Vol. 167, No. 2, 1990 March 16, 1990
BIOCHEMICAL
PRESENCE
OF PANCREATIC MEDULLRRY
Randolph
U. Lewis,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 543-547
TRYPSIN CHROMRFFIN Purnlma
and Wllllam Molecular
Biology
Received
January
12,
IN ADRENAL
CELLS
Ray, Rhonda
Cogulll,
Kruggel
Department,
Laramie,
INHIBITOR
Uniuerslty
of Wyoming,
WY 82071-3944
1990
A bouine pancreatic trypsin inhibitor was isolated from bouine adrenal medullary chromaffin granules. Its N-terminal sequence is: arg-pro-asp-phe-cys-leu-glu-pro-pro-tyr-thr-gly-pro-cys-lys-ala-arg-ile-arg-tyrphe-tyr-asn-ala-lys-ala-gly-leu-cys-gln-thr-phe-ual-tyr-gly-gly-cys-arg-ala-lysarg-asn-asn-phe-lystuhich corresponds precisely with the N-terminus of Bovine Pancreatic Trypsin inhibitor. The presence of this inhibitor in these granules suggests another method of regulating the prohormone proteases present there. fD1990 Academic Press, 1°C.
Adrenal medullary chromaffin cells synthesize, release large quantities of catechlolamines, ATP, various numerous peptides from chromaffin granules the purification of proenkephalin fragments chromaffin non-opioid
granules, peptides
store, proteins
by exocytosis from adrenal
and and
(1). During medullary
we isolated and sequenced seueral opioid and We also obserued and isolated seueral (2,3,4).
other peptides in large amounts which were not part of proenkephalin (5,6,7). These peptides showed no crossreactiuity to antisera directed We have also purified a “trypsin-like against Met- or Leu-enkephalin. enzyme” or proenkephalin processing enzyme from bouine adrenal chromaffin granules. This enzyme cleaues at the paired basic We present here the isolation and residues of proenkephlin (8). sequence granules Pancreatic
of
a protein
which
matches
Trypsin
from the
Inhibitor).
bouine amino
adrenal medullary chromaffin acid sequence of BPTI (Bovine
BPTI is a small
basic protein
with
a MW
of 651 1 which has been found in uarious tissues such as liuer, spleen, lung, parotid gland and pancreas (9,lO). The presence of this inhibitor 0006-291x/90
543
Copyright
Ali rights
0
1990
of reproducrion
by Academic
$1.50
Press, Inc. in my form reserved.
Vol. 167, No. 2, 1990
from bouine adrenal reported previously.
BIOCHEMICAL
medullary
Materials
AND BIOPHYSICAL
chromaffin
RESEARCH COMMUNICATIONS
granules
has
not
been
and Methods
Bouine adrenal glands were collected from the slaughter house, stored in ice and used within two to three hours. After remoual of the cortex, medullae were used to prepare chromaffin granules by differential centrifugation, followed by a sucrose step gradient (8). The granules were then stored at -8OOC until used. The chromaffin granules were thawed, suspended in 50 mM Tris HCI pH 8, and lysed by sonication. The lysate was centrifuged in a JR20 rotor at 18,000 rpm for 1 h. The supernatant was passed through a trypsin inhibitor column to bind the trypsin-like enzyme. The effluent from the column was collected, acidified with 1 M acetic acid, 1 mM HCI and 0.1% BME and applied to a Sephadex G-l 00 column (5 H 100 cm). The column fractions were pooled in fiue groups or peaks on the basis of molecular weight as described by Lewis, et al (1 1). Peak Ill, which corresponds to a molecular weight range of 5,000-l 0,000 daltons, was resolued further by HPLC (High Performance Liquid Chromatography). The HPLC system coupled to a fluorometric detection system was used with pyridine/acetate buffer (0.2M/O.SM, pH 4.00) and 1 -propanol as the organic modifier to elute the peptides and proteins (12). The details of the chromatographic conditions are described in the “Results” Section. All