Download Horse pancreatic ribonuclease Scheffer, Albert Jan

University of Groningen
Horse pancreatic ribonuclease
Scheffer, Albert Jan
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Publication date:
1973
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Citation for published version (APA):
Scheffer, A. J. (1973). Horse pancreatic ribonuclease s.n.
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8. SUMMARY
The.exocrine.pancreatic
ribonucleases forn a group of relatively
rapíd1y evolving proteins.
Their disiribution
anong the vertebrate
species is exceptionally
unequal;
an acceptable explanation of this distribution
by a Dossíb1e physiological
function has been suggested ón1y'for
aninals with ruminant or ruminant-1ike digestive
systens.
Many ribonucleases are glycosidated
to a greater or lesser
ext ent .
Bovine ribonuclease is one of the most thoroughly studied enzymes. Comparative studies with ribonugleases derived
from other species nay yield valuable information
on their
phylogeny and structure-function
relationships,
and on the
specificity
requirements for the types of oligosaccharide
s ide-chain attached.
Crude horse ribonuclease was isolated
fron fresh tissue by
acid extraction,
phenol extraction
a n d a r n n o n i u ms u l f a t e
fractionation
(4.1). Further purification
of the enzyme
was found to be difficult.
After trying many modifications,
we succeeded by using repeated chronatography on CM-cel1u1ose with three different
gradient systens (4.2). The
purity
of the preparations was checked by disc ge1 elecand "staining"
trophoresis
for ribonucleolytic
activity,
and by N-terrninal analys is
(4.3 , 4. 5); the specific
activity was found to be an unreliable
criterion
for purity.
Pure horse ribonuclease was both chromatographically
and electrophoretically
h e t e r o g e n e o u s . W e h a v e r n a d ep l a u s ible that the heterogeneity
resides in the carbohydrate
moiety (4. 8) .
The amino acid composition of horse ribonuclease has the
characteristics
conmon to the other known pancreatic ribonucleases. Tryptophan is absent. The carbohydrate noiety
contains glucosanine and hexoses (4.4). Determination of
the molecular welght was difficult
because of the carboby a conbination of methods we found a
hydrate noietyi
value of 14,000 for the nolecular weight of the protein
noi.ety and about 18,000 for the average nolecular weight
(4.7). Horse ribonuclease has the
of the glycoprotein
usual N-terninus
lysine,
but its C-terminus is threonine
instead of the usual valine.
With spectrophotometric
tiwe found only two abnormal- tyrosines
tration
and three
normal ones (4.6).
tto
We derived
four
qeries
nf
nontids5
from
horse
ribonuclease
(chapter 5) : rc (;;i;;i;^"í;";hynotryptic
digesti.on after
perfornic
acid oxidation),
T Itryptic
cleavage after perforrnic acid oxidation),
CN (cyanogen bronide cleavage followed by perfornic
acid oxidation),
and AET (tryptié
iiydrolysis
after reduction and aminoethvlation).
Sóme secondary cleavages were perforned with thermolysin,
chyrnotryptil:
and cyanogen bromide. Peptides were isolatecl and pu:
rified
by ge1 filtration,
chrornatography on various ion
exchangers, and column electrophoresis.
The latter
methocl
proved particularly
useful for glycopeptides
(3.5.5).
For
the detection of-peptides
in colrrmn eluates we used mainly
spectrophotornetric rnonitoring, manual ninhydrin analys is ,and automated analysis with a considerably modified peptide analyser.that
has been described at some length (3.
5.7). The purity
of the peptides was checked by high vo1tage paper electrophoresis,
amino acid analysis and dansylation.
Peptide sequences were determined-by conventional nethods: two versions of the Dansyl-Edrnan technique
and a nrodification
o f t h e d i r e c t E d n a n d e s r a d a t i o n f 3 . ó ') .
Only the AET-peptides covered the entiie
s"q.l"n.è.
Several of the CN-peptides had arisen frorn aspecific
cleavages, especially
to the right
of tyrosine residues. All
peptides were positioned by sequence homology and overlappings; all but four peptide bonds were overlapped by
one or nore peptides (6.2). Alnost al1 amide groups were
l o c a t e d u n e q u i v o c a l l y ( 6 . 1) .
Horse ribonuclease consists of a polypeptide chain containing
125 anino acid re idues and carrying three oligosaccharide side-chains
in positions
21, 34, and 62. part
of Asn-2.1 occurs in the carbohydrate-free
forn (6.4). All
three sites conforn to the postulated recognition
triplet
for glycosidation;
site 21-23 is the first
attachment site
in any glycoprotein
found to contain proline
(7.3).
The C-terminus of horse ribonuclease contains two additional
arnino acids, representing the only instance of a
C-terminal addition
in pancreatic ribonucleases observed
thus far. Another peculiarity
unprecedented in pancreatic
ribonucleases
is the deletion
of residue 39. Including
deletion
and addition,
horse ribonuc lease differs
in 35
positions
fron the bovine enzyme (7 .1). All substitutions,
the addition,
and the oligosaccharide
residues can be acconmodated in the three-dinensional
nodel of bovine ribonuclease-S without alterating
the folding of the backbone.
The nodel built
of the loop containing the deletion offered an elegant explanation for the deviating
titration
behaviour of horse ribonuclease:
the shortening and consequent distortion
of this loop prevents Tyr-92 fron forming a hydrogen bridge between its phenolic hydroxyl group
and the carbonyl oxygen of residue 37 (7.2).
127
Comparison of all ribonuclease sequences elucidated partly
to conthus far provided the infornation
or èonpletely
tree and a hypothetical
evolutionary
a preliminary
struct
riboamino acid sequence of narnmalian pancreatic
ancestral
nuclease (7 .1) .
),n1\à \6
b
Qn A-
@
Fig.
B.
Ancestral
128
mammal