Download A Brief Review of Growth Hormone (GH) and Introduction to

A Brief Review of Growth Hormone (GH) and Introduction to
Shibayagi’s Rat Growth Hormone (GH) ELISA KIT
Katsumi WAKABAYASHI, Ph. D.
Prof. Emer., Gunma Univ.
Technical Consultant, Shibayagi, Co., Ltd.
1. What is GH?
Growth hormone (GH, Somatoropic hormone, STH, Somatotropin) a simple protein
hormone mainly produced in acido-philic cells, somatotrophs, which are present in the largest
population. GH positive cells are found in the brain and lymphoid tissues. In human, A
protein with high homology to GH named GH2 is found in the placental tissue.
GH receptors of WSbox type are distributed among liver, kidney, adipocytes, muscle,
thymus, etc., that belongs to the cytokine receptor group passing the cell membrane once.
Such a wide distribution of GH receptor indicates multiple action of GH on various tissues.
2. Structure and nature of GH
Rat GH is consisted of 190 amino-acids with the molecular weight of 21,777. The
amino-acid sequence including signal peptide is shown below (PubMed, Protein PO1244).
1 maadsqtpwl ltfsllcllw pqeagalpam plsslfanav lraqhlhqla
51 adtykefera yipegqrysi qnaqaafcfs etipaptgke eaqqrtdmel
101 lrfsllliqs wlgpvqflsr iftnslmfgt sdrvyeklkd leegiqalmq
151 eledgsprig qilkqtydkf danmrsddal lknygllscf kkdlhkaety
201 lrvmkcrrfa esscaf
Signal peptide is shown in red letters. The underlined parts indicate helix structure. There
is a discrepancy about N-terminal amino acid read from DNA sequence. Here, N-terminal
amino acid is written as leucine (1,2), while some reports told (3,4) that it is phenylalanine (f)
instead of leucine (l).
However, Dr. A. F. Parlow who provided NIDDK rGH I-6, a highly
purified preparation of radioiodination for RIA stated the nature of the preparation as follow:
“Chemical characterization of NIDDK-rGH-I-6, by analytic sephadex gel filtration, revealed
it to be 95-100% monomeric. A similar degree of homogeneity was established by
polyacrylamide gel and SDS-gel electrophoresis. In addition, NH2-terminal analysis by the
dansyl method revealed only a single amino acid, leucine.”
GH has disulfide bonds in two positions, 78-189 that forms a big loop, and 206-214 of a
small loop. The big loop is indispensable for GH bioactivity.
Ref: Mouse GH structure (PO6880)
1 matdsrtswl ltvsllcllw pqeasafpam plsslfsnav lraqhlhqla
51 adtykefera yipegqrysi qnaqaafcfs etipaptgke eaqqrtdmel
101 lrfsllliqs wlgpvqflsr iftnslmfgt sdrvyeklkd leegiqalmq
1
151 eledgsprvg qilkqtydkf danmrsddal lknygllscf kkdlhkaety
201 lrvmkcrrfv esscaf
（Only two amino acids (valine) shown in bold letters are different from rat GH sequence.
N-terminal amino acid is same in some reports.）
GH is hydrophobic and difficult to be dissolved in water around neutral pH, however soluble
under basic pH. In order to prepare the clear neutral solution, first dissolve GH in 0.01M
Sodium bicarbonate, and then dilute it with neutral phosphate buffer (according to the
instruction paper for NIDDK Rat GH RIA Kit).
Isoform of human GH
In human, the gene of GH is located in GH locus of chromosome no.17 with other 4 related
genes. These 5 genes are similar to each other in base sequence. Furthermore, in the process
of transcription, further isoforms are generated. This means that many isoforms of GH are
present in the pituitary gland. GH found in the pituitary is called GH1, but here I will write
only “GH”. Mutation and deficit of the gene will cause defect syndrome or lilliputian syndrome.
(Please see isoforms generated in the transcription process in the last part of this text.)
