954
BIOCHEMICAL SOCIETY TRANSACTIONS
Acid-Soluble Ribonucleotides in the Brine Shrimp ( h e r n i a sulinu)
PETER J. CONROY and JAMES T. NODES
Department of Biochemistry, Brunel University, Uxbridge, Middx. UB8 3PH, U.K.
The brine shrimp (Artemia salina) has a worldwide distribution, commonly inhabiting
evaporating pools of high salinity. The undeveloped cysts contain significant amounts of
two unusual guanosine-containing nucleotides. Finamore & Warner (1963, 1965) have
identified these as GppppG and GpppG. Both are found in the acid-soluble fraction and
can be purified by ion-exchange chromatography. The present preliminary investigation
was undertaken to examine the qualitative distribution and quantitative concentrations
of the free ribonucleotide pools in the brine shrimp at different stages in its development.
Separation and identification of nucleotides was carried out on (i) undeveloped cysts,
(ii) cystsrehydrated withartificialsea-water at 32~0.5”Cfor
24hand (iii) matureshrimps.
The method ofextraction was essentially that described by Finamore &Warner (1965).
A 20g portion of the desiccated and treated cysts (Great Salt Lake Eggs, Sanders, Ogden,
Utah, U S A . ) or a 2.71g portion of mature shrimps was used in the extraction method.
The undiluted soluble fractions were applied directly to a Dowex AG (X10; formate
form) column (1 cm x 15cm) for preliminary chromatography. Nucleotides were
separated by the use of the extended-gradient formate-formic acid system introduced by
Hurlbert et al. (1954) and subsequently modified by Nodes & Reid (1964). The hyperbolic mixing gradient was generated in a 500ml mixing chamber and 10 ml samples were
collected. Plotted elution profiles of Ez60wereused to identify peak tubes for rechromatography and pooled fractions were freeze-dried. Ammonium formate was sublimed off,
and each sample was taken up in lOml of water and applied to a Dowex AG (X10)
column (1 cm x 10cm). Nucleotides were eluted by an ammonium formate solution
adjusted to pH5 and Sml samples were collected. The nucleotide components were
characterized by elution position, molar extinction ratios and wavelength of maximum
absorbance.
The following free nucleotides were identified (Table 1) and are expressed in quantitative terms in Table 2: AMP, ADP, ATP, GMP, GDP, GpppG, GTP, UMP, UDP,
‘UDP-X’ (eluted in the same position as UDP-glucose). In contrast with the results
obtained by Finamore & Warner (1965), the occurrence of UMP, UDP and ‘UDP-X’
was detected in this study. The mononucleotide UMP is eluted with GMP in the preliminary chromatography and is separated on rechromatography ; UMP constitutes
2.5%of the total nucleotide pool. UMP could not be detected in the adult shrimp.
The ‘UDP-X’ concentration declines on rehydration from 10.5 % to zero, being
Table 1 . Elution positions and spectral data for nucleotides rechromatographed on
1 cm x lOcm Dowex AG (X10) columns with an ammonium formate system, 5 m l samples
being used
Elution
Extinction ratios (pH5.0)
position
(tube no.)
Nucleot ide
E ~ ~ O / E ZE 2~7 5OI E 2 6 0
E~,oIEz~o
Amax. (nm)
50-60
0.81
0.40
0.15
258-259
AMP
0.80
0.41
60-69
0.18
258
ADP
0.15
260-261
0.82
0.38
69-80
ATP
0.67
253
1.18
0.78
38-50
GMP
253-254
0.67
61-71
1.16
0.75
GDP
0.66
120-1 3 1
1.16
0.75
254
GTP
0.64
254
72-80
1.15
0.73
GPPPG
0.74
0.64
17-23
260-2 61
UMP
0.39
0.72
0.65
263
3 5-44
0.40
UDP
0.72
0.60
58-66
262-263
‘UDPX’
0.38
1973
539th MEETING, UXBRIDGE
955
Table 2. Acid-soluble ribonucleotide concentrations in the brine shrimp at different stages
in its development
Units are expressed as nmol (at 260nm with 1 cm light-path) of the material as if present
in a 1 ml volume per g of material and as percentage of total nucleotides.
Hydrated cysts
Dry cysts
r
Nucleotide
AMP
ADP
ATP
GMP
GDP
GTP
GPPPG
UMP
UDP
‘UDPX‘
CMP
(% of
(% of
total)
5.0
(nmol/ml
per g)
56.4
0
0
0
141.2
214.5
59.8
177.0
104.4
19.4
142.0
82.5
23.2
6.5
18.3
11.3
2.1
18.7
8.9
341.6
0
25.8
0
40’
5.0
660*
50
(nmol/ml
per g)
40*
0
Adult shrimps
7
-
0
0
122.7
0
0
0
total)
4.25
0
10.7
0
9.25
0
0
0
(nmol/ml
per g)
821
1019
1359
0
1087
0
0
0
(% of
total)
10.1
12.5
16.8
0
13.4
0
0
0
1910
1402
23.6
17.3
512
6.3
* Estimated value.
paralleled by a relative and absolute rise in that of UDP. This would be consistent with
the observations made by Clegg (1964), who maintains that the disaccharide trehalose
supplies the glucosyl-building blocks required for glycogen biosynthesis during
embryogenesis. Polysaccharide and glycerol biosynthesis temporarily ceases when the
nauplius has vacated the cyst. As development proceeds the relative contribution made
to the total nucleotide pool by the nucleoside triphosphates declines and that of the
diphosphates increases. In the dry cysts the former account for 29.6% and the latter
34.1 %. Similarly for hydrated cysts thecorrespondingvalues are 10.7 and 35 %.A similar
relative order occurs in the adult shrimps, namely 16.8 and 66.6%.
