Enkephalin degradation by human erythrocytes and hemolysates studied using 1H NMR spectroscopy

ARCHIVES Vol.

OF BIOCHEMISTRY

242,

No.

2, November

Enkephalin JAMIE

AND

BIOPHYSICS

1, pp. 5X-522,1985

Degradation by Human Erythrocytes and Hemolysates Studied Using ‘H NMR Spectroscopy

I. VANDENBERG,

Department

of Biochemistry, Received

GLENN University

April

F. KING,

of Sydney,

Sydney,

2, 1985, and in revised

PHILIP

AND

form

New

South

June

W. KUCHEL’ Wales 2006, Australia

12,1985

High resolution (400 MHz) ‘H spin-echo NMR spectroscopy was used to monitor the degradation of leucine-enkephalin, and peptide fragments of it, by human erythrocytes and hemolysates. We showed that leucine-enkephalin is rapidly degraded by the cytosolic peptidases of the human erythrocyte, and we have elucidated the most probable pathway of degradation. Computer simulations of the proposed pathway, using a model incorporating the experimentally derived steady-state kinetic parameters obtained for the individual enzyme steps, showed close agreement with the experimental results. From a methodological perspective, the work demonstrates the value of ‘H spin-echo NMR spectroscopy for rapidly elucidating, both qualitatively and quantitatively, an entire peptide-degradation pathway as it operates in situ. o 19% Academic press, inc.

neurotransmitter response, the presence of enkephalins in peripheral tissues such as blood, gut, and the adrenal gland (6), and the absence of enkephalinase from some of these tissues [e.g., blood; (‘7)], suggests that other proteolytic enzymes may play the major role in the termination of the more general antinociceptive activity of the enkephalins. In fact, Coletti-Previero and co-workers (7) have shown that an enkephalin-degrading aminopeptidase (EC 3.4.11.11) is mainly responsible for the short plasma half-life of 2-2.5 min for the enkephalins (2); however, their calculated plasma enkephalin half-life of 3.0 min based on the steady-state kinetic parameters of the purified enzyme indicates that this enzyme does not account totally for the enkephalin-degrading activity of the blood. Thus, since human erythrocytes contain a large range of peptidase activities (8) and these cells have been shown to be able to transport small peptides (9-11) we considered that they may play a role in the degradation of circulating enkephalins and/or their break-down products. This study was an examination of this hypothesis, and the work illustrates the use of ‘H

The “enkephalins” are two endogenous pentapeptides (Tyr-Gly-Gly-Phe-Leu and Tyr-Gly-Gly-Phe-Met) with opiate agonist activity (1). Much of the interest in them is due to their antinociceptive action, and hence their possible clinical use as analgesics. However, the enkephalins have only a very transient antinociceptive action, being rapidly degraded by the proteolytic activities of the blood and brain (2, 3). It is generally considered that the enzyme enkephalinase, a dipeptidylcarboxypeptidase capable of cleaving enkephalins at the Gly-Phe bond, is responsible for the specific deactivation of the pentapeptides in the central nervous system; the parallel anatomical distribution of enkephalinase and opiate receptors in the central nervous system (4) suggests that the enkephalins are not only antinociceptive hormones, but that they are also neurotransmitters capable of inhibiting neurones conducting pain impulses (5). Although enkephalinase may be of prime importance in terminating the central 1 Author dressed.

to whom

correspondence

should

be ad-

515

0003-9861/85

$3.00

Copyright Q 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

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KING,

NMR in the analysis of the degradation pathway of biologically active peptides in situ. EXPERIMENTAL