soluents were distilled ouer ninhydrin. Radioimmunoassays were performed by the procedure described by Lewis et.al. (13). Anti-serum was purchased from lmmuno Nuclear (Stillwater, MN), and 1251-Leu-enkephalin was obtained from New England Nuclear ( Boston, MA). Samples were digested with trypsin (Sigma Chemical Co., St. Louis, MO.) and carboxypeptidase 8 (Boehringer) prior to use. The HPLC purified peptides were hydrodyzed in uacuo using 6M HCI at 155OC for 35 min as described by Lookhart et al (14). The precolumn deriuatization method using o-phthalaldehyde and reuersed phase HPLC was used for amino acid analysis (15). A Spectraphysics 8700 soluent delivery system using a fluorometer (Schoeffel/ Kratos 950) with an Rlltech 3u Cl8 column (4.6 x 150 cm) from Rainin were used. Buffer “A” contained 50mM sodium acetate pH 5.9 and buffer “B” contained 50 mM sodium acetate with 80% methanol and 1% tetrahydrofuran. The amino acid sequencing was done on the Applied Biosystems (RBI) 470 A gas phase protein sequencer. Approximately 500 p mole of peptide was used for this analysis. Sequencing was done according to the method described by Hewick et al (16). Phenylthiohydantoin amino acids were identified by HPLC on the RBI 120 A system using the Brownlee ODS column. 544
Vol. 167, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Besults The acid extractable according
to size on a Sephadex
corresponding pumped eluted
to a range
I-propanol
was run from
and 34 were
pooled,
were
eluted
with
20-40%
in 35 min.
propanol
indicated
euaporated
column
buffer
symmetric
uacuum,
washed
a clean
Cl8 column
and “pulsed
resuspended The peptides
loading
the sample
and eluting
in lml
33 of
in 150 min and
These fractions
were
times
with
to concentrate.
it with
were
and protein
eluted
three
and
Fractions
peak which
off”
pooled
at pH 4.00.
in 150 min.
of O-20% 1-propanol
a pure peptide.
were
The peptides
pyridine
on to a 5u Cl8 column.
under
Fractions,
(E.M.Science).
concentrated,
a gradient
into fiue peaks
daltons,
O-40% propanol
uacuum
A single
diuided
G-100 column.
in 0.5 M acetate/
The gradient
fl and loaded
were
of 5,000 to 10,000
onto a Cl8 cartridge with
Buffer
peptides
at 11.25% then
water,
applied
to
This was done by
a step gradient
of 40%
propanol. Amino purified
acid analysis
peptide.
Amino acid sequencing
in Fig. 1. The sequence sequence
data base.
homology
with
agrees
was tested The protein
Bouine
and the sequence this data
and sequencing
trypsin
uery
closely
revealed
sequence
on the
the sequence against
by
shown
the protein
data bank indicated
(Fig.1.)
reported with
performed
for homology
inhibitor
of BPTI were
were
100%
Amino acid analysis Kassell
et al (17) and
our amino acid analysis
data.
Discussion The presence and a trypsin-like
of both the bouine
enzyme
181 in bouine
pancreatic adrenal
trypsin medullary
arg-pro-asp-phe-cys-leu-glu-pro-pro-tyr-thr-glypro-cys-iys-aia-arg-ii~-arg-tyr-phe-tyr-asn-aiaiys-aia-giy-ieu-cys-gin-thr-phe-uai-tyr-giy-giycys-arg-aia-iys-arg-asn-esn-phe-iysFigure 1. Sequence of Adrenal 545
BPTI Peptide.
inhibitor chromaffin
Vol.
167,
No.
granules
suggest
function.
Protein
forms
BIOCHEMICAL
2, 1990
the inhibitor
tissues
Although
the presence
reported
in seueral
chromaffin
gene
a bouine
from
the BPTI coding
granuales genomic
region
BPTI may
be produced
polypeptide
precursor
inhibitors
a lysate
why prior
actiuity.
fraction
has high biological
to their
biological
secretory
trypsin
aCtiUatiOn
of trypsinogen granules
medullary
chromaffin
proenkephalin
that
data).
action
have
granules.
must
a similar
which
form
from
of
a larger function
[19].