In human and monkey, placental lactogen (PL) is coded as one of GH-related genes in the
GH locus, and has high homology to GH and has potent GH-like action and weak
prolactin-like action. It is also called placental growth hormone or GH2. You can see amino
acid sequence of 22kD isoform 1 of GH2 in the last part of this text. Please compare it with
GH isoform 1.
Rat or mouse PL gene, on the other hand, locates on different chromosome to that of GH,
and is thought to be derived from prolactin gene because of its potent prolactin-like action.
3. Biological action of GH and regulation of secretion
GH acts on liver, kidney, fibroblast, and thymus epithelial cells. In the liver, GH causes the
gene expression and production of insulin-like growth factor-1 (IGF-1, somatomedin C), and
IGF-1 enhances the growth through causing proliferation of cartilage cells, biosynthesis of
chondroitin sulfate, hyperplasia and proliferation of various tissue cells, and protein
anabolism, and enhances secretion of thymulin from thymus cells (5).
Metabolic effect of GH is biphasic. Administration of GH tentatively shows insulin-like
effect (suppression of amino acid decomposition, enhancement of protein synthesis, decrease
of blood glucose, amino acids and free fatty acids), but later it causes increase of free fatty
acids owing to degradation of fat in adipose tissue, raising blood glucose, and shows insulin
antagonistic actions such as suppression of glycolysis, glycogen content increase in muscle,
and lowering insulin sensitivity in peripheral tissue (6,7).
GH has also prolactin like actions such as retention of Na, K, Mg, Ca, and P, enhancement
of Ca absorption in small intestine, development of mammary gland, enhancement of milk
secretion.
2
Blood GH levels and related diseases
Human diseases and other factors showing low blood GH levels: Pituitary dysfunction such as
Sheehan syndrome, thyroid dysfunction, obesity, Cushing syndrome, hypoglycemia,
pregnancy, treatment with corticosteroids.
Human diseases showing high blood GH levels: acromegary, pituitary gigantism, anorexia
nervosa, acute porphyria
Factors to increase blood GH levels
GHRH and ghrelin bind their receptors and activate Gs protein (stimulatory G protein)
that activate PKA via cAMP, and with increased intracellular calcium ion, enhances GH
production and secretion.
Thyroid hormones, cortisol, and retinoic acid bind their nuclear receptors, which control
transcription to effect GH gene expression.
Glucagon, vasopressin, 1-deoxy-D-glucose administration, administration of amino acids
such as arginine etc., protein intake, TF5, β−endorphin, L-DOPA, and stimulation of
epinephrine α-receptor also promote GH secretion.
Physiological conditions that cause GH secretion are hypoglycemia, stress (pyrexia, trauma,
hemorrhage, ether anesthesia, psychic unrest), fasting, exercise, and non-REM sleep, etc.
Factors to suppress GH secretion
Somatostatin (SRIF) binds its receptor changes intracellular cAMP and calcium ion
concentration via Gi protein (inhibitory G protein).
Activin, stimulation of epinephrine β-receptor, glucose, free fatty acid, corticoid
administration, high concentration of IGF-1, and high concentration of GH suppress GH
secretion.
Physiological conditions that suppress GH secretion are hyperglycemia, increased blood
fatty acid level, and REM sleep, etc.
4. Important notes in GH assay
Human GH measurement is much complex due to molecular heterogeneity and the
presence of GH binding protein. As specific assay system for each isomer of GH were not
available, physiological significance of isomer populations has not been clarified. Most
isomers of GH are thought to be those produced by transcription of whole exons.
Little has been known about rat GH isoforms, and so far, we have to observe physiological,
pharmacological and pathological changes of the measurable levels of GH using the assay
system available.
Measurement of GH content in the gland
According to my own measurement, GH content in adult male rat pituitary is around
300~350μgxRP-2/gland or 600~700 IU/gland (8). For RP-2, refer to the section of Standard
preparation of GH”.
3
In estimation of the pituitary hormone contents, their complete extraction from the gland is
indispensable. All the hormones are stored in secretory granules, and some are hydrophobic.
