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Myo-inositol phosphates in a phosphoinositide complex from kidney
BIOCHIMICA ET BIOPHYSICA ACTA
681
BBA 5 5 0 5 1
M Y O - I N O S I T O L P H O S P H A T E S IN A P H O S P H O I N O S I T I D E FROM K I D N E Y
COMPLEX
F. A N D R A D E AND C. (~. H U G G I N S
Department of Biochemistry, Tulane University School of Medicine, New Orleans, La. ( U.S.A .) (Received J a n u a r y 31st, I964)
SUMMARY
I. Chemical studies on the "phosphatido-peptide fraction" from kidney have shown that this material more properly should be called a phosphoinositide complex. 2. Glycerophosphate, inositol mono-, di- and triphosphate were separated from alkaline hydrolysates by means of paper chromatography. 3- Six fractions were obtained by cohtmn chromatography from the deacylated phosphoinositide complex and identified as inositol monophosphate, glycerophosphate, inositol diphosphate, glycerylphosphoryl inositol, glycerylphosphoryl inositol phosphate, and glycerylphosphoryl inositol diphosphate. 4. These phosphates, presumably as the inositides, are believed to be responsible for the high metabolic activity of this fraction as measured by radiophosphorus. INTRODUCTION
Myo-inositol meta-diphosphate was first reported by FOLCH1,2 as being present in a diphosphoinositide from beef brain. Evidence showing that FOLCH'S beef-brain diphosphoinositide contains more than one type of inositol phosphate has been reported by several groups of investigators 3,4, all of whom have shown the presence of inositol mono-, di- and triphosphate in this fraction. Recently, BROCKERHOFFAND BALLOU8,7 in a series of publications have sho~n that beef-brain phosphoinositides (FOLCH'Sdiphosphoinositide) exists as a complex of substances containing L-phosphatidyl-Lmyo-inositol, L-phosphatidyl-L-myo-inositol 4-phosphate and L-phosphatidyl-L-myoinositol 4,5-diphosphate. Furthet more, these structures appear to be interrelated in such a way as to suggest a step-wise phosphorylation of the monophosphoinositide in the biosynthesis of di- and triphosphoinositides 8,9. The term "phosphatido-peptides" was introduced into the literature by FOLCH1° in 1952 to characterize a protein-bound complex lipid fraction which he had obtained from a lipid-free trypsin-resistant residue of beef brain by extraction with acidified chloroform-methanol. LEBARON AND FOLCH11 suggested that this fraction represented a hitherto unreported form of tissue phosphorus containing esterfied fatty acids, inositol diphosphate, glycerol and amino acids in polypeptide chains. HUGGINS AND COHNTM in 1959 isolated a very similar fraction from kidney cortex using FOLCH'S extraction method. Distribution studies by these latter investigators have revealed Biochim. Biophys. Acta, 84 (I964) 681-693
()~2
1;. ANI)ICAI)E, ('. ~,. t I U ( ; ( ; I N S
this fraction to be present in various amounts in br,dn, heart, liver, lung, spleen, pancreas, intestine, saliwtry glands and tumor tissue. ['sing kidney tissm~. ]lI'(;tilNs ANt) (_'OHNr' have shown that the specific activity . f the l~hosl~h
MI,71"HOI)S A N D I..'XPI.H¢IMENTAI. PI¢OCEI)URE
A nalytical methods Phosphorus was determined by the method of F'ISKF AND SUBBAI~,ow l:~ aS modified by BARTLETT2°. Glycerol was determined according to Hax..XHaX axl) ()LL].;Y"~ and a-amino nitrogen was determined by the ninhydrin procedure 2" using leucine for the standard curve. Myo-inositol was assayed by the microbiological procedure "a with Kloeckera brevis wtr. apicnlata and also bv the spectrophotometric procedure as described by LEBARON et al. 24.
Chromatographic methods Descending paper chromatography of the products from alkaline hydrolysis was carried out for 3 ° h at 3 °° on Whatman 3 MM paper according to the procedure described by 1)ESJOBERT AND -PETEK 2's using the solvent system n-propanol -ammonia water (5:4:1, v/v). More effective separation of the different isomers of inositol diand triphosphate could be obtained if the chromatograms were. run for 5° h ; however, under these conditions, inositol monophosphate and glycerophosphate were cluted ldiockim, t~iopkys...lcta, 84 (19o4) (JSi t,q3
KIDNEY PHOSPHOINOSITIDE COMPLEX
683
from the paper. Localization of phosphate esters was accomplished using the HANESISHERWOOD spray as modified by BANDURSKI AND AXELROD26 and also by radioactive scanning of developed strips on which the phosphate esters were labeled with 3~.p. Samples containing approx. 200/zg of phosphorus were applied as a band over 15 cm and subjected to descending chromatography. The separated phosphate esters were eluted from the paper strips with o.1% NH4OH. Column chromatography of the deacylated material was carried out according to the method of BROCKERHOFF AND BALLOUs using Dowex-I (C1-), and gradient elution ~ith 0.4 M LiC1. The pattern of elution was followed by assaying an aliquot from each tube for total phosphorus and radioactivity.
Materials Inositol di- and triphosphate were prepared by acid hydrolysis of phytic acid according to KERR AND KFOCRYz7 and inositol monophosphate was obtained from California Corporation for Biochemical Research. Beef-brain phosphoinositide was prepared according to FOLCH°-. The crude inositide was precipitated eight times from chloroform with methanol and then dialyzed for 48 h against running water at 4 °. Radiophosphorus (Na2Ha~PO,) was obtained from Oak Ridge National Laboratory. Myo-inosose-2 was reduced to i2-3H!inositol with tritium by the New England Nuclear Corporation. The final [2-3Hlinositol was recrystallized two times from hot water and ethanol and had a melting point of 219-221 ° (uncorr.). It had a 3H content of 63.3 mC/mmole and gave 1.o2. lO8 counts/min per mg when counted at infinite thinness,in a windowless gas-flow counter. For incorporation of radioisotopes kidney slices were incubated as described previously t2 to which 5/~C of Na2H32PO4 or 2/~C of [2-3H]inositol were added per ml of incubation mixture. Scanning for the location of radioactive areas on paper chromatograms was accomplished with a Vanguard automatic scanner, Model 80o. Radioactive samples were also plated in stainless-steel planchets and counted in a windowless gas-flow G-M tube using a Baird-Atomic Model 135 Scaler.