of
This could
inhibitor
column,
trypsin-like the eluted
As proteases be controlled
mediate according
is the presence
of a
which
premature
preuents
protein
from
inhibitory
This could
proteins
the mature
the column
1191. This inhibitory
or other
showed
which
little
from
in the pancreas
might
A BPTI
a trypsin
One such example
inhibitor
chromaffin
elution
their
need.
before.
proteolysis
uery
(unpublished
processes,
reported
physiological
through
shows
after
actiuity
adrenal
processing
unnecessary
granules While
in bouine
that
One suggested
to passing
of chromaffin
prOteaSe
many
[211.
spleen,
nor immediately
proteolytic
is to preuent
be the reason
preceded
has been
glands,
was isolated
codon indicating through
in multiple
inhibitor
parotid
has not been
uras neither
by a termination
trypsin
presence
library
a key physiological
and microorganisms.
namely
their
COMMUNICATIONS
and present
pancreatic
tissues,
RESEARCH
haue
plants
of bouine
followed
these
in animals,
118,19,201,
medullary
may
are ubiquitous
bovine
and pancreas
BIOPHYSICAL
protein
inhibitors
in various
liuer,
AND
slow
role or stop
bouine
in the adrenal processing
of
by a trypsin-like
are cleaued
enzyme. The peptide 47 a.a. and that bouine data
determination, this
sequence
pancreatic beyond
peptide
we haue
that
isolated
was sequenced
matched
with
trypsin
inhibitor
which
residue
was not sufficient
but the amino containing
acid
the first
sequence 546
the first
47 a-a. sequence
has 58 a.a.
composition
the complete
through
to make data
of
The sequence an accurate is consistent
of BPTI.
with
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Wrinkler, C., S.W. (1982) In: The secretory granule. Poisner AM. Trifaro, J.M. teds) Elseuier Biomedical Press. Amsterdam. pp 3-60. Lewis, R.U., Ray, P., Blather, R., Stern, A. (1983) Biochem. Biophys. Res. Commun. 112:229-234. Micanouic, R., Ray, P., Kruggel, W., Lewis, R.U. (1984) Biochem. Biophys. Res. Commun. 118:299-303. Micanouic, R., Ray, P., Kruggel, W., Lewis, R.U. (1984) Peptides 5:853-856. Brandau, Cl., Ray, P., Stern, A., Lewis, R.U. (1984) Int. J. Peptide Protein Research. 25:238-241. Lewis, R., Micanouic, R., Ray, P., Blacker, R., Stern, A. (1984) Arch. Biochem. Biophys. 230:154-l 57. Micanouic, R., Ray, P., Kruggel, W., Lewis, R.U. (1985) Neuroendocrinology 41 :197-200. Euangelista, R., Ray, P., Lewis, R.U. (1982)Biochem. and Biophys. Res. Commun. 106:895-902. Kunitz, M. (1974) J. Gen. Physiol. 30:31 l-320. Laskwoski, M., Kato, I. (1980) Ann. Reu. of Biochem. 49:599-626. Lewis, R.U., Stern, R.S., Rossier, J., Stein, S., Udenfriend, S. (1979) Biochem. and Biophys. Res. Commun. 89:822-824. Lewis, R.U. (1979) Anal. Biochem. 98:142-l 45. Lewis, R.U. (1982) Rdu. Biochem. Psychopharmacol. 33:167-l 74. Lookhart, G.L., et. al. (1982) Journal of Biochemical and Biophysical Methods. 7:15-23. Jones, B.N., Paibo, S., and Stein, S. (1981) J. Liquid Chromatography 4:565-586. Hewiick, R.M., Hunkapillar, M.W., Hard, L.E. Dreyer, W.J. (1981) J. Biol. Chem. 256:7990-7997. Kassel, B., Radioeuic, M., Rnsfield, M.J., Laskowski, M. (1965) Biochem. Biophys. Res. Commun. 18:255-258. Kunitz, M. (1947) Gen. Physiol. 30:31 l-320. Laskowsi, M., and Kato, I. (1980) Ann. Rev. Biochem. 49:593-626. Kraut, H., Frey, E., and Bauer, E.Z. (1928) Physiol. Chem. 175, 97. Anderson, S. and Kingston, B. Proc. Natl. Rcad. Sci. USA 80, 6838-42.
