189

The role of assembly in insulin's biosynthesis

Guy Dodson*t and Don Steiner
Insulin is synthesised as a single-chain precursor,
preproinsulin, that contains an N-terminal signal sequence and
a connecting peptide linking the A and B chains of the insulin
molecule. Nascent proinsulin is directed into the regulated
secretory pathway, converted to insulin and stored as
microcrystals. These processes exploit assembly to the zinccontaining hexamer. Structural, chemical and genetic studies,
and experiments with transgenic animals and transfected cells,
are providing new details about the molecular events in
insulin's biosynthesis.

Addresses
*National Institute of Medical Research, Mill Hill, London, NW7 1AA,
UK; e-mail: r-sumray@nimr.mrc.ac.uk
*Chemistry Department, Universityof York, York YO1 5DD, UK
e-mail: myers@yorvic.york.ac.uk
~+TheHoward Hughes Medical Institute and Department of
Biochemistry and Molecular Biology, Universityof Chicago, Chicago,
Illinois 60637, USA
Current Opinion in Structural Biology 1998, 8:189-194
http://biomednet.com/elecref/0959440X00800189
Current Biology Ltd ISSN 0959-440X

Introduction
Since the discovery of insulin in 1921 by Banting and Best,
a wealth of chemical, biochemical and structural knowledge about the hormone and its biosynthetic precursor,
proinsulin, has become available. Thus, its biological and
physiological effects have been characterised, its solution
properties described in detail, its amino acid sequence and
three-dimensional structure determined, and its chemical
and recombinant synthesis established. Analyses of biological and structural research have identified the residues
involved in the molecule's self-assembly and those
involved in its binding to its receptor. (For reviews of
insulin's biology, chemistry and structure see [1-3].) It has
been possible to take the implications of the hormone's
structure further. Electron microscopic studies, timecourse experiments and chemical reactivity, as well as the
important development of research with transgenic mice
and transfected cells, together with an understanding of
insulin and proinsulin assembly, have allowed us to
deduce in detail some of the fundamental chemical and
physical events that occur during the hormone's biosynthesis, and their locations in the cell. Thus, we can now
describe many aspects of insulin's biosynthesis at both the
molecular level, and the cellular level.
In this review, we shall relate the structure and assembly
of insulin to the major events of its biosynthesis. T h e s e
begin with preproinsulin, its processing to proinsulin,
which is then folded and subsequently assembles to a
hexamer, facilitating transport. Hexameric proinsulin is
converted to insulin, which then forms microcrystals in the

storage residues; uhimately it is released from the 13 cell

into the blood.
Insulin's s t r u c t u r e a n d a s s e m b l y
Although insulin circulates in the serum and binds to its
receptor as a monomer, it forms dimers at micromolar concentrations, and in the presence of zinc ions it further
assembles to hexamers [1]. T h e hormone's solution and
crystal structures and its pattern of assembly are illustrated
in Figure 1. T h e insulin monomer itself consists of two
chains, an A chain of 21 amino acids and a B chain of 30
amino acids. T h e B chain has a central helical segment
between residues B9 and B19, from which the N and C
termini extend as strands. T h e A chain has an N-terminal
helix, A1-A8, linked to an antiparallel C-terminal helix,
A12-A20. T h e two chains are joined by two disulphide
bonds, AT-B7 and A20-B19, which clamp the A chain
helices at each end of the central B chain helix. Note that
the A chain N terminus and the B chain C terminus are
both located on the molecule's surface, about 8 A apart.
Figure 2 shows the A chain N terminus and B chain C terminus, which in proinsulin are linked by a connecting peptide. T h e structures of the termini in the monomer
(Figure 2a) and the hexamer (Figure 2b) are essentially
the same. T h e solution structure of insulin monomers
(Figure 2c) is also essentially identical to those of insulin
monomers within the dimer and hexamer as determined
by X-ray analysis [4"].
As is indicated in Figure 2, the insulin monomer has two
extensive nonpolar surfaces. One surface is flat, well
defined and mainly aromatic; it is buried upon dimer formation. T h e other is more extensive and complex and it
has some flexibility. This extensive surface is buried when
the dimers further assemble to hexamers in the presence
of zinc ions [1]. Proinsulin and insulin have essentially the
same association behaviour; thus, any significant stable
contacts made by the connecting peptide must be limited
to the surface and not buried in the hexamer (see Figure 3;
[5,6]). T h e closeness of the two junctions also requires that
the connecting polypeptide chain linking them takes a
complex course on the insulin hexamer surface.
Spectroscopic and N M R studies indicate that there is no
convincing evidence for secondary structure in the connecting polypeptidc, and neither X-ray analysis nor N M R
studies have been successful in defining its probably
mobile structure. We shall see, however, that proinsulin's
self assembly is beautifully exploited in the different
stages of biosynthesis.
Insulin's b i o s y n t h e s i s in t h e cell
T h e biosynthesis of insulin has been studied for many
years, and a good deal has been written on the various
celhdar, physiological and structural aspects [2,3,5,6].