PROCEDURES

Materials Sodium 2,2-dimethyl-2-silapentane 5sulfonate (DSS)’ and tetramethylsilane (TMS) were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. 2HzO (99.75%)was obtained from the Australian Institute of Nuclear Science and Engineering, Lucas Heights, New South Wales, Australia. Nicotinamide was from Calbiochem, San Diego, California. Carbogen (Oz:COz, 19:l) was from Commonwealth Industrial Gases, Alexandria, New South Wales, Australia. [2,3,4,6,6-2H]Glucose was from Merck Sharp & Dohme, Pointe Claire-Dorval, Quebec. Cbz-Leu-Gly, Cbz-Phe-Leu, Gly-Gly-Phe-Leu, PheLeu, and Tyr-Gly-Gly were obtained from Sigma Chemical Company, St. Louis, Missouri. Pyrogen-free cotton wool was supplied by Tuta Laboratories Pty. Ltd., Lane Cove, New South Wales, Australia. Leucineenkephalin was purchased from Vega Biochemicals, Tucson, Arizona. All other materials were AR grade. Sample preparation Leucocyte-free human erythrocyte suspensions were prepared from freshly drawn venous blood as described previously (9). Hematocrits (Hc) were generally in the range 0.60-0.80 for transport experiments or 0.40-0.60 for lysate studies. Red cell membranes were prepared by repeated centrifugal washing in 5 mM phosphate buffer, pH 8.0 (12). Peptide substrates were added to erythrocyte preparations (lysates or cells) from stock solutions whose pH values had been adjusted to lie within the range 7.2-7.4 (the physiological pH range). The solutions for transport studies were also made isoosmotic with red cells, since it has been shown that ‘H spin-echo NMR signal amplitudes of metabolites in red cell suspensions are dependent on the osmotic pressure of the bathing medium (13). Osmotic pressures were measured using a vapor pressure osmometer (Wescor Instruments, Model 51OOC, Logan, Utah). Samples of 500-600 ~1 final volume, in 5 mm o.d. NMR tubes (52’7-PP, Wilmad, Buena, Calif.), were used for the ‘H NMR experiments. Addition of [2,3,4,6,6-2Hlglucose for cellular energy requirements and nicotinamide to preserve nicotinamide nucleotides was as described previously (9). Zero reaction-time was taken as the time of substrate addition and NMR

* Abbreviations used: DSS, sodium 2,2-dimethyl-2silapentane 5-sulfonate; TMS, tetramethylsilane; Hc, hematocrit; HGSE, homogated spin-echo pulse sequence; DAP II, dipeptidyl aminopeptidase II; CANP, calcium-activated neutral endopeptidase; Cbz, benzyloxycarbonyl.

AND

KUCHEL

measurements were normally begun within 3 min for lysates and 5 min for cell suspensions. 400 MHz ‘H NMR spectra were acquired on a Bruker WM-400 spectrometer operated at 37°C in the Fourier transform mode, and using the homogated spin-echo (HGSE) pulse sequence (9), viz.: r,-900

- 72 - 180” - rz - acquisition.

This pulse sequence involves selective irradiation of the water resonance during the period r1 (0.5-s duration) followed by the usual spin-echo pulse sequence (14) with r2 = 60 ms. Irradiation of the water resonance dramatically reduces digitizer input, allowing a higher receiver gain to be used on the spectrometer which increases the sensitivity. Application of the spin-echo pulse sequence to biological samples has been described elsewhere (15); the efficacy of this technique arises from its ability to filter from the spectrum the multitude of proton resonances arising from large immobile species, such as proteins and membrane phospholipids, on the basis of their faster spin-spin (l/T.) relaxation rates. The resulting spectrum contains, in general, only resonances from small mobile metabolites such as the peptides and amino acids of interest in this work. Spectral data were averaged in 8192 memory locations over a spectral width of 5000 Hz, thus giving a repetition time of 1.44 s. Routinely, 32-128 transients (lysates) or 128-256 transients (whole cell suspensions) were averaged for each spectrum. Samples were spun at 20 Hz to average out inhomogeneities in the bulk magnetic susceptibility of the solutions and tubes. DSS (0.2% w/v in 2H20) which was present in a coaxial capillary within the NMR tubes was used as the chemical shift (6) reference at 0.000 ppm; the signal from the 2H20 was used for field-frequency locking. The quaternary ammonium methyl resonance of erythrocyte ergothioneine was used as an internal frequency and intensity reference; the concentration of this compound is time-invariant (16). Numerical methods. Peak amplitudes measured from each spectrum were converted to concentrations by use of calibration curves (17) or by calculating the extinction coefficients of resonances using the conservation of mass conditions (11). Kinetic parameters for all hydrolytic reactions were obtained by nonlinear least-squares regression of the appropriate integrated form of the Michaelis-Menten equation onto progress curves for product or substrate. Equation [l] is the integrated equation for a non-product-inhibited Michaelis-Menten enzyme, expressed in terms of product concentration: t is the time of reaction, [P] is the product concentration at this time, [Sk is the initial substrate concentration, and K, and V,, are the Michaelis-Menten parameters;