Preparation of the phosphoinositide complex The phosphoinositide complex was isolated by the procedure described by HUGGINS AND COHN 12, and the crude extract was partitioned against 0.2 volume of 0.2 M NaHCO v The resulting lower phase (organic plus the interracial fluffy layer which redissolved on the addition of 0.2 volume of methanol) were used as the starting material for these studies. In some instances, we have used the technique described by LEBAROX et al. 14 because it was found to be more adaptable for large-scale preparations of this fraction. This latter procedure differs from that described by HOGGINS ANn COH.',"1~in the solvents used for the extraction of the other phosphorus-containing fractions, but it is similar in that both procedures use acidified chloroform-methanol for the extraction of the crude fraction. The composition of this fraction using either method does not differ as far as total phosphorus, inositol or glycerol is concerned. The crude fraction so obtained, which represented 5 % of the total tissue phosphorus, had the following composition : 3.3% phosphorus, 7.3% glycerol and 16.8% inositol. The esterified f a t t y acid/ Biochim. Biophys. Acta, 84 (1964) 681-693
{)~4
F. A N I ) R A I ) E , C. (;. H U ( ; ( i l N S
phosphorus molar ratio was 1.8 for the crude flaction, l)ata ()btained when variou, species and organ systems were analyzed for the different components , f tilt, ph,~sl~h.inositide complex indicate that there is a characteristic reproducibility in regard t~, the different organ systems and species analyzed. Chromatograt)hic techniques using silicic acid-impregnated glass fiber paper "~sand thin-lay'er c h r o m a t , g r a p h y "u were not successful in resolving the complex into components - t h e r than im>itides and ~,nly ttmse areas containing phosphorus and inositol were ninhydrin p.sitive. The crude fraction was subjected to dialysis in older to, establish tile presence ~r absence of fiee amino acids or other small dialyzabh~ components. Dialysis was carried out at room temperature fi~r 48 h against three changes of distilled water. "l'al)le I presents the values before and after dialysis, it can be seen that there is a los> . f aptm~x. 5 o ° . of the free ~t-amino nitrogen; however, it can also be s e e n t h a t ;ipproxinaatvly the same molar amount of phost)horus and inositol were ;tls~ dialvzabh.. TABLE
1
IIIAI.YSIN OF PHOSPttOINO.SITII)I.I COMPI.EX T h e r e p o r t e d values are represt:n|~'d i t s / r a m i e s per g wet w t . . f (onstltl.'nts
Total phosphorus Glycerol I nositol u-Amino nitrogen (before hydrolysis) Phosphorus/inositol ratio
ltcfore dialysis (#moles)
.-1.1t,'r dialysis [tt~'loh's )
z.,s -.o ".3 o.35 t.-'
-'.~, 13, -'. I o. t 7 I.-
tissue. Loss Omloles ) ~.2 o. 4 o.2 o. 18
Alkaline hydrolysis of pig-kidney phosphoinositide comple:c 7 g of crude fraction were suspended in 25o ml of o. 5 N Na()H and refluxed for 45 min. At the end of this time complete solution was obtained. The dark brown solution was then worked up according to tile procedure descrihed by (~RADO ..~,ND BALLOt;3 for base hydrolysis of beef-brain phosphoinositide. The resulting aqueous layer was made basic by the addition of cyclohexylamine and then concentrated to a small volume. The concentrate which contained no Pi was the starting material for separations by descending paper chromatography. Mild alkaline deacylation of the crude fraction was carried out according to the fl~llowing procedure: 2 g of crude fraction in 5 ° m l of chlorofi~rm-nmthanol (2 :I, v/v) containing o.I2 N N a O H were swirled for IO min at 25 °. At the end of this time, 5 g of Dowex-5o (H ') in 2o ml of water were added to the reaction. The resulting aqueous phase was separated ; washed two times with an equal w)lume of ethyl ether and passed through a l)owex-5o ( H ' ) column containing approx. 15 g of resin. The column was washed with 5o'!,~, ethanol according to BROCKERHOFF AND BALI.OU 6 and the eluate was made basic with cyclohexylainine and concentrated to a small w)lume. This served as the starting material for separation of the phosphate esters hy column c h r o m a t o g r a p h y using l)owex-I (C1-) as described by, BROCKEmmVF axI) BaLLOU~. Under these conditions, 95~}{~ of the phosphorus became water soluble. H t o c h i m . t ¢ i o p h y s . ..1eta, S, t (rgO.;) (~81.-693
685
KIDNEY PHOSPHOINOSITIDE COMPLEX RESULTS
Alkaline hydrolysis of pig-kidney phosphoionsitide complex The results in Fig. I were obtained by descending paper chromatography of alkaline hydrolysates of 32p_ and 3H-labeled phosphoinositide complex of pig-kidney cortex• The upper tracing represents the products from Na,Hs2po4-1abeled material and the lower tracing from [2-ZH]inositol-labeled material. Six separate fractions were obtained with glycerophosphate (Fraction F) being used to calculate the relative mobilities of the other fractions. The data in Table II were obtained in the process of studies designed to characterize the six fractions represented in Fig. I. In these experiments large-scale preparations of the crude fraction from pig-kidney cortex were mixed with 32P-labeled material, the latter being used to facilitate localization of the different fractions. After alkaline hydrolysis and separation by descending chromatography, the different fractions were eluted from the paper. The pooled eluates from several papers were lyophilized and subjected to acid hydrolysis in a sealed tube with 6 N HC1 at i i o ° for 24 h. The phosphorus/inositol molar ratios in the hydrolysates were 3.06 and 3.z A
B
A 3 2 p
/-': ;'" '_
:" I ~ I]
'-
I. I •I
|
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~=i
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~-
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..........
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',
. . .. . . .i - - - . ;-x,-. .--i• . . ., . ;~_.• *---.- I . 7-i--1--',-'-: -!-
. . . . . . . .
-:~-'-'-~
~--
--~,--~ ....
~--~-.-J--'--~--
~--,~ -t-"-- ~-t':,~
:.
'
!' ',_ L - L - I - - ' - r ~ - ' , ; i - - [ - l - -I
;- J
_L_.L_. ~ ; ' ,
i o
F
~--'.-'~- ~-~
,-
I
:/~i..-Z_ L7-I-, -jE -7 ~-~-'-Fr,, ,_'xl
S,oo0or0, I
E
:--,-.gN;~,.,-4--i--$-Ji-.-'.,---i--J;--i---~--.
"~ l}_~_,.o~,to, -r~
O
C
i %
i
I i%
l
I- I ~ - - I !
I
1
I
[
i ,p
6p
Fig. i. T h e d i s t r i b u t i o n of r a d i o a c t i v i t y on d e s c e n d i n g c h r o m a t o g r a m s o b t a i n e d b y p a p e r chrom a t o g r a p h y of a n alkaline h y d r o l y s a t e of t h e p h o s p h o i n o s i t i d e c o m p l e x from k i d n e y cortex. S a m p l e s were applied a t Position A prior to s e p a r a t i o n by d e s c e n d i n g c h r o m a t o g r a p h y . T h e t r a c i n g s were o b t a i n e d u s i n g a V a n g u a r d a u t o scanner. U p p e r t r a c i n g from s2P-labeled m a t e r i a l ; lower t r a c i n g from [2-SH]inositol-labeled material. Lower p h a s e is a d i a g r a m m a t i c r e p r e s e n t a t i o n of m o v e m e n t of s t a n d a r d s in which I P a = inositol t r i p h o s p h a t e , IP 2 - inositol d i p h o s p h a t e , I P -- inositol m o n o p h o s p h a t e , G P = g l y c e r o p h o s p h a t e .
for Fractions A and B; 2.26 and 1.54 for Fractions C and D and 0.96 for Fraction E. Fraction F did not contain inositol but did contain glycerol and, since glycerol is partially destroyed in 6 N HC1 (ref. 2I), we have used 2 N HC1 routinely for hydrolysis in the measurement of glycerol. Suitable aliquots of the a'P-labeled eluates were mixed with standard compounds and rechromatographed by descending chromatography or subjected to separation by high-voltage electrophoresis. The radioactive areas were found to correspond to the areas revealed by the H A N E S - I S H E R W O O D spray. It is Biochim. Biophys. Acta, 84 (1964) 681-693
t.o
e~
a:
~, i .o
(;
hydrolysis
i.o
Alkaline
0.72
E
F
0. 4
C
o.6
o. I 5
B
D
0.02
R,
A
Fraction
TABLE
I1
and
obtained
eluates
chronlatography
trace
o.6i
o
o
o
o
o
Glycerol fltmoles)
from pooled
paper
o
o
g 16
o. 19
o. 11
0.28
o. 17
Mvo-inositol (lonolcs) •
descending
i. 15
0.57
I. l x
o.29
o.26
0.87
0.53
Phospltorus (ttmoles,;
from several
carried
---
0.96
x.54
2.26
3.1
3.06
Phosphorus/ inositol ratio
according
experiments,
out
--
0.93
....
-- ]
--
-
Phosphorus/ glycerol ratio
leg
described
--
274
328
92o
86.,,
143o
4260
with
-
--
120 900
IO7 5oo
to 7 880
the
92 750
149 4 ° 0
[a-ttlJMj,o-inositol counts/rain per g tissue
in t e x t
agtivitv'Specific counts/rain" per /*g phosphorus
migration o f c o m P0u_ml migration of glycerophosphate.
to procedures
24
I2
23
II
18
ii
phosphorusT°tal placed on paper (%)
data
representing
unknown that
phosphate
diphos-
diphos-
triphos-
triphos-
glycerophosphate
nlyo-inositol
myo-inositol phate
myo-inositol phate
myo-inositol phate
myo-inositol phate
('omposttitm
COMPONENTS O B T A I N E D BY D E S C E N D I N G PAPER C H R O M A T O G R A P H Y OF T H E P R O D U C T S OF A L K A L I N E H Y D R O L Y S I S OF s2p_ AND [2-all~INOSITOI--LADELI':I) PHOSPHOINOSITIDI., COMPI.FX OF K I D N E Y CORTEX
;=
>
> 7.
oc
687
KIDNEY PHOSPHOINOSITIDE COMPLEX
believed that Fractions C and D are contaminated with inositol triphosphate and inositol monophosphate, respectively. The percentage distribution of the phosphorus under these circumstances was calculated after digesting the papers in perchloric acid according to DAWSON3° and then assaying for phosphorus. We have used in our chromatography tank a trough beneath the paper to collect the phosphorus-containing material which had a mobility greater than glycero-phosphate. These data showed that almost 30% was present in Fractions A and B (inositol triphosphate); I I % in Fractions C and D (inositol diphosphate); 23% as inositol monophosphate in Fraction E; 12 % as glycerophosphate and 24% being eluted from the paper. The specific activity data with the 3zP-labeled material was very interesting in that the greater the degree of phosphorylation the higher the specific activity. This would, therefore, be similar to the data found by t~ROCKERHOFF AND BALLOU 7,s, ELLIS AND H A W T H O R N E s, DITTMER AND DAWSON 4
and WAGNER et al. al in brain tissue.