190

Macromolecular assemblages

Figure 1

BIO

B13

.J

B13

BIO

Monomers

~L

Dimer

Zn2+

Hexamer
CurrentOpinionin StructuralBiology

The pattern of assembly of insulin monomers, dimers and hexamers. Only the backbone and selected sidechains important in assembly are shown.
The B10 histidine that coordinates the two central zinc ions is indicated; this residue always associates with zinc in the hexamers [5]. Six B13
glutamate residues are buried in the hexamer within hydrogen bonding distance of each other. Their unfavourable interactions are important in
hexamers released into the blood.

Insulin is synthesised in the 18 cells of the islets of

Langerhans. T h e 13cells are characterised by two features
associated with cells that export proteins - - they have
rough endoplasmic reticular surfaces and well-defined
storage vesicles. In this case, the vesicles typically contain microcrystals of packaged insulin. 18cells have another important feature - - in almost all animals they are rich
in zinc and calcium. Biochemical experiments and
radioisotopic chase studies have determined the location
and duration of the different stages of the process from
the initial event of polypeptide synthesis to the appearance of crystalline insulin in a granule within the vesicle
(Figure 3). These studies also show the corresponding
molecular events that occur during biosynthesis [5]. T h e
final exocytotic release of insulin from the 13-cell storage
vesicles into the blood is an energy-dependent process
governed by the sugar level in the blood.

The ribosomal synthesis of proinsulin

T h e synthesis of the insulin molecule depends on the
normal mechanisms for exported proteins [2]. Its messenger RNA contains sequences that correspond to a
leader or signal sequence that is responsible for stabilising the ribosome on the rough endoplasmic reticulum
(RER) membrane. This is followed by a sequence that
translates into the B chain and then a sequence that
translates into the 30-35 amino acid connecting peptide
segment, which links to the following A-chain sequence.
Thus insulin is directed into the endoplasmic volume as
a single-chain molecule of some 110 amino acids,
referred to as preproinsulin. The signal sequence is

thought to be immediately removed to generate proinsulin. In the special environment of the ER, proinsulin is
able to fold quickly into its correct three-dimensional
structure, or it may fold while anchored to the membrane, as this could help localise the nascent molecules
conveniently for assembly, for example as discussed in
[7] with respect to haemagglutinin folding.
The nature of the environment in the ER is only beginning to be understood in detail. There is evidence (from
the reactivity of proinsulin to iodination and disulphide
reduction) that proinsulin assembly to dimers and hexamers could occur here, though questions as to the availability of zinc are unsettled [8,9]. The iodination studies
exploited the altered folding properties of mutant SerB5
proinsulin that allow it to be iodinated in the ER [9].
Incidentally, this behaviour suggests that BS, normally a
histidine residue, is important in folding and hexamer
formation. It may further imply that the hexamer uses
HisB5 in zinc binding in vivo as has been observcd in the
insulin crystal that contains four zinc ions. Possibly this
extra zinc interaction will further stabilise the assembly in
the vesicle [10].