t = PI - Km - ln(l - Pl4Sl0) V mu

PI

‘H

NMR

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The corresponding equation for substrate can be obtained by replacing [P] with ([Sk - [S]). Regression of these equations onto the data was performed using the method recently described by Vandenberg et al (11); this technique essentially renders Eq. [l] explicit in [PI. The kinetic parameters thus obtained were incorporated into a computer model to simulate possible degradation pathways. Numerical integration for the biochemical simulations was performed using the program BIOSSIM (18); all enzymes in the model were assumed to obey simple Michaelis-Menten kinetics. To derive the unitary rate constants for the biochemical simulations from the steady-state kinetic parameters, all “on” rate constants were assumed to be 1 X lo6 Me1 s-i and the concentrations of intracellular peptidases were set equal to 1.0 pM [e.g., (9)]. RESULTS

The single pulse ‘H NMR spectrum of leucine-enkephalin, with the assignment of all peaks, is shown in Fig. 1. Where possible the peaks were assigned using standard

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ERYTHROCYTES

517

techniques, i.e., analysis of chemical shifts, calculation of integrals, analysis of splitting patterns and spin decoupling experiments [see e.g., (19)]. The H* resonances of leucine, phenylalanine, and tyrosine were assigned unambiguously using pH titrations and difference decoupling experiments (20). The spectrum of leucine-enkephalin obtained with the HGSE pulse sequence was also assigned, since this sequence was used in the analysis of the degradation of the peptide in erythrocytes. HGSE spectra of tyrosine, glycine, phenylalanine, and leutine revealed that at least one set of resonances from each of the amino acids was well resolved from their counterpart resonances in the pentapeptide. This therefore meant that it was possible to monitor, simultaneously, resonances from both the substrate and products in the degradation of leucine-enkephalin. For example, the

6 H

\5

4’

/4

$H:2 H,tbi;HECONH-CH;-CONH-CHFCONH-kH%ONH-kH%Q-

DO

i

?I

5

..f I

7.2

6.0

4.8

3.6

2.4

1.2

6 (ppd

FIG. 1. The primary structure and ‘H NMR single-pulse spectrum liter in 2Hz0, pH 7.3). NMR: 128 transients in 16k data locations. numbered on the spectrum correspond to the resonances of the structure. Other assignments are *, impurity from the purification formamide); t*, impurity from the purification process (acetate); 0.000 ppm).

of leucine-enkephalin (20 mmol/ Spectral assignments: Peaks protons labeled on the primary process (possibly NJ-dimethyl*:*, TMS (external reference at

0.0

518

VANDENBERG,

KING,

AND

KUCHEL

relevant sections of the HGSE spectra of tyrosine, leucine, and leucine-enkephalin are shown in Fig. 2. Figure 3 shows the ‘H NMR HGSE spectrum of a hemolysate (Hc = 0.40) 52 min after the addition of leucine-enkephalin (10 mmol/liter cell water). The most prominent spectral changes (contrast with Figs. 1 and 2) over the period of incubation are the large increase in the amplitude of the free tyrosine resonances (c and e) and the concomitant diminution in amplitude of the leucine-enkephalin tyrosyl resonances (d and f); this indicates that aminopeptidase-catalyzed cleavage of the Tyr-Gly bond is the initial step in degradation of the pentapeptide by hemolysates. Other important spectral changes include the appearance of resonances due to free leutine (j) and phenylalanine (a), and a large FIG. 3. A ‘H NMR HGSE spectrum of a hemolysate (Hc = 0.40) 52 min after the addition of leucine-enkephalin (final concentration = 10 mmol/liter cell water). NMR: 128 transients/spectrum in 8k data locations. Spectral assignments: a, phenylalanine H’; b, phenylalanyl H* of leucine-enkephalin; c, tyrosine H6; d, tyrosyl H* of leucine-enkephalin; e, tyrosine H’; f, tyrosyl H’ of leucine-enkephalin; g, glycyl H” of GlyGly; h, glycyl H” of Gly-Gly-Phe-Leu; i, glycine H”; j, leucine H6; k, leucyl H* of leucine-enkephalin/GlyGly-Phe-Leu.

