Chemistry Reference
In-Depth Information
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The preparation of crystalline concanavalin A: 'Stir 100 grams of fi nely pow-
dered jack bean meal with 500 cc. of 32 - per - cent acetone and fi lter in an ice
chest for 4 or 5 hours, or overnight. Place the residue in a beaker, stir with
500 cc. of 30 - per - cent alcohol and fi lter at room temperature. Now extract the
concanavalin A from the residue with 400 cc. of a solution containing 1%
sodium chloride and 0.1- per - cent neutral phosphate and fi lter. Extract the
residue with 250 cc. of 5- per - cent sodium chloride and fi lter. Combine the two
last fi ltrates, add toluene and dialyze against distilled water in three collodion
bags for 24 hours. Centrifuge off the crystals of concanavalin A and wash once
with 10-per-cent sodium chloride solution. Dissolve the crystals in a small
amount of 0.1 N hydrochloric acid. After standing for about one-half hour add
a small quantity of neutral phosphate solution and then exactly enough 0.1 N
sodium hydroxide to neutralize the hydrochloric acid previously added. Dialyze
the material for several hours and centrifuge off the crystals of concanavalin A'
[J.B. Sumner, S.F. Howell. The identifi cation of the hemagglutinin of the jack
bean with concanavalin A.
J Bact
1936;
32
, 227-237]. In a previous publication
[J.B. Sumner. The globulins of the jack bean,
Canavalia ensiformis.
J Biol Chem
1919;
37
, 137-142] the crystals were characterized as follows: 'Dr. A.C. Gill has
been kind enough to examine them, and has provisionally declared them to be
bisphenoidal in shape, probably belonging to the rhombic system, optically
biaxial, with a large optical angle, and negative in optical character.'
nitrogen of Asn14 to the sugar and lets Asp208 fl ip into its position for contact
via acquisition of the Ala207- Asp208
cis
- peptide bond. Obviously, Ca
2+
acts as
mastermind of structural rearrangements to optimize ligand contact.
The
-sandwich fold is not only encountered in the leguminous agglutinins.
Lectins involved in quality control of folding of nascent glycoproteins (calnexin,
calreticulin) and in further sorting and transport of maturing glycoproteins in the
endoplasmic reticulum - Golgi intermediate compartment (ERGIC) such as ERGIC-
53 share this general structure, together with other proteins to be mentioned below
(please see Chapter 6.3 for details on quality control). The conspicuous changes
in the positions and/or number of Ca
2+
-binding sites are signs for independent
evolutionary ancestry of this apparently useful compact fold. This assumption is
backed by a lack of sequence homology among respective proteins (please see also
below). Going into details of the number of bound cations and their positioning,
the endoplasmic-reticulum-resident calnexin has only one putative Ca
2+
- binding
site. Ca
2+
is coordinated to Ser75, Asp118 and Asp437 (Figure 16.1b). It is located
rather far away from the site interacting with
β
-
D
-glucose, as this ligand accepts
hydrogen bonding with Tyr165, Lys167, Tyr186 and Glu217 [4]. The role of the
calcium ion may in this case thus be more indirect than for ConA.
α