150000 I
'1
I
E
70
N 1500( c
500(
/i
~ >
3000 1000
~1 \~,
\ I' //.~, / \\
\./
/\izA~
g .o_ "o n-o
500
0
,
, /~,
/\
\
/..,,,', / ~
/ \ / A'
30
,'/\~ 20 3~ 0
o
~b ~
30 4o ~o do ~o ~o
0 .c
Tube number
Fig. 2. Mild alkaline h y d r o l y s i s of saP-labeled p h o s p h o i n o s i t i d e complex. T h e h y d r o l y s a t e w a s applied to a D o w e x - I X-8 (20o-4o0 mesh, Cl--form) c o l u m n a n d s e p a r a t e d by g r a d i e n t elution with 25o ml distilled w a t e r in t h e m i x i n g c h a m b e r a n d o. 4 M I.iC1 in t h e reservoir, l o - m l f r a c t i o n s were collected. Analysis for p h o s p h o r u s a n d r a d i o a c t i v i t y were carried o u t on suitable aliquots.
Mild alkaline hydrolysis of pig-kidney phosphoinositide complex Six separate fractions were obtained by column chromatography of the watersoluble products by deacylation of 32P-labeled phosphoinositide complex (see Fig. 2). These fractions were dried in vacuo, extracted with absolute ethanol, converted to the acid form with Dowex-5o (H +) and then quickly neutralized with cyclohexylamine. In Table III the specific activity (counts/min per fig P) for these six fractions is presented. Again it is evident that as the ratio phosphorus/inositol increases, there is an increase in the metabolic activity of the phosphorus atoms in these components. The data reported in Table III represented studies designed to characterize these six fractions and it is of interest that each fraction eluted from the column contained a-amino nitrogen. Fraction VI representing 7% of the total phosphorus was found to have a phosphorus/inositol ratio of 3.3 and a phosphorus/glycerol ratio of 3.1. Analysis for glycerol in this fraction before hydrolysis by the periodate oxidation method revealed o.17 ffmoles and 0.34 ffmoles after hydrolysis. Fraction VI was Biochim, Biophys. Acta, 84 (I964) 68x-693
t~a
0o
Z
Z
e~
:g
0.24
0.3, |
IV
V
o.2o
0.40
III
VI
1.37
o.31
1
II
Fraction
Phosphorus (#moles)
0.06
o.I 5
o. 11
0.30
0.28
0.46
Myoinositol (pmolt~s)
o.00
o
o. t I
0.28
0.32
1.25
Glycerol (pmoles)
0. 3
0. 5
t. 1
0. 41
0.62
1.2
u-Amino nitrogen (14moles)
3-3
2.'2
2.2
i .3
1.1
2. 9
Phosphorus/ inositol ratio
m
3" I
---
2.2
~.4
0.97
1.I
l)hosphorus] glycerol ratio
7
l,
8
t4
1I
48
Total phosphorus phtced on column (°o)
m
.
337 °
2942
rc~77
S41
732
t44o
Specific activity (counts/rain per t~g phosphorus)
_
glycerylphosporyl diphosphate
phos-
myo-inositol phos-
nlyo-inositol
I n y o - i n o s i t o l di-
myo-inositol diphosphate
glycerylphosphoryl phate
glycerol~hosphate, phate, l'i
myo-inositol
(see T a b l e I V ) glycerylphosphoryl
uncertain
('omposition
Data expressed as pmoles per g wet wt. kidney cortex from which the crude fraction was obtained. I)eacvlation and c o l u m n c h r o m a t o g r a p h . v accomplished according to procedures described in the text. All samples elutcd from the c o l u m n were found to be n i n h v d r i n positive.
COMPONENTS OBTAINED BY COLI,:7~IN CHROMATOGRAPHY OF THE DIgACYI,ATED PRODUCTS OF THE aaI)-LAHELED PHOSPIIOINOSITIDE COMPLEX ON DO~.VEX-I ( e l - )
TABLE IlI
>
>
689
KIDNEY" P H O S P H O I N O S I T I D E COMPLEX
refluxed with 0.5 N NaOH for 45 min and the products of this hydrolysis were separated by descending paper chromatography. Inositol triphosphate was the only discernible phosphorus-containing material obtained. Finally, when Fraction VI was incubated with a specific phosphomonoesterase (Escherichia toll, Worthington Biochemicals Corporation), Pi was liberated. Fraction V, which represented 12% of the total phosphorus was found to have a phosphorus/inositol ratio of 2.2 and to be free of glycerol. Co-chromatography with standard inositol diphosphate using descending paper chromatography showed the presence of only those phosphorus-containing areas of the different isomers of inositol diphosphate. Furthermore, Fraction V could not be separated from inositol diphosphate standard by means of high voltage electrophoresis. Fraction IV, containing 8% of the phosphorus, had a phosphorus/ inositol ratio of 2.2 and a phosphorus/glycerol ratio of 2.1. Analysis for glycerol by the periodate oxidation method 21 revealed twice as much glycerol after acid hydrolysis as was found before hydrolysis. After hydrolysis with 0.5 N NaOH under reflux for 45 min, only inositol diphosphate and a trace of glycerophosphate was found. Fraction III was found to have a phosphorus/inositol ratio of 1.3 and a phosphorus/glycerol ratio of I. 4 and contained 14% of the total phosphorus. There was only a slight TABLE IV SEPARATION
OF THE ALKALINE HYDROLYSIS PRODUCTS OF FRACTION BY DESCENDING PAPER CHROMATOGRAPHY
I
F r a c t i o n I r e p r e s e n t s t h e first f r a c t i o n o b t a i n e d b y c o l u m n c h r o m a t o g r a p h y o f t h e d e a c y l a t e d p h o s p h o i n o s i t i d e c o m p l e x . (See T a b l e I I I ) . S o l v e n t s y s t e m : n - p r o p a n o l - a m m o n i a - H 2 0 (5:4:1, v / v ) 23 h. F o r d e f i n i t i o n Rg see T a b l e If. Phosphorus (l~moles)
Myo-
inositol (l~moles)
Glycerol (l~moles)
Phox#horus] inositol catio
Phosphorus/ glycerol ratio
Ninhvdrin .
haNgS" I.qHERWOOD
spot No.
Rg
i
0.03
0.80
0.27
o
2.96
---
negative
positive
2
x .o
o.92
o
o.9o
--
1 .o2
negative
positive
3
1-4
o.22
o
o.43
--
o.51
positive
positive
4
L7
o
o
o
--
--
positive
negative
increase in the apparent glycerol after total acid hydrolysis. Descending paper chromatography revealed the presence of glycerophosphate and inositol phosphate. Fraction II contained 12% of the total phosphorus with a phosphorus/inositol and phosphorus/glycerol ratio of I.I and 0.97 , respectively. It is of interest to point out the similarity of the specific activity of this fraction with that of Fraction I n . Three times as much glycerol was found after total acid hydrolysis which in all probability is a contaminant from Fraction I which has a high content of glycerol. Fraction lI was found to have the same mobility as a standard preparation of glycerylphosphoryl myo-inositol by high-voltage electrophoresis. Fraction I was rechromatographed on Dowex-i (C1-) with the same results as those obtained initially, that is, no further separation. To answer the question of column overload, excess Dowex-I (C1-) was added until all the phosphorus was adsorbed and none could be extracted by washing with water. The slurry was then placed into a column and eluted with a gradient of 0. 4 M LiC1 with similar results as described above. Fraction I was found to contain Biochim. Biophys. Acta, 84 (i96,t) 6 8 t - 6 9 3
(3()0
F. ANI')RADF, C. G. HI.'G(;INS
48~{, of the total phosphorus with a specific activity intermediate between that of Fractions 11 and IX:. A phosphorus/inositol ratio of 3.o and a ptmsphorus/glyeerol ratio of I.I were obtained. It is of interest that on a molar basis, there was as much glycerol as there was re-amino nitrogen. The material in Fraction I was refluxed with o. 5 N NaOH for 45 rain and then separated by descending chrmnatography for 23 h at 3 o°. Four distinct components were separated (see Table I V) and partially identitied as follows: (I) inositol trit~hosl~hate (phost)llorus/inositol molar ratio 3.o), (2) glycerophosphate, (3) unknown (contains glycerol and phost)horus in a ratio of 1.94), (4) unknown (contains only ninhydrin-positive material). Infrared spectroscopy
The infrared spectra of the cyclobexylamine salts of the different inositol phosl~hate standards and of those isolated and purified from the phosphoinositide complex were analyzed in an lnfracord Spectrophotometer. In the non-hydrolyzed fraction, absorption peaks were observed at 36oo, 34oo-315o, 297o, 17oo and 14oo cm--L and were interpreted as indicating the presence of OH, NH, CH, C = O , phosphates and ester phosphates, respectively. The individual inositol phosphates isolated from hydrolysates of the phosphoinositide complex showed absorption bands characteristic of OH, CH, l)host)hate and l)host)hate ester groups. DISCUSSION The term "t)hosphatido-peptides '' has been used by I.EBARoN AND FOIX'Hit to represent that material extracted from "lipid-free" tissue by the use of acidified c.hloroform -methanol. On the basis of the chemical findings described in the present paper, it is believed that this fraction could be more correctly labeled as a i)hosphoinositide complex in so far as kidney tissue is concerned. This belief is supported by the data of I)ITTMER AND DAWSON4 who tlave used an acidified chloroform-methanol solution to extract polyptmst)hoinositide from beef brain. In our study of the phosphoinositide complex from kidney cortex, as well as other orgtul systems, the reproducibility of the products of extraction has bee.n found to be independent of the procedures used to obtain the other phosphorus-containing fractions. Using silicic acid-mlt)regnated glass fiber paper chromatograt~hy and thin-layer chromatograt)hy, only inositides could be demonstrated in this fraction. Silicic acid column chromatography a" has also been used to separate the inositides in the crude fraction. In the latter, three fractions were eluted; all of which were fimnd to contain myo-inositol phosphates after alkaline hydrolysis. Alkaline hydrolysis of tile t)hosphoinositide complex was accomplished by refluxing for 45 rain in o.5 N Na()H, which, according to BRowx et al. ss and BROCKERHOVV AND BaI.I.Ot:6 would cleaw~ the fatty ackls from the lipid component as well as result in the hydrolysis of the diester t)hosphate bond between glycerol and inositol. Such hydrolysis conditions would be expected to y M d inositol phosphate and glycerophosphate with the former predominating when the parent phospholipid is more highly phosphorylatedaL These hydrolysis components of the phosphoinositide complex as their cyclohexylamine salts were resolved by descending pat)er chromatography and identified as inositol mono-, di- and triphosphate and glycerophosphate. Buwhim. Biophvs..