7.8

s.3 lfl

-0.1

6 bpm) FIG. 2. HGSE spectra of ?&O solutions of (A) leutine-enkephalin (20 mmol/liter), (B) tyrosine (2.5 mmol/liter), and (C) leucine (20 mmol/liter). NMR: 123 transients in 16k data locations. Spectral assignments: a, tyrosyl H6; b, tyrosyl H’, c, leucyl H6; d, TMS (external reference); e, tyrosine H*; f, tyrosine I-P; g, leucine H”; h, DSS (external reference); *, phenylalanyl resonances. The resolution of the amino acid resonances from their counterpart resonances in leucineenkephalin is highlighted by the dotted lines.

peak indicating accumulation of Gly-Gly (g). The changes in concentrations of products with time, as calculated from the peak amplitudes of the NMR resonances, are illustrated in Fig. 4. To elucidate unambiguously the pathway of leucine-enkephalin degradation by human erythrocytes it was necessary to monitor independently the hydrolysis of PheLeu and Gly-Gly-Phe-Leu in hemolysates so that the resulting kinetic parameters for these steps could be incorporated into the computer model (see Discussion). In the spectra acquired from experiments monitoring the hydrolysis of these peptides in hemolysates, the leucyl/leucine H6 resonances were not fully inverted but appeared dispersive. Therefore, the spectra from these reactions were processed with

OF

ENKEPHALIN

DEGRADATION

60

TIME

BY

(min)

HUMAN

120

180

FIG. 4. Product-versus-time progress curves from a ‘H NMR experiment monitoring of leucine-enkephalin. Experimental conditions are described in the legend to Fig. represent the experimentally obtained concentrations of (A), tyrosine; (O), Gly-Gly, The solid lines depict a simulation of this experiment using the computer model degradation pathway as illustrated in Fig. 6 (see Discussion); the various products the simulation were (A) tyrosine, (B) Gly-Gly, (C) leucine, and (D) Phe-Leu.

a line broadening factor of 10 Hz prior to Fourier transformation to achieve a relatively flat baseline. The concentrations of substrate and products indicated by the spectra were estimated using the extinction-coefficient method described previously (11) and the steady-state kinetic parameters for the hydrolytic reactions were derived from the progress curves as detailed under Numerical Methods; these parameters are summarized in Table I.

519

ERYTHROCYTES

the degradation 3. The symbols and (B), leucine. of the proposed monitored in

and leucine, are released almost simultaneously. There are two degradative pathways which could account for this observation: First, a carboxypeptidase may sequentially cleave the leucine and phenylalanine residues from the C-terminus of Gly-GlyPhe-Leu; if the second step were very much faster than the first then the two amino acids would appear to be released simultaneously. The hydrolysis of Gly-Gly by human erythrocytes is very slow (23) and

DISCUSSION

Due to the unique ability of NMR to monitor simultaneously all the products of the degradation of oligopeptides in real time, it is possible to determine the most probable degradation network of leucineenkephalin in hemolysates from a single experiment. As illustrated in Fig. 4, incubation of leucine-enkephalin with hemolysates results in the rapid appearance of tyrosine; this parallels the results found in other tissues where aminopeptidase-catalyzed cleavage of the Tyr-Gly bond is the initial step in enkephalin degradation (2, 21,22). Following this step, the three other major products, Gly-Gly, phenylalanine,

TABLE

I

MICHAELIS-MENTEN KINETIC PARAMETERS FOR THE HYDROLYSIS OF LEIJCINE-ENKEPHALIN, AND PEPTIDE FRAGMENTS OF IT, IN HEMOLYSATES

Substrate Tyr?Gly-Gly-Phe-Leu Gly-Gly-FPhe-Leu Ph&Leu

J-L (mmol/l) 0.4” 2.1 f 0.5 0.82 f 0.04

V (mmIrh/l packed cells) 22.9 f 1.0 22.4 f 2.4 28.7 f 0.4

Note. The arrows indicate the step for which kinetic parameters were obtained. Values are means f 1 SD. a Coletti-Previero et al, 1981.