In my laboratory, we tested various methods for the simple and complete extraction of
hormones (8).
We found that prolactin was the most difficult to extract, and that immunoreactivty of GH
was lost if high concentration of ethanol is present under neutral pH, and that simple
homogenization with neutral phosphate buffer is not enough even for glycoprotein hormones.
As our conclusion, we found two methods: 1) to homogenize the gland with neutral
phosphate buffered saline (PBS) containing 1~2% urea followed by freezing and thawing, and
then centrifugation, and 2) to sonicate （20watt, 10sec.x 3times）the gland with PBS
containing about 0.1% Triton X-100, followed by freezing and thawing, then centrifugation.
The centrifugation at 4,000rpm for 15 minutes will give a clear supernatant fluid.
Measurement of blood GH levels
Periodical secretion of GH
GH secretion has been known to be periodical (9), i.e., the level rises and falls acutely with
certain interval. Therefore, if blood samples are collected at random timing, the assay values
will show a big variation as is seen in the assay data in the basic data section.
According to a report (10) where continuous samplings were made from male adult rats,
interval of GH peaks from the clock hour of 0:00 to 12:00 was 2.93±0.1 hour (mean±SE) ,
and 2.85±0.06 hour from 12:00 to 24:00. This means that GH is released periodically with
about 3 hours’ interval. The width of the peak at the foot was reported to be about 2 hours,
and this meant the width of trough is about 1 hour. The shape of the peaks was variable, and
showed individual variation. As rats are nocturnal animals, from 20:00 to 8:00 (corresponds to
their active period), the width of the trough seems to be wider, and height of peaks seems to be
lower compared with those in the daytime (please, refer to the original report in internet.).
We must be careful in planning of blood sampling in our experiments. Observation with
serial sampling may be necessary.
Influence of stress and anesthesia on blood GH level
There are some reports telling changes in GH level caused by stress and anesthesia,
suggesting that, in blood sampling for GH assay, we should be careful not to give animals
unnecessary stress and avoid some anesthetics.
Cold exposure for 5 minutes caused significant decrease in GH blood level to 43% of the
onset level, and GH level was also decreased by 5 minutes’ immobilization stress (12).
Eight hours’ immobilization a day for 10 days decreased GH levels , and GH level was
further decreased by injection of GnRH+TRH during this period (13).
GH level was increased by administration of ketamine(100mg/kg)＋Xylazine(10mg/kg) (15).
Standard preparations of GH
NIDDK rGH RP-2 is provided as a standard preparation for rat GH RIA. It is a component
4
of NIDDK rat GH RIA kit. RP-2 has the purity equal to highly purified preparation, NIDDK
rGH I series, and is added protective protein with buffer components. By adding fixed volume
of purified water, we make up a standard solution containing a fixed amount of purified rat
GH.
Rat GH ELISA KIT provided by Shibayagi uses the standard calibrated using RP-2. So, the
assay value is expressed as NIDDK rGH RP-2 equivalent.
Statement about purity of NIDDK rGH: The biological potency of NIDDK-rGH-I-6 is 2.0 IU
(Bovine GH) per mg. Contamination of this preparation with rPRL is 0.07%, with rFSH is
0.02%, with rLH is 0.2% and with rTSH is 0.004% (wt/wt), as determined by
radioimmunoassay.（from Instruction paper for NIDDK rat GH RIA kit）
Introduction to
Shibayagi’s Rat Growth Hormone (GH) ELISA KIT
This is an ELISA (Enzyme Linked ImmunoSorbent Assay) kit for measurement of rat GH
with high sensitivity using Sandwich assay principle.
[Advantage]
(1) Rapid assay (total reaction time: 5 hours.).
(2) A small sample volume (5 μl).
(3) An ecologically excellent preservative is used.
(4) Every reagent is provided in liquid form and ready to use.
(5) Excellent precision and reproducibility.