-Iota, 84 (ngO.0 t',?,x-093
KIDNEY PHOSPHOINOSITIDE COMPLEX
69I
The criteria used to establish the chemical constitution of these resolved compounds were: (a) phosphorus/inositol ratio, (b) phosphorus/glycerol ratio, when applicable, (c) their migration on descending paper chromatography, (d) their migration on highvoltage paper electrophoresis, (e) co-chromatography with standard compounds, (f) use of the isotopes Na2Hs2po 4 and [2-SH]inositol. Infrared spectra of standard compounds when compared with those of the resolved compounds served as additional evidence. The hydrolysates resulting from mild alkaline hydrolysis (deacylation) which produce very little rupture of diester phosphate bonds was separated into six fractions by elution from Dowex-I (C1-) with 0.4 M LiC1. Chemical analysis of the individual fractions provided information suggesting the identity of five of these fractions as glycerylphosphoryl myo-inositol, glycerophosphate and inositol monophosphate, glycerylphosphoryl myo-inositol phosphate, inositol diphosphate and glycerylphosphoryl myo-inositol diphosphate. It is not possible at the present time to explain the presence of inositol mono- and diphosphate and glycerophosphate as components of the deacylated material. The criteria used for this recognition included: phosphorus /inositol ratios, phosphorus/glycerol ratios, migration on descending paper chromatography, migration on high-voltage electrophoresis, glycerol determinations before and after hydrolysis and co-chromatography and electrophoresis with standard compounds when available. Fraction I, which was obtained from the deacylated fractions and which represented 50% of the total phosphorus and almost 30% of the total a-amino nitrogen, was not completely characterized. Alkaline hydrolysis of this fraction followed by descending paper chromatography allowed the identification of inositol triphosphate as a major component. In addition, a second major component which contained 2 moles of glycerol per mole of phosphorus was also found. Although the latter was not identified, it is conceivable that glycerylphosphoryl glycerol might be present. An interesting feature concerning the data obtained with column chromatography of the deacylated phosphoinositide complex is the presence of amino acids in each fraction eluted from the column. No evidence is available at present to indicate the relation of these amino acids to the phosphate components since it is possible that they were elated from the column at varying ionic strengths of the LiC1 used for the gradient elution and, thus, their presence in each peak may be fortuitous. Their presence, however, in all fractions must be explained. On tile assumption that glycerol is the only acylated compound in the original material, the present study would indicate that the phosphoinositide complex from kidney cortex is composed of: L-phosphatidyl-L-myo-inositol, L-phosphatidyl-Lmyo-inositol phosphate, L-phosphatidyl-L-myo-inositoldiphosphate, plus an unknown lipid compound which contained glycerol, phosphorus and amino acids, presumably in peptide linkage. The phosphoinositide complex from kidney, pancreas and submaxillary glands has been previously reported as showing a rapid incorporation of Na2H~2PO4 (ref. I2, I5,I6), both in vivo and in vitro. In the latter two tissues, the metabolic activity has been shown to increase when secretion was induced by electrical stimulation or by administration of acetylcholine or epinephrine. Furthermore, it is of interest to note that the specific activity of the phosphorus increases as one increases the degree of phosphorylation of inositol. Recently, GARBUSet al. ~ have shown an inositol component in kidney mitochondria with a high metabolic activity which could conceivably Biochim. Biophys. Acta, 84 (t964) 681-693
692
I:. A N I ) R A D E , C. (;. HU(;(;INS
be similar to tim compounds we are describing from kidney. This is fltrther strengthened by the report of Ht,(;~;Lxs AND COHN12 that mitochondria from kidney contain almost two-thirds of the total tissue complex. Thus, it is believed that the inositol polyphosphates, primarily those in the monoester linkage in the parent coml~ound, are responsible for the relatively high metabolic activity in the l~hosphoinositide complex from the kidney. ()n the other hand, in our studies with secretion it appears that the diester phosphate atom is stimulated when secretion is initiated either electricall\or by acetylcholine or epinephrine. investigations concerning the chemical constitution of this complex in several organ systems are being continued in our efforts to establish whether the complex exists as a chemical entity iir whether it is a non-specific chenaical mixture and to understand more clearly the role of the constituents in the economy of the cell.
ACKNOWLEDGEMENTS
This investigation was supported in part by Public Health Service Research Grant No. o6152 from the National Cancer Institute and Public Health Service (;rant No. I-GS77 and American Cancer Society Grant No. IN24 D. The experimental data in this paper are taken from a thesis submitted by F. Axt~I~.~1)e to the Graduate School of Tulane University, in partial fulfilment of the requirements for the degree of Doctor of Philosophy. One of us (F.A.) was the recipient of a Rockefeller Foundation fellowship I96I-63 from the Delmrtment of Biochemistry, Faculty of Medicine, Javeriana University, Bogota, D. E. Colombia, also present address. The culture of Kloeckera brevis w m apiculata was obtained from l)r. I). J. HANartAN, l)epartment of Biochemistry, University of Washingeton, Seattle, Washington, and the sample of myo-inosose-e was ohtained from I)r. G. I{I:I)OI.PH, Department of Biochemistry, Vanderbilt University, Nashville, Tennessee. REFERENCES x j . FOLCH, J. Biol. Chem., 146 11942 ) 352 j . FOLCH, J. Biol. Chem., 177 (I949) 497. s C. GRADO AND C. E. BALLOU, J. Biol. Chem., 236 11961 ) .54. 4 j . C. DITTMER ANn R. M. C. DAWSON, Biochem. J., 81 ( i 9 6 1 ) .535. R. B. ELLIS, T. (;ALLIARD AND j. x . HAWTHORNE, Btochem. J., 8,s (19()3) I25. 6 It. BROCKERHOFF AND C. E. BALLOU, J. Biol. (7hem., 231) (190I) I 9 o 7. 7 H. BROCKERtIOFF AND C. E. BALLOU, .]. Biol. Chem., 237 1t962 ) 49s H. BROCKERHOFF AND C. E. BALLOU, J. Biol. Chem., 237 (19(~2) 17~ 4. 9 R. G. ELLIS AND J. N. |]'AWTHORNE, Biochem. J., 84 11062 ) IOP. ~o j . FOLlZH, in IV. I). M c E L R o v AND ]{. (;LASS, l'hosphorus Metabolisnz, Vol. 2, J o h n s I l o p k i n s P r e s s , B a l t i m o r e , 1952, p. 186. xx F. N. LEBARON AND J. Fot.ctt, .[. Neurochem., 1 (1951') t o t . 12 C. G. HUGGINS AND |). V. COHM, J. Biol. Chem., 234 (t959) 257. xa F. N. [,EBARON, J. I'. KIgTLER AND (;. ]IAUSER, Biochim. Biophys. Acta, 44 (19'~o) 17o. 14 V. N. I,EBARON, G. IIAUSER AND E. E. R u I z , Biochim. Biophys. Acta, 6o 11962) 338. a5 C. G. ltUGGINS, Nature, 184 11959) 1412. ts H . B. BURFORD AND C. G. I-IUGGINS, ,.Int. J. l~hysiol., 205 (I903) 23.5. 17 F. ANDRADE AND ('~. (;. HUGGINg, Federation Proc., 22 11963) 414 . xs F. ANDRADE AND ('. (;-. HUGGINg, Biochim. Biophys. Acta, 84 (1904) 98. 19 C. H. FISKE AND V. SUm*AROw, J. Biol. Chem., 66 (1925) 37.5. ~0 R. (;. BARTLETT, J. Biol. Chem., 234 (1959) 466. zl D. J. ]-IANAHAN AND J. N. OLLEY, J. Biol. Chem., 23I (1958) ~SI3. 22 R. J. BLOCK AND K. B. WVlSS, Amino Acid Handbook, T h o m a s , S p r i n g f i e h l , I950, p. 29.