The transport of proinsulin into the Golgi

The vesicular transfer of proinsulin from the ER brings it
to the Golgi, an aqueous environment containing zinc and
calcium [11]. Thus, from here on, the solution conditions
will favour formation of the zinc-containing proinsulin
hexamer [8]. The surface structure of the proinsulin hexamer is quite different to that of insulin as the schematic in

The role of assembly in insulin's biosynthesis Dodson and Steiner

191

Figure 2

(a)

(b)
B30

A1-3

(c)

B30

A1-3

B30

A1-3

'iS-.

f
Nonpolar surfaces
(crystal)

Nonpolar surfaces
(crystal)

Nonpolar surfaces
(solution)

Monomer extracted from

hexamer

Hexamer

Monomer

Current Opinion in Structural Biology

The insulin monomer and hexamer structures [1]. The N terminus of the A chain (A1-3) and the B chain (B30) are drawn with van der Waals' radii.
These are the sites of the connecting peptide junctions in proinsulin. (a) The crystal monomer isolated from the hexamer. (b) The two-zinc insulin
hexamer with the selected monomer highlighted. (c) The solution structure of the insulin monomer. On each side of the molecule are the two
nonpolar surfaces that are buried by assembly to the dimer and hexamer [3]. The two-zinc hexamer is viewed perpendicular to its threefold axis; the
monomers are viewed in the same direction.

Figure 3 shows, although they do share the same organisation and internal structure. This explains their profoundly
different solubilities in the presence of zinc in which the
proinsulin hexamer is soluble and the insulin hexamer is
insoluble [11,12]. Probably, the extra four to six carboxylate groups in each proinsulin monomer are arranged to
form metal ion binding sites [5]. This solubility favours the
transport of concentrated proinsulin (=10%)into the Golgi
apparatus in which it is sequestered into evaginations that
develop and bud off"into storage vesicles [5,6].
Expcriments on the sccretion of dimeric mutant proinsulins
in transgenic animals suggest that for efficient sorting proinsulin must have a low affinity for the insulin receptor. This
affinity will be provided by the proinsulin connecting peptide and additionally by the hexamer structure [13,14].
Similar studies on monomeric proinsulin using AtT-20 transfected pituitary cells have provided fllrther evidence for thc
sorting of the dimeric AspB10 proinsulin into the constitutive secretory pathway [15]. Experiments with these cells
using a monomeric insulin mutant (SerB9 --+ Asp) indicate
that there is normal sorting [161. An explanation for this may
be that AspB9 insulin has only 30% of its receptor binding

ability, which will rednce the receptor's capacity to divert

the mutant proinsulin from the regulated secretory pathway.
It also follows that normal sorting can be efficient without
presence of the hexamer. In the presence of zinc, calcium
and various additives, however, this mutant can form hexamers and this possibility should be considered [17]. More
flmdamentally these transfected pituitary cells do not
express high levels of insulin and are not enriched with zinc,
which presumably means that there may not be hexamers.
Their absence will probably affect the secretory events in
the Golgi and perturb the normal concentration, precipitation and crystallisation events that occur in the Golgi and
storage vesicles of the ~ cell.
In another experiment, a single chain insulin in which A1
was directly linked to B30 was used. This proinsulin analogue is closely related to the A1-B29 cross-linked insulin
that forms hexamers and crystallises readily [18-20]. This
molecule, essentially a truncated proinsulin from which
the connecting peptide segment has been deleted, has
very low binding affinity for the receptor and thus will sort
efficiently. It would be interesting to measure the efficiency of this truncated proinsulin's biosynthesis and

192

Macromolecular assemblages

Figure 3
Cellular events

Molecular events

l I

Reduced unfolded
preproinsulin

RER

SS bond formation,
proinsulinfolding

Proinsulin
(SS bond
formation)

~W\ _~
acids
"~
Jf Amino
Transfer
q/ I .... / RNA ATP,GTP,Mg++
"~ Enzymes
10-20 min

MV

i
~
%

t ...... Antimycinblocks
O ....

O O
O

~1~""Transferstep1 (energydependent)

o
Proinsulin O O O
O

Formationof zinc proinsulinhexamers

o o

-- Transferstep 2
20 min

zinc insulinhexamer
with releasedC peptides
Precipitation begins

Membrane"~
bound }, ....
proteases 3
Zinc++'~-----~ ~ I

-k
Crystal formation

Mainly insulin
(crystalloid) .