[Components]
Reagents
Amounts
(A)
Anti-GH-coated plate
96 wells(8x12) / 1 plate
(B)
Standard rat GH solution (20 ng/ml)
100μl / 1 vial
(C)
Buffer solution
60ml/１ vial
(D)
Biotin-conjugated anti-GH
100μl/ 1 vial
(E)
Peroxidase-conjugated streptavidin
100μl/ 1 vial
(F)
Chromogenic substrate reagent(TMB)
12ml/ 1 vial
(H) Reaction stopper (1M H2SO4)
12ml/ 1 vial
(I)
100ml/ 1 bottle
Concentrated washing buffer(10x)
[Assay sample]
Rat serum or plasma: 5 μl in the standard procedure.
The volume of assay sample can be applied in the range of 5 ~ 25μl.
5
In such case the final volume to be added to the well should be adjusted to 50μl using
assay buffer (C) as sample + buffer = 50μl.
[Assay range]
31.3 ~ 2000 pg/ml
This range corresponds to 313 ~ 20000 pg/ml of original serum/plasma level in the
standard procedure.
[Summary of Assay Procedure]
Antibody-coated 96 well plate
↓
Washing 3 times
↓
Buffer 45 μl + Sample 5 μl
or
Standard
(diluted sample 50μl)
50μl
↓
Shaking, then reaction for 2 hours at 20~25C
↓
Washing 3 times
↓
Biotin-conjugated anti-GH
50μl
↓
Shaking, then reaction for 2 hours at 20~25C
↓
Washing 3 times
↓
Peroxidase-conjugated avidin
50μl
↓
Shaking, then reaction for 30 mins at 20~25C
↓
Washing 3 times
↓
Chromogenic substrate solution
50μl
↓
Shaking, then reaction for 30 mins. at 20~25C
↓
Reaction stopper
1M H2SO4
50μl
6
↓
Shaking, then measurement of absorbance
at 450nm(sub. 620nm)
[Important notice in the treatments]
1. Treatment of assay samples
(1) Use serum or plasma samples obtained by ordinary standard method.
(2) Turbid samples or those containing insoluble materials should be centrifuged before
assay and remove those materials.
(3) Measure the samples as soon as possible after sampling.
2. Storage of assay samples.
If assay samples have to be stored for a long period, freeze samples and store below
–35oC. Avoid repeated freezing and thawing.
3. Influence of interfering substances
If presence of interfering substances is suspected, examine by a dilution test using
more than 2 points.
[Assay range and standard curve]
A model standard curve
Low concentration area extended
Stan dard c u rve
Standard curve
2.1
0.300
1.8
Abs.450( ⊿620)nm
Abs.450(⊿620)nm
0.250
1.5
1.2
0.9
0.6
0.200
0.150
0.100
0.050
0.3
0.000
0.0
0
500
1000
1500
2000
0
100
R at G H (pg/ml)
Rat GH (pg/ml)
[Storage condition]
Store the kit at 2~8C.
200
Do not freeze.
[Term of validity]
Six months from production. Expiration date is indicated on the container.
7
300
[Unit of package]
96 wells/1 plate
[Product code]
AKRGH-010
Basic Data
Assay precision
Wells
Sample A
Sample B
1
262
864
2
247
837
3
250
813
4
258
775
5
251
780
6
257
800
7
254
771
8
270
779
Mean
256
802
SD
7.19
33.5
CV (%)
2.8
4.2
Unit: pg/ml
Reproducibility
Samples
Day 1
Day 2
Day 3
Day 4
mean
SD
CV (%)
E
1626
1576
1615
1561
1595
31.0
1.9
F
412
407
409
401
407
4.50
1.1
G
96.0
97.9
96.1
103
98.3
3.35
3.4
Unit: pg/ml
n=4
Recovery test
Sample C
Added Found
Unit: pg/ml
n=2
Sample D
Recovered
Recovery (%)
Added
Found
Recovered Recovery (%)
0.00
101
-
-
0.00
506
-
-
155
265
164
106
303
822
316
104
192
285
184
95.8
466
949
443
95.1
223
325
224
100
539
1058
552
102
8
Dilution test
Dilution Test
2000
R = 0.999
GH (pg/ml)
1500
1000
500
R = 0.999
0
0.0
0.2
0.4
0.6
0.8
1.0
Dilution factor
n=2
Normal rat sample assay results
Samples: CD(SD)、6 weeks old males, fed ad libitum
Blood sampling: 14：00-15：00
Rat ID
GH ng/ml
1
10.4
2
7.49
3
7.16
4
7.21
5
6.52
6
3.59
mean
7.06
SD
2.17
GH levels after insulin administration
Animals:CD (SD),7 weeks old females, body weight 160-190g
Fasted for 6 hours.