GpppG is eluted before GDP during preliminary chromatography. The tetraphosphate GppppG isolated by Finamore & Warner (1963) could not be recovered on rechromatography of the dry cysts. However, significant quantities of GTP and GMP were
isolated. GppppG breaks down to give equimolar quantities of GTP and GMP. Hence
freeze-drying is probably responsible for hydrolysis of GppppG. Approx. 32 % of the
u.v.-absorbing material in the cold-acid extract can be ascribed to these diguanosine
nucleotides or their breakdown products. Both the tetraphosphate and triphosphate
diguanosine nucleotides are characteristic of dry cysts and are not found in either
hydrated cysts or adult shrimps.
The biochemical function of these compounds may rest in the sparing solubility of
guanine derivatives (40mg/l at 25°C) in contrast with the more readily soluble adenine
nucleotides. The diguanosine derivatives could act as relatively stable reserves that can be
quickly mobilized. Adenosine nucleotides would tend to break down under the extremes
of desiccation to which the cysts are subjected. The pyrophosphate linkage formed between two GDP molecules is similar to that formed between two ADP molecules. Smith
& Khorana (1958) describe a ‘dismutation’ reaction that takes place on incubating ADP
with dicyclohexylcarbodi-imide,yielding mixtures of ATP and AMP. They believe that
AppppA is an initial condensation product of ADP and that hydrolysis occurs between
the first and second or third and fourth phosphate groups to produce a mixture of AMP
and ATP. In the encysted state there is no detectable ADP or ATP, with AMP making
up 5 of total nucleotides (Finamore &Warner, 1965). With rehydration ATP appears
Vol. 1
956
BIOCHEMICAL SOCIETY TRANSACTIONS
and the total adenosine nucleotide pool increases to 15 %and then to 39.4%in the adult.
A concomitant decrease in the guanosine pool occurs, from 59.3 % to 9.25‘x on rehydration and 13.4%in the adult.
Unusual guanosine nucleotides are not confined to the brine shrimp. Cashel & Gallant
(1969) found that amino acid starvation of stringent (re/+)strains of Escherichia coli
causes a rapid accumulation of two unusual guanosine nucleotides called ‘magic spots’ I
and I1 (MSI and MSII). The relaxed strain (rel-) did not exhibit this response. They
postulated that high intracellular concentrations of the MS compounds led to a cessation
of RNA accumulation and to the other characteristics of the stringent response. Cashel
& Kalbacher (1970) have identified MSI as a guanosine tetraphosphate (ppGpp, 3’- or
2’-diphosphoguanosine 5‘-diphosphate) and MSII as a guanosine pentaphosphate.
Haseltine et al. (1972) have synthesized MSI and MSII in vitro on the E. coli ribosome
with GTP (or GDP) and ATP as substrates. These workers report that the difference
between relaxed and stringent strains with respect to MS accumulation during amino
acid starvation is due to an alteration in a protein factor present in the O.SM-NH,CI
ribosomal wash. They conclude that MS accumulation in vitro is the product of a n
idling step in protein synthesis occurring on the ribosomes. Although the structures of
MSI and MSII differ from those of the unusual guanosine nucleotides found in the
brine shrimp, the latter may perform a similar role in response to increasing salinity.
Cashel, M. & Gallant, J. (1969) Nature (London) 221, 838-841
Cashel, M. & Kalbacher, B. (1970)J.Biol.Chem.245,2309-2318
Clegg, J. S. (1964) J . Exp. Biol. 41, 879-892
Finamore, F. J. & Warner, A. H. (1963) J . B i d . Chem. 238, 344348
Finamore, F. J. & Warner, A. H. (1965) Biochim. Biophys. Acta 108, 525-530
Haseltine, W. A., Block, R., Gilbert, W. & Weber, K. (1972) Nurure(London)238,381-384
Hurlbert, R . B., Schmitz, H., Brumm, A. F. & Potter, V. R. (1954) J . Biol. Chem. 209,23-39
Nodes, J. R. & Reid, E. (1964) Brit.J . Cnncer 17, 745-774
Smith, M. & Khorana, H. G. (1958) J . Amer. Chem. Soc. 80, 1141-1145
Quantitative Measurement of Transplantation (HE-A) Antigen Sites on
Peripheral Human Lymphocytes
ARNOLD R. SANDERSON and KEN I. WELSH
Mclndoe Research Unit, Queen Victoria Hospital, East Grinstead, Sussrx RH19 3 DZ,
U.K .
The two linked HL-A genetic loci in man determine the peripheral occurrence, on all
nucleated cells of a heterozygous individual, of four HL-A antigens (Kissmeyer-Nielsen
& Thorsby, 1970). Immunity to these antigens is principally responsible for graft rejection between genetically dissimilar individuals. Although there are no reliable estimates
of the absolute number of HL-A sites for any single specificity on a given cell type, the
relative antigen contents of various tissues have been compared (Berah et al., 1970) by
careful adsorption of HL-A antisera on various numbers of different cell types.
We here present details of absolute site number for a single HL-A specificity determined by the first (LA) HL-A locus, present on peripheral blood lymphocytes. Specific
HL-A2 antibody is purified after adsorption on and elution from cells. After iodination
with lZ5I,the site number can be calculated from determination of the saturation level
of a given number of lymphocytes with labelled globulin.
Lymphocytes were prepared from defibrinated blood by the Ficoll-Isopaque technique (Boyum, 1968). HL-A typing was performed on Falcon microplates
(Scientific Supplies, London E.C.1, U.K.), Trypan Blue exclusion being used as the
end-point determination of viability (Terasaki & McLelland, 1964).
HL-A2 antibodies in sera having well-characterized specificity were adsorbed on cells
1973