Biochim. Biophys. ,4 cta, 84 (I964) 681 ( ; 9 3
KIDNEY PHOSPHOINOSITIDE COMPLEX
693
23 E. E. S.'~ELL, in P. GYORGYI, Vitamin Methods, Vol. I, Academic Press, New York, i95 o, p. 445,4 F. N. LEBARO.'% J. FOLCH AND E. ROTHLEDER, Federation Proc., I6 (r957) 2o9. t5 A. DESJOBERT AND F. PETEK, Bull. Soc. Chim. Biol., 38 (I956) 87I. ,2 R. S. BA,'~DURSKX AND B. AXELROD, J. Biol. Chem., 193 (I95 I) 4o5 • ,7 S. E. KERR AND G. A. KFOURY, Arch. Biochem. Biophys., 96 (I962) 347. 28 j . G. HAMILTON AND J. E. MULDREY, J. Amer. Oil Chemists" Soc., 38 (196x) 55z. 2, W. D. SKIT)MORE, AND C. ENTE~MAN, J. Lipid. Res., 3 (r962) 47I. ~0 R. M. C. DAWSON, Biochem. J., 75 (I96o) 43. s t H. WAGYER, A. LlSSAU, J. HOLZL AND L. HORnAMMER, J. Lipid. Res., 3 (I962) I77. s2 D. J. HA.'~AHAN, J. C. DITTMER, E. WARASmMA, J. Biol. Chem., 228 (1957) 685. s2 I). M. BROWN, G. E. HALL AND R. I,ETTERS, .]. Chem. Soc., (I959) 3547s4 j . GARBUS, I-t. F. I)ELucA, M. E. I.OOMANS AYD F. M. STROXG, J. Biol. Chem., 238 (1963) 59.
Biochim. Biophys. Acta, 84 (i964) 681-693
681
BBA 5 5 0 5 1
M Y O - I N O S I T O L P H O S P H A T E S IN A P H O S P H O I N O S I T I D E FROM K I D N E Y
COMPLEX
F. A N D R A D E AND C. (~. H U G G I N S
Department of Biochemistry, Tulane University School of Medicine, New Orleans, La. ( U.S.A .) (Received J a n u a r y 31st, I964)
SUMMARY
I. Chemical studies on the "phosphatido-peptide fraction" from kidney have shown that this material more properly should be called a phosphoinositide complex. 2. Glycerophosphate, inositol mono-, di- and triphosphate were separated from alkaline hydrolysates by means of paper chromatography. 3- Six fractions were obtained by cohtmn chromatography from the deacylated phosphoinositide complex and identified as inositol monophosphate, glycerophosphate, inositol diphosphate, glycerylphosphoryl inositol, glycerylphosphoryl inositol phosphate, and glycerylphosphoryl inositol diphosphate. 4. These phosphates, presumably as the inositides, are believed to be responsible for the high metabolic activity of this fraction as measured by radiophosphorus. INTRODUCTION
Myo-inositol meta-diphosphate was first reported by FOLCH1,2 as being present in a diphosphoinositide from beef brain. Evidence showing that FOLCH'S beef-brain diphosphoinositide contains more than one type of inositol phosphate has been reported by several groups of investigators 3,4, all of whom have shown the presence of inositol mono-, di- and triphosphate in this fraction. Recently, BROCKERHOFFAND BALLOU8,7 in a series of publications have sho~n that beef-brain phosphoinositides (FOLCH'Sdiphosphoinositide) exists as a complex of substances containing L-phosphatidyl-Lmyo-inositol, L-phosphatidyl-L-myo-inositol 4-phosphate and L-phosphatidyl-L-myoinositol 4,5-diphosphate. Furthet more, these structures appear to be interrelated in such a way as to suggest a step-wise phosphorylation of the monophosphoinositide in the biosynthesis of di- and triphosphoinositides 8,9. The term "phosphatido-peptides" was introduced into the literature by FOLCH1° in 1952 to characterize a protein-bound complex lipid fraction which he had obtained from a lipid-free trypsin-resistant residue of beef brain by extraction with acidified chloroform-methanol. LEBARON AND FOLCH11 suggested that this fraction represented a hitherto unreported form of tissue phosphorus containing esterfied fatty acids, inositol diphosphate, glycerol and amino acids in polypeptide chains. HUGGINS AND COHNTM in 1959 isolated a very similar fraction from kidney cortex using FOLCH'S extraction method. Distribution studies by these latter investigators have revealed Biochim. Biophys. Acta, 84 (I964) 681-693
()~2
1;. ANI)ICAI)E, ('. ~,. t I U ( ; ( ; I N S
this fraction to be present in various amounts in br,dn, heart, liver, lung, spleen, pancreas, intestine, saliwtry glands and tumor tissue. ['sing kidney tissm~. ]lI'(;tilNs ANt) (_'OHNr' have shown that the specific activity . f the l~hosl~h
MI,71"HOI)S A N D I..'XPI.H¢IMENTAI. PI¢OCEI)URE
A nalytical methods Phosphorus was determined by the method of F'ISKF AND SUBBAI~,ow l:~ aS modified by BARTLETT2°. Glycerol was determined according to Hax..XHaX axl) ()LL].;Y"~ and a-amino nitrogen was determined by the ninhydrin procedure 2" using leucine for the standard curve. Myo-inositol was assayed by the microbiological procedure "a with Kloeckera brevis wtr. apicnlata and also bv the spectrophotometric procedure as described by LEBARON et al. 24.
Chromatographic methods Descending paper chromatography of the products from alkaline hydrolysis was carried out for 3 ° h at 3 °° on Whatman 3 MM paper according to the procedure described by 1)ESJOBERT AND -PETEK 2's using the solvent system n-propanol -ammonia water (5:4:1, v/v). More effective separation of the different isomers of inositol diand triphosphate could be obtained if the chromatograms were. run for 5° h ; however, under these conditions, inositol monophosphate and glycerophosphate were cluted ldiockim, t~iopkys...lcta, 84 (19o4) (JSi t,q3
KIDNEY PHOSPHOINOSITIDE COMPLEX
683
from the paper. Localization of phosphate esters was accomplished using the HANESISHERWOOD spray as modified by BANDURSKI AND AXELROD26 and also by radioactive scanning of developed strips on which the phosphate esters were labeled with 3~.p. Samples containing approx. 200/zg of phosphorus were applied as a band over 15 cm and subjected to descending chromatography. The separated phosphate esters were eluted from the paper strips with o.1% NH4OH. Column chromatography of the deacylated material was carried out according to the method of BROCKERHOFF AND BALLOUs using Dowex-I (C1-), and gradient elution ~ith 0.4 M LiC1. The pattern of elution was followed by assaying an aliquot from each tube for total phosphorus and radioactivity.
Materials Inositol di- and triphosphate were prepared by acid hydrolysis of phytic acid according to KERR AND KFOCRYz7 and inositol monophosphate was obtained from California Corporation for Biochemical Research. Beef-brain phosphoinositide was prepared according to FOLCH°-. The crude inositide was precipitated eight times from chloroform with methanol and then dialyzed for 48 h against running water at 4 °. Radiophosphorus (Na2Ha~PO,) was obtained from Oak Ridge National Laboratory. Myo-inosose-2 was reduced to i2-3H!inositol with tritium by the New England Nuclear Corporation. The final [2-3Hlinositol was recrystallized two times from hot water and ethanol and had a melting point of 219-221 ° (uncorr.). It had a 3H content of 63.3 mC/mmole and gave 1.o2. lO8 counts/min per mg when counted at infinite thinness,in a windowless gas-flow counter. For incorporation of radioisotopes kidney slices were incubated as described previously t2 to which 5/~C of Na2H32PO4 or 2/~C of [2-3H]inositol were added per ml of incubation mixture. Scanning for the location of radioactive areas on paper chromatograms was accomplished with a Vanguard automatic scanner, Model 80o. Radioactive samples were also plated in stainless-steel planchets and counted in a windowless gas-flow G-M tube using a Baird-Atomic Model 135 Scaler.