520

VANDENBERG,

KING,

it is therefore not surprising that Gly-Gly accumulated; Guyong et aZ. (24) also found that prolonged incubation of leucine-enkephalin in mouse corpus striatum resulted in the accumulation of Gly-Gly. However, no carboxypeptidase activity has been detected in human erythrocytes (8). This was confirmed in the present study when incubation of specific substrates for carboxypeptidase A (Cbz-Phe-Leu and CbzLeu-Gly) and carboxypeptidase B (hippuryl-Arg) with hemolysates revealed no activity (spectra not shown). It therefore appears that the release of leucine and phenylalanine must occur via a non-carboxypeptidase-dependent route. The second possibility for the pathway of Gly-Gly-Phe-Leu degradation is that the Gly-Phe bond is cleaved with the resulting Gly-Gly accumulating and the Phe-Leu being rapidly hydrolyzed by aminopeptidase; this enzyme has a high affinity for similar peptides with bulky side chains (25). Peptidases capable of cleaving GlyGly-Phe-Leu at the Gly-Phe bond include dipeptidyl aminopeptidase II (DAP II; EC 3.4.14.2) and DAP III (EC 3.4.14.3), both of which have been isolated from human erythrocytes by Pontremoli and co-workers (8). Calcium-activated neutral endopeptidase (CANP; EC 3.4.22.17), which is also present in human red cells (26), although having a greater affinity for larger peptides, is also capable of cleaving the enkephalins at the Gly-Phe bond, with or without prior removal of the tyrosine residue (27). In summary, Fig. 5 shows all possible degradation pathways for leucine-enkephalin, with the framed products marking out what we consider to be the most probable catabolic pathway. This putative degradation pathway is shown in detail in Fig. 6; to test its validity, we attempted to obtain steady-state kinetic parameters for the individual steps of the pathway, which could then be incorporated into a computer model capable of simulating the system. It was evident from the spectra obtained when Gly-Gly-Phe-Leu was incubated with hemolysates that the initial cleavage was at the Gly-Phe bond, with subsequent rapid hydrolysis of Phe-Leu and accumu-

AND

KUCHEL

._.\,.‘.v ,.,.”,/’ ‘.\.%\ #/”,.Al. ,.’%. “‘*&.”,.,!-. ‘\L F vr,“’ FIG. 5. Schematic representation of all the possible degradation pathways for leucine-enkephalin. The various proteolytic activities represented in the diagram are: --+ aminopeptidase; .-+ carboxypeptidase; --*calcium-activated neutral protease; dipeptidylaminopeptidases II and III; ---.-..dipeptidase/aminopeptidase. The framed products mark out the most likely degradative route in hemolysates.

lation of Gly-Gly. Kinetic parameters were obtained only for the Gly-Phe cleavage from this experiment; these are shown in Table I along with the steady-state kinetic parameters for Phe-Leu hydrolysis obtained from experiments in which this dipeptide alone was incubated with hemolysates. The rapidity of tyrosine release from leucine-enkephalin incubated with hemolysates made it impossible to obtain accurate estimates of the steady-state kinetic parameters for this step. However, since the Km for the aminopeptidase-catalyzed cleavage of Tyr-Gly-Gly in hemolysates [2.11 f 0.08 mmol/liter; (ll)] is similar to that obtained for plasma aminopeptidasecatalyzed hydrolysis of the same tripeptide (2.4 f 0.7 mmol/liter; Vandenberg, King, and Kuchel, unpublished results) we can consider the enzymes to be kinetically similar; thus, we can use the Km value of 0.4 mmol/liter obtained for plasma aminopeptidase-catalyzed hydrolysis of leucineenkephalin (7) as an approximate value for the same catalysis by the erythrocyte enzyme. The V,, for the reaction is easily obtained from the initial rate of tyrosine release, since the initial leucine-enkephalin

‘H

NMR

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ENKEPHALIN

DEGRADATION

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ERYTHROCYTES

521

SIOW

hydrolysis

FIG. 6. The chemical reaction scheme used for computer simulation of the proposed enkephalin degradation-pathway. The abbreviations used for the various enzymes involved are A, aminopeptidase; X, dipeptidylaminopeptidase/CANP; D, dipeptidase/aminopeptidase. The values of all parameters were calculated as explained under Numerical Methods: ki = ka = ks = 1.00 X lo6 mol.liter-‘as-‘; k-, = 3.9363 X lOa s-l; & = 6.3611 s-i; ke3 = 2.0938 X lo3 s-l; k4 = 6.2222 SC’; km5 = 8.1203 X 102 ss’; ks = 7.9722 s-‘; [A& = [Xl, = [D& = 1.00 X 10m6 mol. liter-‘. This consistent set of unitary rate constants and enzyme concentrations yields the steady-state kinetic parameters listed in Table I.