547
BIOCHEMICAL
PRESENCE
OF PANCREATIC MEDULLRRY
Randolph
U. Lewis,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 543-547
TRYPSIN CHROMRFFIN Purnlma
and Wllllam Molecular
Biology
Received
January
12,
IN ADRENAL
CELLS
Ray, Rhonda
Cogulll,
Kruggel
Department,
Laramie,
INHIBITOR
Uniuerslty
of Wyoming,
WY 82071-3944
1990
A bouine pancreatic trypsin inhibitor was isolated from bouine adrenal medullary chromaffin granules. Its N-terminal sequence is: arg-pro-asp-phe-cys-leu-glu-pro-pro-tyr-thr-gly-pro-cys-lys-ala-arg-ile-arg-tyrphe-tyr-asn-ala-lys-ala-gly-leu-cys-gln-thr-phe-ual-tyr-gly-gly-cys-arg-ala-lysarg-asn-asn-phe-lystuhich corresponds precisely with the N-terminus of Bovine Pancreatic Trypsin inhibitor. The presence of this inhibitor in these granules suggests another method of regulating the prohormone proteases present there. fD1990 Academic Press, 1°C.
Adrenal medullary chromaffin cells synthesize, release large quantities of catechlolamines, ATP, various numerous peptides from chromaffin granules the purification of proenkephalin fragments chromaffin non-opioid
granules, peptides
store, proteins
by exocytosis from adrenal
and and
(1). During medullary
we isolated and sequenced seueral opioid and We also obserued and isolated seueral (2,3,4).
other peptides in large amounts which were not part of proenkephalin (5,6,7). These peptides showed no crossreactiuity to antisera directed We have also purified a “trypsin-like against Met- or Leu-enkephalin. enzyme” or proenkephalin processing enzyme from bouine adrenal chromaffin granules. This enzyme cleaues at the paired basic We present here the isolation and residues of proenkephlin (8). sequence granules Pancreatic
of
a protein
which
matches
Trypsin
from the
Inhibitor).
bouine amino
adrenal medullary chromaffin acid sequence of BPTI (Bovine
BPTI is a small
basic protein
with
a MW
of 651 1 which has been found in uarious tissues such as liuer, spleen, lung, parotid gland and pancreas (9,lO). The presence of this inhibitor 0006-291x/90
543
Copyright
Ali rights
0
1990
of reproducrion
by Academic
$1.50
Press, Inc. in my form reserved.
Vol. 167, No. 2, 1990
from bouine adrenal reported previously.
BIOCHEMICAL
medullary
Materials
AND BIOPHYSICAL
chromaffin
RESEARCH COMMUNICATIONS
granules
has
not
been
and Methods
Bouine adrenal glands were collected from the slaughter house, stored in ice and used within two to three hours. After remoual of the cortex, medullae were used to prepare chromaffin granules by differential centrifugation, followed by a sucrose step gradient (8). The granules were then stored at -8OOC until used. The chromaffin granules were thawed, suspended in 50 mM Tris HCI pH 8, and lysed by sonication. The lysate was centrifuged in a JR20 rotor at 18,000 rpm for 1 h. The supernatant was passed through a trypsin inhibitor column to bind the trypsin-like enzyme. The effluent from the column was collected, acidified with 1 M acetic acid, 1 mM HCI and 0.1% BME and applied to a Sephadex G-l 00 column (5 H 100 cm). The column fractions were pooled in fiue groups or peaks on the basis of molecular weight as described by Lewis, et al (1 1). Peak Ill, which corresponds to a molecular weight range of 5,000-l 0,000 daltons, was resolued further by HPLC (High Performance Liquid Chromatography). The HPLC system coupled to a fluorometric detection system was used with pyridine/acetate buffer (0.2M/O.SM, pH 4.00) and 1 -propanol as the organic modifier to elute the peptides and proteins (12). The details of the chromatographic conditions are described in the “Results” Section. All soluents