Download Renin Precursor Synthesis and Renin

Clinical Scfence(1979) 5'1,1099-1 10s
Renin precursor synthesis and renin-binding proteins in the
mouse
K. POULSEN
Institutefor Biochemistry, The Royal Dental College, Copenhagen, Denmark
Summary
1. The precursor synthesis of renin, the storage
form in the kidney and the submaxillary gland, and
the molecular nature of the forms in plasma were
studied in the mouse.
2. Renin is synthesized as a precursor (pre-prorenin) with a molecular weight of 50 000.
3. Renin is stored in the submaxillary gland and
the kidneys as fully active renin with a molecular
weight of 40 000.
4. The predominant form of renin in plasma is
the active mol. wt. 40000 form. High-molecularweight forms of renin (800000 and 70000) are
also present in plasma.
5. Pure 'Wabelled mol. wt. 40 000 renin binds,
after a change in the tertiary structure, to the
plasma protease inhibitors q-macroglobulin, intercetrypsin inhibitor and q-antithrombin. It binds
also to lipoprotein and an unidentified plasma
protein. No binding was seen to more than 50 other
studied plasma proteins.
6. The high-molecular-weight forms of renin in
plasma may be complexes of renin with plasma
protease inhibitors and lipoprotein.
Key words: lipoproteins, protease inhibitors, renin.
Introduction
High-molecular-weight forms of renin which are
partly inactive are found in plasma of many
species, including the .mouse (Nielsen, Malling &
Poulsen, 1978). Such renin forms can either be
precursors of renin or renin-protein complexes. In
order to assess the nature of these renin forms, the
Correspondence: Dr K. Poulsen, Institute for Biochemistry, The Royal Dental College, Jagtvej 160,2100
Copenhagen 0, Denmark.
biosynthesis, the storage form and the binding of
renin to other proteins were investigated.
The mouse was chosen for these studies because
of the availability of pure s u b m d a r y renin and its
specific antibody, which were essential tools for
elucidation of the molecular changes of renin from
synthesis to elimination.
Results and discussion
Proteins are synthesized in the cytoplasma of the
cell by the ribosomes, which use RNA as a
messenger for the amino acid sequence. Ribosomes
from one species and messenger RNA from
another can interact and produce a protein with the
correct structure. This is used in the so-called cellfree translation systems. The two most commonly
used systems are the wheat-germ system (Rowe,
Moen, Davidson, Byers, Bornstein & Palmiter,
1978) and the reticulo-lysate system (Housman,
Jacobs-Lorena, Rajbhandary & Lodish, 1970).
Both are characterized by having all the potentials
for synthesizing proteins, and they translate messenger RNA added to them. Furthermore, an
important feature is that they are derived from nonsecretory tissues, which means that they are not
processing the proteins synthesized. If a messenger
RNA from a different species is translated in these
cell-free systems, they are left in the solution in the
molecular form coded for.
In order to study the molecular form in which
renin is synthesized, the messenger RNA species
from renin-rich submaxillary glands of the mouse
were purified and translated in the two cell-free
translation systems mentioned above. (Poulsen,
Vuust, Lykkegaard, Nielsen & Lund, 1979b). The
messenger RNA from the submaxillary gland were
purified from other RNA species, but there is no
means to purify specifically that particular messenger RNA which codes for renin. Renin will be
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K.Poulsen
synthesized together with all other proteins synthesized in the submaxillary gland. However, the
synthesized renin was purified by precipitation with
a specific antibody against pure submaxillary renin.
In order to visualize the synthesized renin
[35S]methioninewas used in the protein synthesis.
The molecular weight was determined by sodium
dodecyl sulphate-polyacrylamide
gel electrophoresis.
In both assay systems renin was synthesized
with a molecular weight of 50 000, which is 10 OOO
greater than the fully enzymatic active mol. wt.
40000 renin (Poulsen et al., 1979b). Control
experiments demonstrated that it was displaced
from the antibody by pure submaxillary renin and
that it was not precipitated by non-immune yglobulin.
This mol. wt. 50 000 renin form represents most
likely a pre-prorenin. It is unlikely that it is derived
from an even larger precursor, since the two cellfree systems used gave the same molecular weight,
and it is very unlikely that they contain the
necessary processing enzymes.