+
~ . ~
C ~eP'tide ~p
(space)

~
'~.
~

~
I

I
@

1
~'

- -

ature

granules

30-120 min
0

~. Membrane

f-"~ |"~ recycling?

,~ ,

. |

Hours-days
I

PmlaSmb:nae ~
Emiocytosi ~ Y e d p ~ P n ~ n e d n l )
(Exocytosis)

nt
Secreted products
Insulin
} 94%
C peptide
Proinsulin 'lt _60/0
intermediates "
Zn++
Others?

CurrentOpinionin StructuralBiology

A schematic representation of the molecular events (left) and cellular events (right) that occur in insulin's biosynthesis. The asterisk indicates the
bound or (C peptide) free connecting peptide. RER, rough endoplasmic reticulum.

transport through the ER and Golgi compared to that of

normal proinsulin.
Experiments on sulfhydryl accessibility of proinsulin in
pancreatic 13 cells add further direct evidence about zinc

localisation and its effects on proinsnlin assembly. Huang

and Arvan [8] have shown that zinc protects proinsulin
from reduction in the Golgi but not in the ER, consistent
with the idea that proinsulin hexamers are stabilised both
by zinc coordination to the B10 histidines and by zinc

The role of assembly in insulin's biosynthesis Dodson and Steiner

binding at other sites [5,12]. T h e s e observations are in conflict with those derived from the iodination experiments of
Schmitz et a/. [9].

193

vesicles often have amorphous contents although somctimcs therc are poorly formed crystals, as is illustrated in
Figure 4. This shows that crystallisation has been compromiscd by the presence of mutated insulins [13].

Storage granule formation

It is apparent from radiolabelling and electron microscopic
studies that the conversion of the proinsulin to the insulin
starts at the stage of vesicle formation [2,3]. T h e connecting peptide that links the A and B chains then floats flee as
the so called C peptide (see Figure 3), T h e cleavage is carried out by membrane-associated enzymes [21]. T h e presence of zinc ions and the stability of the hexamer makes it
likely that this hcxameric organisation will be retained
throughout the conversion process. Figure 2 shows that the
sites of cleavage at the A chain N terminus and B chain C
terminus jtmctions are about as exposed in the hexamer as
in the monomer, though they will be less flexible. Support
for conversion of the hcxamer is given by the ability of
trypsin to convert dimeric and hexameric proinsulin at the
same rate [,5]. Electron micmgraphs sometimes show precipitating material at the perimeter of the vesicle, next to
the membrane [22]. This material is presumably newly
converted hexameric insulin, which is insoluble in the presence of zinc ions. This is in total contrast to the pronounced
solubility of proinsulin hexamers [12].
T h e r e is a further advantage with retaining the hexamcr:
the six insulin molectdes form an oblate spheroid that
readily fl)rms close-packed arrays that favour crystal
growth. T h u s on precipitation in the vesicle, insulin hexamers tend to form microcrystals, behaviour that has been
duplicated in laboratory experiments [23-2,5]. Storage vesicles of insulins that are dimeric or monomeric, that is,
those of the hagfish or guinea pig, respectivcb; do not contain crystals but rather electron dense amorphous precipitates [,5,6,22]. Equally, in transgenic mice that express
dimeric insulins and native hcxameric insulins, the storage
Figure 4

Current Opinion in Structural Biology

Electron micrographs of ~ cells of (a) normal and (b) HisB10Asp

proinsulin transgenic mice. Pancreas pieces were fixed in Kanovsky's
paraformaldehyde-glutaraldehyde, postfixed in 1% osmium tetraoxide
and embedded in Epon. Bars represent one micron.