were distilled ouer ninhydrin. Radioimmunoassays were performed by the procedure described by Lewis et.al. (13). Anti-serum was purchased from lmmuno Nuclear (Stillwater, MN), and 1251-Leu-enkephalin was obtained from New England Nuclear ( Boston, MA). Samples were digested with trypsin (Sigma Chemical Co., St. Louis, MO.) and carboxypeptidase 8 (Boehringer) prior to use. The HPLC purified peptides were hydrodyzed in uacuo using 6M HCI at 155OC for 35 min as described by Lookhart et al (14). The precolumn deriuatization method using o-phthalaldehyde and reuersed phase HPLC was used for amino acid analysis (15). A Spectraphysics 8700 soluent delivery system using a fluorometer (Schoeffel/ Kratos 950) with an Rlltech 3u Cl8 column (4.6 x 150 cm) from Rainin were used. Buffer “A” contained 50mM sodium acetate pH 5.9 and buffer “B” contained 50 mM sodium acetate with 80% methanol and 1% tetrahydrofuran. The amino acid sequencing was done on the Applied Biosystems (RBI) 470 A gas phase protein sequencer. Approximately 500 p mole of peptide was used for this analysis. Sequencing was done according to the method described by Hewick et al (16). Phenylthiohydantoin amino acids were identified by HPLC on the RBI 120 A system using the Brownlee ODS column. 544
Vol. 167, No. 2, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Besults The acid extractable according
to size on a Sephadex
corresponding pumped eluted
to a range
I-propanol
was run from
and 34 were
pooled,
were
eluted
with
20-40%
in 35 min.
propanol
indicated
euaporated
column
buffer
symmetric
uacuum,
washed
a clean
Cl8 column
and “pulsed
resuspended The peptides
loading
the sample
and eluting
in lml
33 of
in 150 min and
These fractions
were
times
with
to concentrate.
it with
were
and protein
eluted
three
and
Fractions
peak which
off”
pooled
at pH 4.00.
in 150 min.
of O-20% 1-propanol
a pure peptide.
were
The peptides
pyridine
on to a 5u Cl8 column.
under
Fractions,
(E.M.Science).
concentrated,
a gradient
into fiue peaks
daltons,
O-40% propanol
uacuum
A single
diuided
G-100 column.
in 0.5 M acetate/
The gradient
fl and loaded
were
of 5,000 to 10,000
onto a Cl8 cartridge with
Buffer
peptides
at 11.25% then
water,
applied
to
This was done by
a step gradient
of 40%
propanol. Amino purified
acid analysis
peptide.
Amino acid sequencing
in Fig. 1. The sequence sequence
data base.
homology
with
agrees
was tested The protein
Bouine
and the sequence this data
and sequencing
trypsin
uery
closely
revealed
sequence
on the
the sequence against
by
shown
the protein
data bank indicated
(Fig.1.)
reported with
performed
for homology
inhibitor
of BPTI were
were
100%
Amino acid analysis Kassell
et al (17) and
our amino acid analysis
data.
Discussion The presence and a trypsin-like
of both the bouine
enzyme
181 in bouine
pancreatic adrenal
trypsin medullary
arg-pro-asp-phe-cys-leu-glu-pro-pro-tyr-thr-glypro-cys-iys-aia-arg-ii~-arg-tyr-phe-tyr-asn-aiaiys-aia-giy-ieu-cys-gin-thr-phe-uai-tyr-giy-giycys-arg-aia-iys-arg-asn-esn-phe-iysFigure 1. Sequence of Adrenal 545
BPTI Peptide.
inhibitor chromaffin
Vol.
167,
No.
granules
suggest
function.
Protein
forms
BIOCHEMICAL
2, 1990
the inhibitor
tissues
Although
the presence
reported
in seueral
chromaffin
gene
a bouine
from
the BPTI coding
granuales genomic
region
BPTI may
be produced
polypeptide
precursor
inhibitors
a lysate
why prior
actiuity.
fraction
has high biological
to their
biological
secretory
trypsin
aCtiUatiOn
of trypsinogen granules
medullary
chromaffin
proenkephalin
that
data).
action
have
granules.
must
a similar
which
form
from
of
a larger function
[19].