Preparation of the phosphoinositide complex The phosphoinositide complex was isolated by the procedure described by HUGGINS AND COHN 12, and the crude extract was partitioned against 0.2 volume of 0.2 M NaHCO v The resulting lower phase (organic plus the interracial fluffy layer which redissolved on the addition of 0.2 volume of methanol) were used as the starting material for these studies. In some instances, we have used the technique described by LEBAROX et al. 14 because it was found to be more adaptable for large-scale preparations of this fraction. This latter procedure differs from that described by HOGGINS ANn COH.',"1~in the solvents used for the extraction of the other phosphorus-containing fractions, but it is similar in that both procedures use acidified chloroform-methanol for the extraction of the crude fraction. The composition of this fraction using either method does not differ as far as total phosphorus, inositol or glycerol is concerned. The crude fraction so obtained, which represented 5 % of the total tissue phosphorus, had the following composition : 3.3% phosphorus, 7.3% glycerol and 16.8% inositol. The esterified f a t t y acid/ Biochim. Biophys. Acta, 84 (1964) 681-693
{)~4
F. A N I ) R A I ) E , C. (;. H U ( ; ( i l N S
phosphorus molar ratio was 1.8 for the crude flaction, l)ata ()btained when variou, species and organ systems were analyzed for the different components , f tilt, ph,~sl~h.inositide complex indicate that there is a characteristic reproducibility in regard t~, the different organ systems and species analyzed. Chromatograt)hic techniques using silicic acid-impregnated glass fiber paper "~sand thin-lay'er c h r o m a t , g r a p h y "u were not successful in resolving the complex into components - t h e r than im>itides and ~,nly ttmse areas containing phosphorus and inositol were ninhydrin p.sitive. The crude fraction was subjected to dialysis in older to, establish tile presence ~r absence of fiee amino acids or other small dialyzabh~ components. Dialysis was carried out at room temperature fi~r 48 h against three changes of distilled water. "l'al)le I presents the values before and after dialysis, it can be seen that there is a los> . f aptm~x. 5 o ° . of the free ~t-amino nitrogen; however, it can also be s e e n t h a t ;ipproxinaatvly the same molar amount of phost)horus and inositol were ;tls~ dialvzabh.. TABLE
1
IIIAI.YSIN OF PHOSPttOINO.SITII)I.I COMPI.EX T h e r e p o r t e d values are represt:n|~'d i t s / r a m i e s per g wet w t . . f (onstltl.'nts
Total phosphorus Glycerol I nositol u-Amino nitrogen (before hydrolysis) Phosphorus/inositol ratio
ltcfore dialysis (#moles)
.-1.1t,'r dialysis [tt~'loh's )
z.,s -.o ".3 o.35 t.-'
-'.~, 13, -'. I o. t 7 I.-
tissue. Loss Omloles ) ~.2 o. 4 o.2 o. 18
Alkaline hydrolysis of pig-kidney phosphoinositide comple:c 7 g of crude fraction were suspended in 25o ml of o. 5 N Na()H and refluxed for 45 min. At the end of this time complete solution was obtained. The dark brown solution was then worked up according to tile procedure descrihed by (~RADO ..~,ND BALLOt;3 for base hydrolysis of beef-brain phosphoinositide. The resulting aqueous layer was made basic by the addition of cyclohexylamine and then concentrated to a small volume. The concentrate which contained no Pi was the starting material for separations by descending paper chromatography. Mild alkaline deacylation of the crude fraction was carried out according to the fl~llowing procedure: 2 g of crude fraction in 5 ° m l of chlorofi~rm-nmthanol (2 :I, v/v) containing o.I2 N N a O H were swirled for IO min at 25 °. At the end of this time, 5 g of Dowex-5o (H ') in 2o ml of water were added to the reaction. The resulting aqueous phase was separated ; washed two times with an equal w)lume of ethyl ether and passed through a l)owex-5o ( H ' ) column containing approx. 15 g of resin. The column was washed with 5o'!,~, ethanol according to BROCKERHOFF AND BALI.OU 6 and the eluate was made basic with cyclohexylainine and concentrated to a small w)lume. This served as the starting material for separation of the phosphate esters hy column c h r o m a t o g r a p h y using l)owex-I (C1-) as described by, BROCKEmmVF axI) BaLLOU~. Under these conditions, 95~}{~ of the phosphorus became water soluble. H t o c h i m . t ¢ i o p h y s . ..1eta, S, t (rgO.;) (~81.-693
685
KIDNEY PHOSPHOINOSITIDE COMPLEX RESULTS
Alkaline hydrolysis of pig-kidney phosphoionsitide complex The results in Fig. I were obtained by descending paper chromatography of alkaline hydrolysates of 32p_ and 3H-labeled phosphoinositide complex of pig-kidney cortex• The upper tracing represents the products from Na,Hs2po4-1abeled material and the lower tracing from [2-ZH]inositol-labeled material. Six separate fractions were obtained with glycerophosphate (Fraction F) being used to calculate the relative mobilities of the other fractions. The data in Table II were obtained in the process of studies designed to characterize the six fractions represented in Fig. I. In these experiments large-scale preparations of the crude fraction from pig-kidney cortex were mixed with 32P-labeled material, the latter being used to facilitate localization of the different fractions. After alkaline hydrolysis and separation by descending chromatography, the different fractions were eluted from the paper. The pooled eluates from several papers were lyophilized and subjected to acid hydrolysis in a sealed tube with 6 N HC1 at i i o ° for 24 h. The phosphorus/inositol molar ratios in the hydrolysates were 3.06 and 3.z A
B
A 3 2 p
/-': ;'" '_
:" I ~ I]
'-
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~=i
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',
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--~,--~ ....
~--~-.-J--'--~--
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,-
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i
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Fig. i. T h e d i s t r i b u t i o n of r a d i o a c t i v i t y on d e s c e n d i n g c h r o m a t o g r a m s o b t a i n e d b y p a p e r chrom a t o g r a p h y of a n alkaline h y d r o l y s a t e of t h e p h o s p h o i n o s i t i d e c o m p l e x from k i d n e y cortex. S a m p l e s were applied a t Position A prior to s e p a r a t i o n by d e s c e n d i n g c h r o m a t o g r a p h y . T h e t r a c i n g s were o b t a i n e d u s i n g a V a n g u a r d a u t o scanner. U p p e r t r a c i n g from s2P-labeled m a t e r i a l ; lower t r a c i n g from [2-SH]inositol-labeled material. Lower p h a s e is a d i a g r a m m a t i c r e p r e s e n t a t i o n of m o v e m e n t of s t a n d a r d s in which I P a = inositol t r i p h o s p h a t e , IP 2 - inositol d i p h o s p h a t e , I P -- inositol m o n o p h o s p h a t e , G P = g l y c e r o p h o s p h a t e .
for Fractions A and B; 2.26 and 1.54 for Fractions C and D and 0.96 for Fraction E. Fraction F did not contain inositol but did contain glycerol and, since glycerol is partially destroyed in 6 N HC1 (ref. 2I), we have used 2 N HC1 routinely for hydrolysis in the measurement of glycerol. Suitable aliquots of the a'P-labeled eluates were mixed with standard compounds and rechromatographed by descending chromatography or subjected to separation by high-voltage electrophoresis. The radioactive areas were found to correspond to the areas revealed by the H A N E S - I S H E R W O O D spray. It is Biochim. Biophys. Acta, 84 (1964) 681-693
t.o
e~
a:
~, i .o
(;
hydrolysis
i.o
Alkaline
0.72
E
F
0. 4
C
o.6
o. I 5
B
D
0.02
R,
A
Fraction
TABLE
I1
and
obtained
eluates
chronlatography
trace
o.6i
o
o
o
o
o
Glycerol fltmoles)
from pooled
paper
o
o
g 16
o. 19
o. 11
0.28
o. 17
Mvo-inositol (lonolcs) •
descending
i. 15
0.57
I. l x
o.29
o.26
0.87
0.53
Phospltorus (ttmoles,;
from several
carried
---
0.96
x.54
2.26
3.1
3.06
Phosphorus/ inositol ratio
according
experiments,
out
--
0.93
....
-- ]
--
-
Phosphorus/ glycerol ratio
leg
described
--
274
328
92o
86.,,
143o
4260
with
-
--
120 900
IO7 5oo
to 7 880
the
92 750
149 4 ° 0
[a-ttlJMj,o-inositol counts/rain per g tissue
in t e x t
agtivitv'Specific counts/rain" per /*g phosphorus
migration o f c o m P0u_ml migration of glycerophosphate.
to procedures
24
I2
23
II
18
ii
phosphorusT°tal placed on paper (%)
data
representing
unknown that
phosphate
diphos-
diphos-
triphos-
triphos-
glycerophosphate
nlyo-inositol
myo-inositol phate
myo-inositol phate
myo-inositol phate
myo-inositol phate
('omposttitm
COMPONENTS O B T A I N E D BY D E S C E N D I N G PAPER C H R O M A T O G R A P H Y OF T H E P R O D U C T S OF A L K A L I N E H Y D R O L Y S I S OF s2p_ AND [2-all~INOSITOI--LADELI':I) PHOSPHOINOSITIDI., COMPI.FX OF K I D N E Y CORTEX
;=
>
> 7.
oc
687
KIDNEY PHOSPHOINOSITIDE COMPLEX
believed that Fractions C and D are contaminated with inositol triphosphate and inositol monophosphate, respectively. The percentage distribution of the phosphorus under these circumstances was calculated after digesting the papers in perchloric acid according to DAWSON3° and then assaying for phosphorus. We have used in our chromatography tank a trough beneath the paper to collect the phosphorus-containing material which had a mobility greater than glycero-phosphate. These data showed that almost 30% was present in Fractions A and B (inositol triphosphate); I I % in Fractions C and D (inositol diphosphate); 23% as inositol monophosphate in Fraction E; 12 % as glycerophosphate and 24% being eluted from the paper. The specific activity data with the 3zP-labeled material was very interesting in that the greater the degree of phosphorylation the higher the specific activity. This would, therefore, be similar to the data found by t~ROCKERHOFF AND BALLOU 7,s, ELLIS AND H A W T H O R N E s, DITTMER AND DAWSON 4
and WAGNER et al. al in brain tissue.