T h e appearance of crystals in a biological process is unexpected and it is reasonable to expect that they have a function. One such function may well be to protect insulin from
fllrther proteolysis by converting enzymes. Another is the
selection of fnlly converted insulin hexamers by the crystalline lattice - - this would help drive the conversion of all
six subunits to about 95% completion. T h e crystal lattice,
however, does not seem to be important for conversion
efficiency. In experiments on transgenic mice, it has been
fi)und that hexameric and dimeric insulins are converted at
much the same rate and efficient3, exen though most of
the vesicles apparently do not contain crystalline material
but rather amorphous precipitates [13]. It may be relevant
that the dimeric AspB10 mutant insulin crystallises in the
presence of zinc ions to form a dodecamer in which six
dimers are arranged around two central zinc ions coordinated in a novel fashion to the B5 histidines [17]. T h e r e is
no evidence from electron micmgraphs [13], however, that
this kind of crystal is present in the storage vesicles
although it is possible that their assembly in solution could
affect or even fawmr conversion.
The release of the active monomeric insulin from the
hexamer

Within the storage vesicle, the insulin hexamer in the crystalline state is very stable [26]. On release into the serum,
however, the insulin microcrystals experience a jump in
pH from = 5.,5 to 7.4. As can be seen in Figure 1, the carboxylic acids of the six GIuB13 residues are packed closely together at the centre of the hexamer. At a pH of = 6
they are hydrogen bonded to each other [11; at the pH of
blood, 7.4, these interactions are increasingly disfavoured
by the deprotonation of the carbnxylic acids and the zinc
coordination is essential to contain their repulsion. At the
same time, the dilution of the zinc and calcium ions in the
serum means that the central zinc ions, which exchange on
a time scale of seconds, disappear. T h e strong mutual
repulsion of the six central B13 carboxylates canses the
hexamers and therefore the crystal to disintegrate rapidly.
T h e rnle of B13 in destabilising the hexamer can be
demonstrated by the consequences of its mutation to glutamine, which is neutral and can be accommodated in the
hexamer core. T h e hexamer forms in the absence of zinc
and has been crystallised and its structure determined [191.
This hexamer, and the crystal, would presumably not disintegrate on release from a storage vesicle and hence
would delay the appearance of the monomer.
Conclusions

In all but a few species, ~ cells contain high levels of zinc

and correspondingly their insulins arc able to form zinccontaining hexamers. T h e r e is a succession of steps in

194

Macromolecular assemblages

insulin's biosynthesis that appear likely to depend on the

structural organisation of the pmhormone and hormone
hexamer. The combined evidence provided by the hexamer structure, its chemical behaviour and its solution properties makes a persuasive case for a role for the hexamer in
many of the biosynthetic processes of the 13cell. The evidence from site-directed proinsulin and insulin mutants in
transgenic animals and transfection experiments have
added greatly to the arguments concerning the role of the
hexamer in the Golgi and storage vesicles. There are, however, still outstanding questions: the nature of the aggregation in the ER, the precise determinants in sorting, the role
of the connecting peptide in transport and conversion, and
the role of insulin crystallisation in the conversion process,
are just some examples.
The mutations in proinsulin and insulin that modify hexamer solubility and stability and those that alter the length
and nature of the connecting peptide need more investigation. These studies should extend to transgenic animals
and ideally should include transfected 13cells in which the
normal proinsulin gene has been deleted, allowing uncomplicated analysis of the effects of the proinsulin mutations
on biosynthesis. The use of chemical reagents that block
the various biosynthetic steps and the exploitation of specific reactions with the proinsulin molecule are starting to
provide a more precise description of its assembly within
the biosx/nthetic process. There are obvious difficulties
with the chemical approach; the different patterns of reaction with disulphide reduction and iodination are evidence
of this. Nonetheless, this approach promises to penetrate
the complexity of the chemical, structural and celhdar factors that govern insulin's biosynthesis.

Acknowledgements

5.

EmdinSF, Dodson GG, Cutfield JF, Cutfield SM: Role of zinc insulin
biosynthesis. Diabetologia 1980, 19:174-182.

6.

Blundell TL, Dodson GG, Hodgkin DH, Mercola DA: Insulin: the
reflection in the crystal and its reflection in chemistry and biology.
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7.

Wilson IA, Skehel JJ, Wiley DC: Structure of the haemagglutinin

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8.