of
This could
inhibitor
column,
trypsin-like the eluted
As proteases be controlled
mediate according
is the presence
of a
which
premature
preuents
protein
from
inhibitory
This could
proteins
the mature
the column
1191. This inhibitory
or other
showed
which
little
from
in the pancreas
might
A BPTI
a trypsin
One such example
inhibitor
chromaffin
elution
their
need.
before.
proteolysis
uery
(unpublished
processes,
reported
physiological
through
shows
after
actiuity
adrenal
processing
unnecessary
granules While
in bouine
that
One suggested
to passing
of chromaffin
prOteaSe
many
[211.
spleen,
nor immediately
proteolytic
is to preuent
be the reason
preceded
has been
glands,
was isolated
codon indicating through
in multiple
inhibitor
parotid
has not been
uras neither
by a termination
trypsin
presence
library
a key physiological
and microorganisms.
namely
their
COMMUNICATIONS
and present
pancreatic
tissues,
RESEARCH
haue
plants
of bouine
followed
these
in animals,
118,19,201,
medullary
may
are ubiquitous
bovine
and pancreas
BIOPHYSICAL
protein
inhibitors
in various
liuer,
AND
slow
role or stop
bouine
in the adrenal processing
of
by a trypsin-like
are cleaued
enzyme. The peptide 47 a.a. and that bouine data
determination, this
sequence
pancreatic beyond
peptide
we haue
that
isolated
was sequenced
matched
with
trypsin
inhibitor
which
residue
was not sufficient
but the amino containing
acid
the first
sequence 546
the first
47 a-a. sequence
has 58 a.a.
composition
the complete
through
to make data
of
The sequence an accurate is consistent
of BPTI.
with
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
References 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Wrinkler, C., S.W. (1982) In: The secretory granule. Poisner AM. Trifaro, J.M. teds) Elseuier Biomedical Press. Amsterdam. pp 3-60. Lewis, R.U., Ray, P., Blather, R., Stern, A. (1983) Biochem. Biophys. Res. Commun. 112:229-234. Micanouic, R., Ray, P., Kruggel, W., Lewis, R.U. (1984) Biochem. Biophys. Res. Commun. 118:299-303. Micanouic, R., Ray, P., Kruggel, W., Lewis, R.U. (1984) Peptides 5:853-856. Brandau, Cl., Ray, P., Stern, A., Lewis, R.U. (1984) Int. J. Peptide Protein Research. 25:238-241. Lewis, R., Micanouic, R., Ray, P., Blacker, R., Stern, A. (1984) Arch. Biochem. Biophys. 230:154-l 57. Micanouic, R., Ray, P., Kruggel, W., Lewis, R.U. (1985) Neuroendocrinology 41 :197-200. Euangelista, R., Ray, P., Lewis, R.U. (1982)Biochem. and Biophys. Res. Commun. 106:895-902. Kunitz, M. (1974) J. Gen. Physiol. 30:31 l-320. Laskwoski, M., Kato, I. (1980) Ann. Reu. of Biochem. 49:599-626. Lewis, R.U., Stern, R.S., Rossier, J., Stein, S., Udenfriend, S. (1979) Biochem. and Biophys. Res. Commun. 89:822-824. Lewis, R.U. (1979) Anal. Biochem. 98:142-l 45. Lewis, R.U. (1982) Rdu. Biochem. Psychopharmacol. 33:167-l 74. Lookhart, G.L., et. al. (1982) Journal of Biochemical and Biophysical Methods. 7:15-23. Jones, B.N., Paibo, S., and Stein, S. (1981) J. Liquid Chromatography 4:565-586. Hewiick, R.M., Hunkapillar, M.W., Hard, L.E. Dreyer, W.J. (1981) J. Biol. Chem. 256:7990-7997. Kassel, B., Radioeuic, M., Rnsfield, M.J., Laskowski, M. (1965) Biochem. Biophys. Res. Commun. 18:255-258. Kunitz, M. (1947) Gen. Physiol. 30:31 l-320. Laskowsi, M., and Kato, I. (1980) Ann. Rev. Biochem. 49:593-626. Kraut, H., Frey, E., and Bauer, E.Z. (1928) Physiol. Chem. 175, 97. Anderson, S. and Kingston, B. Proc. Natl. Rcad. Sci. USA 80, 6838-42.
547