Insulin administered: Human Insulin Ultra Pure(Cell Science Inc),100μIU/100gBW. IV
Assay samples: sera
Assay kit: Rat Growth Hormone (GH) ELISA KIT, Lot.0712
9
S erum G H levels after ins ulin injection
(mean±S E of 5rats )
GH [ng/ml]
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
0
30
60
90
120
150
180
T ime after injection [min]
Crossreactivity
Crossreactivity of this assay kit to prolactin and rat placental lactogen is negligible.
The kit does not crossreact with TSH, LH, nor FSH. Though the N-terminal amino acid is
not fixed, it does not matter because the antibody of the kit recognizes other part.
The kit crossreacts with mouse GH because the antibody recognizes the common amino acid
sequence to both rat and mouse GH. We cannot show the reactivity by percentage because
purified mouse GH preparation. It is possible to measure mouse GH using this kit if the assay
results were expressed as rat GH RP-2 eeuivalent.
Species
Substances examined
Reactivity(%)
r-GH
100
Prolactin
0.02
Placental lactogen
0.02
TSH
－
LH
－
FSH
－
Mouse
GH
＋
Mouse
TSH
－
Rat
＋：Cross-reacted
－：No cross reaction
*Amount of substance：2000pg/ml
10
Assay results for mouse GH
C57BL/6J males,6w
ICR males, 6w
Animal No.
GH ng/ml
Animal No.
GH ng/ml
N232-M1
13.2
N232-M11
19.9
N232-M2
12.4
N232-M12
12.4
N232-M3
11.3
N-232-M13
2.25
N232-M4
14.2
N-232-M14
6.44
N232-M5
14.2
N232-M15
12.8
N232-M6
12.2
N232-M16
5.49
N232-M7
11.6
N232-M17
16.3
N232-M8
16.8
N232-M18
7.12
N232-M9
14.3
N232-M19
5.78
N232-M10
9.84
N232-M20
13.4
Mean
13.0
Mean
10.2
SD
1.96
SD
5.59
GH assay values are expressed as NIDDK rat GH RP-2 equivalent．
References
1) Seeburg,P.H., Shine,J., Martial,J.A., Baxter,J.D. and Goodman,H.M.
Nucleotide sequence and amplification in bacteria of structural gene for rat growth hormone
Nature 270 (5637), 486-494 (1977))
2) Rohn,W.M. and Weigent,D.A.
Cloning and nucleotide sequencing of rat lymphocyte growth hormone cDNA
Neuroimmunomodulation 2 (2), 108-114 (1995)
3) Page,G.S., Smith,S. and Goodman,H.M.
DNA sequence of the rat growth hormone gene: location of the 5' terminus of the growth
hormone mRNA and identification of an internal transposon-like element
Nucleic Acids Res. 9 (9), 2087-2104 (1981)
4) Barta,A., Richards,R.I., Baxter,J.D. and Shine,J.
Primary structure and evolution of rat growth hormone gene
Proc. Natl. Acad. Sci. U.S.A. 78 (8), 4867-4871 (1981)
5)
Casanueva, F.F.
Physiology of growth hormone secretion and action.
Endocrinol Metab Clin North Am 21: 483-517（1992）
6)
Davidson, M.B.
Effect of growth hormone on carbohydrate and lipid metabolism.
