T. G.; Richards, A. M.; Nichols, M. G.; Cuneo, R.; Espiner, E. A.; Livesey, J. H. Metabolic c l ~ rate and plasma half life of alpha-human atrial natriuretic pep¢ide in man. Life Sci. 38:1827-1833: 1986.

Lihat lebih banyak...

Comentários

Copyright © 2017 DADOSPDF Inc.