Huang XF, Arvan P: Intrecellular transport of proinsulin in

pancreatic B-cells. J Biol Chem 1995, 270:2041 ?-20423.

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Schmitz A, Maintz M, Kehle T, Herzog V: In vivo iodination of a

misfolded proinsulin reveals co-localised signals for Bip binding
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10. Smith GD, Swenson DC, Dodson EJ, Dodson GG, Reynolds CD:
Structural stability in the 4Zn human insulin hexamer. Proc Nat/
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14. Steiner DE Tager HS, Nanjo K, Chan SJ, Rubenstein AH: Familial
syndromes of hyperproinsulinemia and hyperinsulinemia with mild
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16. Quinn D, Orci L, RavazzolaM, Moore HPH: Intracellular transport
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Whittingham JL, Xiao B, Brange J, Kaarsholm N, Thogerson H: Insulin
assembly: its modification by protein engineering and ligand
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The authors are grateful to Stephen Mumfurd for preparing the figurcs, and
to Hewson Swift, who made the electron micro~,raphs.

18. Powell SK, Orci L, Craik CS, Moore MHM: Efficient targeting to
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References and recommended reading

19. Bentley GA, Brange J, Dodson EJ, Dodson GG, Wilkinson A J, Xiao B:
The role of B13 glutamic acid in assembly. J Mol Biol 1992,
228:1163-1176.

Papers of particular interest, published within the annual period of review,

have been highlighted as:
of special interest
*" of outstanding interest

20. Derewenda U, Derewenda Z, Dodson EJ, Dodson GG, Bing X,

Markussen J: The X-ray analysis of the single chain A1-B29
peptide linked insulin molecule. J Mol Biol 1991,220:425-433.

1.

Baker EN, Blundell TL, Cutfield JF, Cutfield SM, Dodson EJ, Dodson
GG, Hodgkin DC, Hubbard RE, lsaacs NW, Reynolds CD etaL: The
structure of 2 Zn pig insulin crystals at 1.5A. Philos Trans R Soc
Lond Biol 1988, 319:369-456.

21. Bennet DL, Bailyes EM, Nielsen E, Guest P, Rutherford NG, Arden
SD, Hutton JC: Identification of the type-2 proinsulin processing
endopeptidase as PC2, a member of the eukaryotic subtilisin
family../Bie/Chem 1992, 267:15229-15236

2.

Steiner DF: The biosynthesis of insulin: genetic, evolutionary and

pathophysiologic aspects, In The Harvey Lectures. New York:
Academic Press; 1981,78:191-228.

22. Sato T, Herman L, Fitzgerald PJ: The comparative ultrastructure of

the pancreatic islet of Langerhans. Gen Comp Endocrino/1966,
7:132-157.

3.

Steiner DF, Bell GI, "lager HS, Rubenstein AH: Chemistry and
biosynthesis of the islet hormones. In Endocrinology. Edited by De
Groot L. London: WB Saunders; 1995:1296-1328.

23. Schlichtkrull J: Insulin crystals [PhD Thesis]. Munskgaard: University

of Copenhagen; 1958.

4.

Olsen HB, Ludvigsen S, Kaarsholm NC: Solution structure of an

engineered insulin monomer at neutral pH. Biochemistry 1996,
35:8836-8845.
The solution structure derived from NMR spectroscopy of a mutant insulin
monomer at neutral pH is described. There is great similarity between this
well-defined structure and the molecules in the dimers and T6 hexamers
determined by X-ray analysis. In particular the A and B-chain termini are
seen to be little changed in the monomer, consistent with the thinking that
conversion can occur in the hexamer.

24. Brange J: Galenics of Insulin. Heidelberg: Springer-Verlag; 1987.

25. MichaelJ, Carroll R, Swift HH, Steiner DF: Studies on the molecular
organization of rat insulin secretory granules. J Biol Chem 1987,
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26. Coore HG, Hellman B, Taljedal IB: Preparation and properties of
isolated mammalian insulin storage granules. In The Structure and
Metabolism of the Pancreatic Islets. Edited by Falkmer S, Hellman B,
Taljedal B. Oxford: Pergamon Press; 1970.