150000 I
'1
I
E
70
N 1500( c
500(
/i
~ >
3000 1000
~1 \~,
\ I' //.~, / \\
\./
/\izA~
g .o_ "o n-o
500
0
,
, /~,
/\
\
/..,,,', / ~
/ \ / A'
30
,'/\~ 20 3~ 0
o
~b ~
30 4o ~o do ~o ~o
0 .c
Tube number
Fig. 2. Mild alkaline h y d r o l y s i s of saP-labeled p h o s p h o i n o s i t i d e complex. T h e h y d r o l y s a t e w a s applied to a D o w e x - I X-8 (20o-4o0 mesh, Cl--form) c o l u m n a n d s e p a r a t e d by g r a d i e n t elution with 25o ml distilled w a t e r in t h e m i x i n g c h a m b e r a n d o. 4 M I.iC1 in t h e reservoir, l o - m l f r a c t i o n s were collected. Analysis for p h o s p h o r u s a n d r a d i o a c t i v i t y were carried o u t on suitable aliquots.
Mild alkaline hydrolysis of pig-kidney phosphoinositide complex Six separate fractions were obtained by column chromatography of the watersoluble products by deacylation of 32P-labeled phosphoinositide complex (see Fig. 2). These fractions were dried in vacuo, extracted with absolute ethanol, converted to the acid form with Dowex-5o (H +) and then quickly neutralized with cyclohexylamine. In Table III the specific activity (counts/min per fig P) for these six fractions is presented. Again it is evident that as the ratio phosphorus/inositol increases, there is an increase in the metabolic activity of the phosphorus atoms in these components. The data reported in Table III represented studies designed to characterize these six fractions and it is of interest that each fraction eluted from the column contained a-amino nitrogen. Fraction VI representing 7% of the total phosphorus was found to have a phosphorus/inositol ratio of 3.3 and a phosphorus/glycerol ratio of 3.1. Analysis for glycerol in this fraction before hydrolysis by the periodate oxidation method revealed o.17 ffmoles and 0.34 ffmoles after hydrolysis. Fraction VI was Biochim, Biophys. Acta, 84 (I964) 68x-693
t~a
0o
Z
Z
e~
:g
0.24
0.3, |
IV
V
o.2o
0.40
III
VI
1.37
o.31
1
II
Fraction
Phosphorus (#moles)
0.06
o.I 5
o. 11
0.30
0.28
0.46
Myoinositol (pmolt~s)
o.00
o
o. t I
0.28
0.32
1.25
Glycerol (pmoles)
0. 3
0. 5
t. 1
0. 41
0.62
1.2
u-Amino nitrogen (14moles)
3-3
2.'2
2.2
i .3
1.1
2. 9
Phosphorus/ inositol ratio
m
3" I
---
2.2
~.4
0.97
1.I
l)hosphorus] glycerol ratio
7
l,
8
t4
1I
48
Total phosphorus phtced on column (°o)
m
.
337 °
2942
rc~77
S41
732
t44o
Specific activity (counts/rain per t~g phosphorus)
_
glycerylphosporyl diphosphate
phos-
myo-inositol phos-
nlyo-inositol
I n y o - i n o s i t o l di-
myo-inositol diphosphate
glycerylphosphoryl phate
glycerol~hosphate, phate, l'i
myo-inositol
(see T a b l e I V ) glycerylphosphoryl
uncertain
('omposition
Data expressed as pmoles per g wet wt. kidney cortex from which the crude fraction was obtained. I)eacvlation and c o l u m n c h r o m a t o g r a p h . v accomplished according to procedures described in the text. All samples elutcd from the c o l u m n were found to be n i n h v d r i n positive.
COMPONENTS OBTAINED BY COLI,:7~IN CHROMATOGRAPHY OF THE DIgACYI,ATED PRODUCTS OF THE aaI)-LAHELED PHOSPIIOINOSITIDE COMPLEX ON DO~.VEX-I ( e l - )
TABLE IlI
>
>
689
KIDNEY" P H O S P H O I N O S I T I D E COMPLEX
refluxed with 0.5 N NaOH for 45 min and the products of this hydrolysis were separated by descending paper chromatography. Inositol triphosphate was the only discernible phosphorus-containing material obtained. Finally, when Fraction VI was incubated with a specific phosphomonoesterase (Escherichia toll, Worthington Biochemicals Corporation), Pi was liberated. Fraction V, which represented 12% of the total phosphorus was found to have a phosphorus/inositol ratio of 2.2 and to be free of glycerol. Co-chromatography with standard inositol diphosphate using descending paper chromatography showed the presence of only those phosphorus-containing areas of the different isomers of inositol diphosphate. Furthermore, Fraction V could not be separated from inositol diphosphate standard by means of high voltage electrophoresis. Fraction IV, containing 8% of the phosphorus, had a phosphorus/ inositol ratio of 2.2 and a phosphorus/glycerol ratio of 2.1. Analysis for glycerol by the periodate oxidation method 21 revealed twice as much glycerol after acid hydrolysis as was found before hydrolysis. After hydrolysis with 0.5 N NaOH under reflux for 45 min, only inositol diphosphate and a trace of glycerophosphate was found. Fraction III was found to have a phosphorus/inositol ratio of 1.3 and a phosphorus/glycerol ratio of I. 4 and contained 14% of the total phosphorus. There was only a slight TABLE IV SEPARATION
OF THE ALKALINE HYDROLYSIS PRODUCTS OF FRACTION BY DESCENDING PAPER CHROMATOGRAPHY
I
F r a c t i o n I r e p r e s e n t s t h e first f r a c t i o n o b t a i n e d b y c o l u m n c h r o m a t o g r a p h y o f t h e d e a c y l a t e d p h o s p h o i n o s i t i d e c o m p l e x . (See T a b l e I I I ) . S o l v e n t s y s t e m : n - p r o p a n o l - a m m o n i a - H 2 0 (5:4:1, v / v ) 23 h. F o r d e f i n i t i o n Rg see T a b l e If. Phosphorus (l~moles)
Myo-
inositol (l~moles)
Glycerol (l~moles)
Phox#horus] inositol catio
Phosphorus/ glycerol ratio
Ninhvdrin .
haNgS" I.qHERWOOD
spot No.