concentration (10 mmol/liter = 25 K,) was saturating; the value of I’,,,,, calculated in this manner was 22.9 f 1.0 mmol/h/liter cell water. A consistent set of unitary rate constants was obtained from the steady-state kinetic parameters for each of the individual steps in the proposed pathway (see Numerical Methods) and incorporated into the computer model. The model of the proposed pathway is shown in Fig. 6 with the unitary rate constants given in the legend. A simulation of the experiment depicted in Fig. 4 using this model is shown as bold lines on the same diagram; a close agreement between the computer simulation and the experimental results is evident. The presence of other substrates and products in the incubation mixture could influence the rates of various reactions and thus account for the slight systematic deviation of the simulation from the experimental results. For the human erythrocyte cytosol to have a physiological role in the degradation of the enkephalins, or any biologically active peptides, these peptides must first be transported into the cells. In an experiment in which leucine-enkephalin was incubated with intact red cells, no changes in the NMR spectra were observed over a 2-h period. Furthermore, no leucine-enkephalin degradation was observed when the pentapeptide was incubated with purified erythrocyte membranes. Thus, leucine-enkephalin is neither transported into human erythrocytes nor hydrolyzed on their surface; however, in view of recent evidence indicating that Tyr-Gly-Gly and Gly-Gly-

Gly-Gly are permeable to human erythrocytes (11) then it is most likely that peptide fragments resulting from leucine-enkephalin degradation in plasma (such as Gly-Gly-Phe-Leu) would readily enter these cells. In conclusion, the present series of experiments have revealed a potentially important intraerythrocyte degradation pathway for leucine-enkephalin peptide fragments, although the whole peptide is not degraded by these cells. This work has also demonstrated the value of ‘H spinecho NMR for elucidating an entire degradation pathway in situ with a small number of experiments. These results also introduce the possibility that ‘H NMR can be used to determine the sequence of “unknown” peptides by monitoring the degradation pathway when the peptides are incubated with an appropriate mixture of purified peptidases. ACKNOWLEDGMENTS Project support for P.W.K. was from the Australian National Health and Medical Research Council. G.F.K. received a Commonwealth Postgraduate Research Award and J.I.V. was supported by N.H. & M.R.C. and J. G. Hunter Medical Awards. Mr. W. G. Lowe and Mr. B. T. Bulliman are acknowledged for expert technical and computing assistance, respectively. We also thank Professor Karl Brand for initial discussions on enkephalin degradation. REFERENCES 1. HUGHES, J., SMITH, J. W., KOSTERLITZ, H. W., FOTHERGILL, L. A., MORGAN, B. A., AND MORRIS, H. R. (1975) Nature (lmzdon) 258,577-579.

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2. HAMBROOK, J. M., MORGAN, B. A., RANCE, M. J., AND SMITH, C. F. C. (1976) Nature &o&m) 262, 782-783. 3. MEEK, J. L., YANG, H. Y-T., AND COSTA, E. (1977) NeuropharmecoloSy 16,151-X4. 4. MALFROY, B., SWERTS, J. P., GUYON, A., ROQUES, B. P., AND SCHWARTZ, J. C. (1978) Nature (Lenda) 276,523~526. 5. HENDERSON, G. (1983) Brit. Mea! Bull 39,59+X. 6. CLEMENT-JONES, V., LOWRY, P. J., REES, L. H., AND BESSER, G. M. (1980) Nature (London) 283,295-

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Actu 657,122-127.

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Biophys. Acta 579,20-30. 13. ENDRE, Z. H., KUCHEL, P. W., AND CHAPMAN, B. E. (1984) B&him Biophys. Acta 803, 137144. 14. RABENSTEIN, D. L., AND NAKASHIMA, T. T. (1979)

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25. CHUNG, Y. C., SILK, D. B. A., AND KIM, Y. S. (1979) Clin Sci. 57, l-11. 26. MELLONI, E., SPARATORE, B., SALAMINO, F., MICHETX, M., AND PONTREMOLI, S. (1982) B&hem Biophys. Res. Commun 106,731-740. K. (1983) 27. HIRAO, T., HARA, K., AND TAKAHASHI,

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