11
Endocr Rev 8: 115-131 (1987)
7)
Strobl, J.S., Thomas, M.J.
Human growth hormone.
Pharmacol Rev 46: 1-34 (1994)
8)
Ishikawa, J., Fuse, Y. and Wakabayashi, K.
Choice of extraction procedure for estimation of anterior pituitary hormone content.
Endocrinol Japon 34: 755-767 (1987)
GH secretion has been know to be episodic by Tannenbaum & Martin (1976),
9) Tannenbaum, G. S., and Martin, J. B.
Evidence for an endogenous ultradian rhythm governing growth hormone secretion in the
rat. Endocrinology 98, 562-570. 1976
10) Kimura, F. and Tsai, C.-W.
Ultradian rhythm of growth hormone secretion and sleep in the adult male rat.
J Physiol 353, 305-315 (1987)
11)
Popii, V. and Baumann, G.
Laboratory measurement of growth hormone
Clinical Chemica Acta: 350, 1-16 (2004)
12)
Specific hormonal and neurochemical response to different stresses.
Lenox RH, Kant GJ, Sissions GR., et al.
Neuroendocrinology 30:300-308, 1980
13)
Effects of chronic immobilization stress on pituitary hormone secretion, on hypothalamic
factor levels, and on pituitary responsiveness to LHRH and TRH in female rats.
Du Ruisseau P, Tache Y, Brazeau P, and Collu R.
Neuroendocrinology 29: 90-99, 1979
14)
Changes in serum levels of gonadotropins and testosterone in the male rat in response to
fasting, surgery and ether.
Howland BE, Beaton DB and Jack MI.
Experientia 30:1223-1225, 1974
15) Acute hyperglycemia induced by ketamine/xylazine anesthesia in rats: Mechanism and
implication for preclinical models.
Saha JK, Xia J, Grondin JM, et al.
Exp Biol Med (Maywood) 230: 777-784, 2005
Growth hormone precursors of various animals
Five isomers are known with human GH.
Human isoform 1 (P01241, NP_000506)
12
1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla
51 fdtyqefeea yipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle
101 llrisllliq swlepvqflr svfanslvyg asdsnvydll kdleegiqtl
151 mgrledgspr tgqifkqtys kfdtnshndd allknyglly cfrkdmdkve
201 tflrivqcrs vegscgf
All exons are transcripted, and the molecular weight is 22kDa. This isofom
occupies most part of GH.
Human isoform 2(NP_072053)
1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla
51 fdtyqefnpq tslcfsesip tpsnreetqq ksnlellris llliqswlep
101 vqflrsvfan slvygasdsn vydllkdlee giqtlmgrle dgsprtgqif
151 kqtyskfdtn shnddallkn ygllycfrkd mdkvetflri vqcrsvegsc
201 gf
This isomer is produced by selective splicing of exson No. 3. As result, it 58-72 amino acid
sequence is lost. Occupies about 10% of population.
Human isoform 3 (NP_072054)
1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla
51 fdtyqefnle llrisllliq swlepvqflr svfanslvyg asdsnvydll
101 kdleegiqtl mgrledgspr tgqifkqtys kfdtnshndd allknyglly
151 cfrkdmdkve tflrivqcrs vegscgf
This isomer lacks amino acid sequence corresponding to exon No. 3.
Human isoform 4 (NP_072055)
1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla
51 fdtyqefrle dgsprtgqif kqtyskfdtn shnddallkn ygllycfrkd
101 mdkvetflri vqcrsvegsc gf
This isomer lacks amino acid sequence corresponding to exons No. 3 and No. 4.
Human isoform 5 (NP_072056)
1 mateagrwqp pdwadlqadl qqvrhkltqr
This isomer lost exons No. 3,4,and 5 in the transcription process. Most part for signal
peptide is also lost.
Comparison between Human GH1 ( isoform 1) and Human GH2(placental lactogen) ( isoform
1)（NP_002050）
Upper sequence:GH1，lower sequence:GH2
Different amino acids are shown in red letters.