Other acid proteases, the group to which renin
belongs, e.g. pepsin and chymosin (Foltman,
Pedersen, Jacobsen, Kauffman & Wybrand, 1977)
are also synthesized as a single-chain precursor
polypeptide. Pepsinogen is stored in this precursor
form, and secreted from the cell in that form.
However, renin seems to be stored in both the
submaxillary gland (Nielsen, Lykkegaard & Poulsen, 1979) and the kidney (K. Poulsen, unpublished work) of the mouse as fully enzymically
active mol. wt. 40 OOO renin.
Thus it is most likely that renin is secreted into
the blood as fully active mol. wt. 40 OOO renin. This
is in accordance with the findings that the majority
of renin in mouse plasma is present as fully active
renin. That the molecular weight is 40 000 is
determined by gel chromatography, and that it is
fully active is determined by measuring its specific
enzymatic activity (Nielsen et al., 1978).
However, in plasma of the mouse two distinct
high-molecular-weight forms of renin are present
(Nielsen et al., 1978). One has mol. wt. 800000
and the other 70 000. Such high-molecular-weight
forms of renin are most likely complexes of renin
with other molecules, since they are greater than
the mol. wt. 50000 form in which renin is
sykhesized.
If pure submaxillary renin is mixed with mouse
plasma it does not bind to other proteins. This
facilitates its enzymatic activity in mouse plasma,
but does not offer an explanation for the highmolecular-weight forms.
In order to mimic complex-formation of renin
with plasma proteins 1251-labelled pure submaxillary mol. wt. 40000 renin was mixed with
mouse plasma after different kinds of treatment in
order to provoke binding. Unfolding and subsep e n t refolding of renin by guanidine facilitated
binding to plasma proteins. The binding proteins
nere identified as the plasma protease inhibitors qmacroglobulin, inter-ct-trypsin inhibitor and qantithrombin. Binding to a,- and /],-Lipoproteins
and to an unidentified plasma protein was also
demonstrated. There was no binding to 56 other
tested plasma proteins (Poulsen, Kr011, Nielsen,
Jensenius & Malling, 1979a). The physiological
relevance of the renin-protein complexes is still
unknown.
References
FOLTMAN,B., PEDERSEN,
V.B., JACOBSEN,
H., KAUFFMAN,
D.
& WYBRAND,
G. (1977) The complete amino acid sequence
of prochymosin. Proceedings of the National Academy of
Sciences U S A . , 74,2321-2324.
HOUSMAN,
D. JACOBS-LORENA,
M., RAJBHANDARY,
U.L. &
LODISH,H.E. (1970) Initiation of haemoglobin synthesis by
methionyl-tRNA. Nature (London), 227.9 13-918.
S.& POULSEN,
K. (1979) Renin
NIELSEN,A.H., LYKKEGAARD,
in the mouse submaxillary gland has a molecular weight of
40 000. Biochimica el Biophysica Acta, 516,305-3 13.
NIELSEN,A.H., MALLMG,C. & POULSEN,K. (1978) Characteristics and conversion of high molecular weight forms of
renin in plasma and their uncomplete activation by the
current acid treatment. Biochimica et Biophysica Acta, 534,
246-257.
POULSEN,K.. KRBLL, J., NIELSEN,A.H., JENSENIUS,
J. &
MALLING,C. (1979a) Renin binding proteins in plasma.
Binding of renin to some of the plasma protease inhibitors, to
lipoproteins, and to a non-trypsin-binding unidentified plasma
protein. Biochimica et Biophysica Acta, 577, 1-10.
POULSEN,
K.. VUUST,J., LYKKEGAARD,
S., NIELSEN,A.H. &
LUNO,T. (1979b) Renin is synthesized as a 50000 dalton
single-chain polypeptide in cell-free translation systems.
FEES Letters, 98,135-138.
ROWE, D.W., MOEN, R.C., DAVIDSON,J.M., BYERS, P.M.,
BORNSTEIN,
P. & PALMITER,R.D. (1978) Correlation of
procollagen mRNA levels in normal and transformed chick
embryo fibroblasts with different rates of procollagen
synthesis. Biochemistry. 17, 1581-1590.