1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla
1 maagsrtsll lafgllclsw lqegsafpti plsrlfdnam lrarrlyqla
13
51 fdtyqefeea yipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle
51 ydtyqefeea yilkeqkysf lqnpqtslcf sesiptpsnr vktqqksnle
101 llrisllliq swlepvqflr svfanslvyg asdsnvydll kdleegiqtl
101 llrisllliq swlepvqllr svfanslvyg asdsnvryhl kdleegiqtl
151 mgrledgspr tgqifkqtys kfdtnshndd allknyglly cfrkdmdkve
151 mwrledgspr tgqifnqsys kfdtkshndd allknyglly cfrkdmdkve
201 tflrivqcrs vegscgf
201 tflrivqcrs vegscgf
Chimpanzee
（P58756）
Sequence is same to human GH except one amino acid in the
signal peptide.
1 mapgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla
51 fdtyqefeea yipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle
101 llrisllliq swlepvqflr svfanslvyg asdsnvydll kdleegiqtl
151 mgrledgspr tgqifkqtys kfdtnshndd allknyglly cfrkdmdkve
201 tflrivqcrs vegscgf
Rhesus monkey (AAA20180)
1 maagswtcli laiallclpw lqegsafpti plswlfntav frahhlhkla
51 fdtypkfeea yipkeqkysf lrnpqtslcf sesiptpsnk eetqqksnle
101 llhisllliq swlepvqflr svfanhlvht nsnfdiylyl kkleegiqtl
151 mgrledgspr tgqifketys kydtnshndd tllknyrlly cfrkdmnkve
201 tflrtvrcra vegscgf
Cattle (NP_851339)
1 mmaagprtsl llafallclp wtqvvgafpa mslsglfana vlraqhlhql
51
101
151
201
aadtfkefer
llrisllliq
reledgtpra
ylrvmkcrrf
tyipegqrys iqntqvafcf setipaptgk neaqqksdle
swlgplqfls rvftnslvfg tsdrvyeklk dleegilalm
gqilkqtydk fdtnmrsdda llknygllsc frkdlhktet
geascaf
Pig (NP_999034)
1 mmaagprtsv llafallclp wtqevgalga mplsslfana vlraqhlhql
51 aadtykefdr pyipegqrys iqnaqaafcf setipaptgk deaqqrsdve
101 llrfsllliq swlgpvqfls rvftnslvfg tsdrvyeklk dleegiqalm
151 reledgspra gqilkqtydk fdtnlrsdda llknygllsc fkkdlhkaet
201 ylramkcrrf vesscaf
14
Sheep (NP_001009315)
1 mmaagprtsl llaftllclp wtqvvgafpa mslsglfana vlraqhlhql
51 aadtfkefer tyipegqrys iqntqvafcf setipaptgk neaqqksdle
101 llrisllliq swlgplqfls rvftnslvfg tsdrvyeklk dleegilalm
151 reledvtpra gqilkqtydk fdrnmrsdda llknygllsc frkdlhktet
201 ylrvmkcrrf geascaf
Chicken (CAA35619)
1 mapgswfspl liavvtlglp qeaaatfpam plsnlfanav lraqhlhlla
51 aetykefert yipedqrytn knsqaafcys etipaptgkd daqqksdmel
101 lrfslvliqs wltpvqylsk vftnnlvfgt sdrvfeklkd leegiqalmr
151 eledrsprgp qllrptydkf dihlrnedal lknygllscf kkdlhkvety
201 lkvmkcrrfg esncti
Rainbow trout (AAA49553)
1 mgqvfllmpv llvscflsqg aaienqrlfn iavsrvqhlh llaqkmfndf
51 dgtllpderr qlnkiflldf cnsdsivspv dkhetqkssv lkllhisfrl
101 iesweypsqt liisnslmvr nanqisekls dlkvginlli tgnqdgvlsl
151 ddndsqqlpp ygnyyqnlgg dgnvrrnyel lacfkkdmhk vetyltvakc
201 rksleanctl
(10/02/21)
15