Rg
i
0.03
0.80
0.27
o
2.96
---
negative
positive
2
x .o
o.92
o
o.9o
--
1 .o2
negative
positive
3
1-4
o.22
o
o.43
--
o.51
positive
positive
4
L7
o
o
o
--
--
positive
negative
increase in the apparent glycerol after total acid hydrolysis. Descending paper chromatography revealed the presence of glycerophosphate and inositol phosphate. Fraction II contained 12% of the total phosphorus with a phosphorus/inositol and phosphorus/glycerol ratio of I.I and 0.97 , respectively. It is of interest to point out the similarity of the specific activity of this fraction with that of Fraction I n . Three times as much glycerol was found after total acid hydrolysis which in all probability is a contaminant from Fraction I which has a high content of glycerol. Fraction lI was found to have the same mobility as a standard preparation of glycerylphosphoryl myo-inositol by high-voltage electrophoresis. Fraction I was rechromatographed on Dowex-i (C1-) with the same results as those obtained initially, that is, no further separation. To answer the question of column overload, excess Dowex-I (C1-) was added until all the phosphorus was adsorbed and none could be extracted by washing with water. The slurry was then placed into a column and eluted with a gradient of 0. 4 M LiC1 with similar results as described above. Fraction I was found to contain Biochim. Biophys. Acta, 84 (i96,t) 6 8 t - 6 9 3
(3()0
F. ANI')RADF, C. G. HI.'G(;INS
48~{, of the total phosphorus with a specific activity intermediate between that of Fractions 11 and IX:. A phosphorus/inositol ratio of 3.o and a ptmsphorus/glyeerol ratio of I.I were obtained. It is of interest that on a molar basis, there was as much glycerol as there was re-amino nitrogen. The material in Fraction I was refluxed with o. 5 N NaOH for 45 rain and then separated by descending chrmnatography for 23 h at 3 o°. Four distinct components were separated (see Table I V) and partially identitied as follows: (I) inositol trit~hosl~hate (phost)llorus/inositol molar ratio 3.o), (2) glycerophosphate, (3) unknown (contains glycerol and phost)horus in a ratio of 1.94), (4) unknown (contains only ninhydrin-positive material). Infrared spectroscopy
The infrared spectra of the cyclobexylamine salts of the different inositol phosl~hate standards and of those isolated and purified from the phosphoinositide complex were analyzed in an lnfracord Spectrophotometer. In the non-hydrolyzed fraction, absorption peaks were observed at 36oo, 34oo-315o, 297o, 17oo and 14oo cm--L and were interpreted as indicating the presence of OH, NH, CH, C = O , phosphates and ester phosphates, respectively. The individual inositol phosphates isolated from hydrolysates of the phosphoinositide complex showed absorption bands characteristic of OH, CH, l)host)hate and l)host)hate ester groups. DISCUSSION The term "t)hosphatido-peptides '' has been used by I.EBARoN AND FOIX'Hit to represent that material extracted from "lipid-free" tissue by the use of acidified c.hloroform -methanol. On the basis of the chemical findings described in the present paper, it is believed that this fraction could be more correctly labeled as a i)hosphoinositide complex in so far as kidney tissue is concerned. This belief is supported by the data of I)ITTMER AND DAWSON4 who tlave used an acidified chloroform-methanol solution to extract polyptmst)hoinositide from beef brain. In our study of the phosphoinositide complex from kidney cortex, as well as other orgtul systems, the reproducibility of the products of extraction has bee.n found to be independent of the procedures used to obtain the other phosphorus-containing fractions. Using silicic acid-mlt)regnated glass fiber paper chromatograt~hy and thin-layer chromatograt)hy, only inositides could be demonstrated in this fraction. Silicic acid column chromatography a" has also been used to separate the inositides in the crude fraction. In the latter, three fractions were eluted; all of which were fimnd to contain myo-inositol phosphates after alkaline hydrolysis. Alkaline hydrolysis of tile t)hosphoinositide complex was accomplished by refluxing for 45 rain in o.5 N Na()H, which, according to BRowx et al. ss and BROCKERHOVV AND BaI.I.Ot:6 would cleaw~ the fatty ackls from the lipid component as well as result in the hydrolysis of the diester t)hosphate bond between glycerol and inositol. Such hydrolysis conditions would be expected to y M d inositol phosphate and glycerophosphate with the former predominating when the parent phospholipid is more highly phosphorylatedaL These hydrolysis components of the phosphoinositide complex as their cyclohexylamine salts were resolved by descending pat)er chromatography and identified as inositol mono-, di- and triphosphate and glycerophosphate. Buwhim. Biophvs..-Iota, 84 (ngO.0 t',?,x-093
KIDNEY PHOSPHOINOSITIDE COMPLEX
69I
The criteria used to establish the chemical constitution of these resolved compounds were: (a) phosphorus/inositol ratio, (b) phosphorus/glycerol ratio, when applicable, (c) their migration on descending paper chromatography, (d) their migration on highvoltage paper electrophoresis, (e) co-chromatography with standard compounds, (f) use of the isotopes Na2Hs2po 4 and [2-SH]inositol. Infrared spectra of standard compounds when compared with those of the resolved compounds served as additional evidence. The hydrolysates resulting from mild alkaline hydrolysis (deacylation) which produce very little rupture of diester phosphate bonds was separated into six fractions by elution from Dowex-I (C1-) with 0.4 M LiC1. Chemical analysis of the individual fractions provided information suggesting the identity of five of these fractions as glycerylphosphoryl myo-inositol, glycerophosphate and inositol monophosphate, glycerylphosphoryl myo-inositol phosphate, inositol diphosphate and glycerylphosphoryl myo-inositol diphosphate. It is not possible at the present time to explain the presence of inositol mono- and diphosphate and glycerophosphate as components of the deacylated material. The criteria used for this recognition included: phosphorus /inositol ratios, phosphorus/glycerol ratios, migration on descending paper chromatography, migration on high-voltage electrophoresis, glycerol determinations before and after hydrolysis and co-chromatography and electrophoresis with standard compounds when available. Fraction I, which was obtained from the deacylated fractions and which represented 50% of the total phosphorus and almost 30% of the total a-amino nitrogen, was not completely characterized. Alkaline hydrolysis of this fraction followed by descending paper chromatography allowed the identification of inositol triphosphate as a major component. In addition, a second major component which contained 2 moles of glycerol per mole of phosphorus was also found. Although the latter was not identified, it is conceivable that glycerylphosphoryl glycerol might be present. An interesting feature concerning the data obtained with column chromatography of the deacylated phosphoinositide complex is the presence of amino acids in each fraction eluted from the column. No evidence is available at present to indicate the relation of these amino acids to the phosphate components since it is possible that they were elated from the column at varying ionic strengths of the LiC1 used for the gradient elution and, thus, their presence in each peak may be fortuitous. Their presence, however, in all fractions must be explained. On tile assumption that glycerol is the only acylated compound in the original material, the present study would indicate that the phosphoinositide complex from kidney cortex is composed of: L-phosphatidyl-L-myo-inositol, L-phosphatidyl-Lmyo-inositol phosphate, L-phosphatidyl-L-myo-inositoldiphosphate, plus an unknown lipid compound which contained glycerol, phosphorus and amino acids, presumably in peptide linkage. The phosphoinositide complex from kidney, pancreas and submaxillary glands has been previously reported as showing a rapid incorporation of Na2H~2PO4 (ref. I2, I5,I6), both in vivo and in vitro. In the latter two tissues, the metabolic activity has been shown to increase when secretion was induced by electrical stimulation or by administration of acetylcholine or epinephrine. Furthermore, it is of interest to note that the specific activity of the phosphorus increases as one increases the degree of phosphorylation of inositol. Recently, GARBUSet al. ~ have shown an inositol component in kidney mitochondria with a high metabolic activity which could conceivably Biochim. Biophys. Acta, 84 (t964) 681-693
692
I:. A N I ) R A D E , C. (;. HU(;(;INS
be similar to tim compounds we are describing from kidney. This is fltrther strengthened by the report of Ht,(;~;Lxs AND COHN12 that mitochondria from kidney contain almost two-thirds of the total tissue complex. Thus, it is believed that the inositol polyphosphates, primarily those in the monoester linkage in the parent coml~ound, are responsible for the relatively high metabolic activity in the l~hosphoinositide complex from the kidney. ()n the other hand, in our studies with secretion it appears that the diester phosphate atom is stimulated when secretion is initiated either electricall\or by acetylcholine or epinephrine. investigations concerning the chemical constitution of this complex in several organ systems are being continued in our efforts to establish whether the complex exists as a chemical entity iir whether it is a non-specific chenaical mixture and to understand more clearly the role of the constituents in the economy of the cell.
ACKNOWLEDGEMENTS
This investigation was supported in part by Public Health Service Research Grant No. o6152 from the National Cancer Institute and Public Health Service (;rant No. I-GS77 and American Cancer Society Grant No. IN24 D. The experimental data in this paper are taken from a thesis submitted by F. Axt~I~.~1)e to the Graduate School of Tulane University, in partial fulfilment of the requirements for the degree of Doctor of Philosophy. One of us (F.A.) was the recipient of a Rockefeller Foundation fellowship I96I-63 from the Delmrtment of Biochemistry, Faculty of Medicine, Javeriana University, Bogota, D. E. Colombia, also present address. The culture of Kloeckera brevis w m apiculata was obtained from l)r. I). J. HANartAN, l)epartment of Biochemistry, University of Washingeton, Seattle, Washington, and the sample of myo-inosose-e was ohtained from I)r. G. I{I:I)OI.PH, Department of Biochemistry, Vanderbilt University, Nashville, Tennessee. REFERENCES x j . FOLCH, J. Biol. Chem., 146 11942 ) 352 j . FOLCH, J. Biol. Chem., 177 (I949) 497. s C. GRADO AND C. E. BALLOU, J. Biol. Chem., 236 11961 ) .54. 4 j . C. DITTMER ANn R. M. C. DAWSON, Biochem. J., 81 ( i 9 6 1 ) .535. R. B. ELLIS, T. (;ALLIARD AND j. x . HAWTHORNE, Btochem. J., 8,s (19()3) I25. 6 It. BROCKERHOFF AND C. E. BALLOU, J. Biol. (7hem., 231) (190I) I 9 o 7. 7 H. BROCKERtIOFF AND C. E. BALLOU, .]. Biol. Chem., 237 1t962 ) 49s H. BROCKERHOFF AND C. E. BALLOU, J. Biol. Chem., 237 (19(~2) 17~ 4. 9 R. G. ELLIS AND J. N. |]'AWTHORNE, Biochem. J., 84 11062 ) IOP. ~o j . FOLlZH, in IV. I). M c E L R o v AND ]{. (;LASS, l'hosphorus Metabolisnz, Vol. 2, J o h n s I l o p k i n s P r e s s , B a l t i m o r e , 1952, p. 186. xx F. N. LEBARON AND J. Fot.ctt, .[. Neurochem., 1 (1951') t o t . 12 C. G. HUGGINS AND |). V. COHM, J. Biol. Chem., 234 (t959) 257. xa F. N. [,EBARON, J. I'. KIgTLER AND (;. ]IAUSER, Biochim. Biophys. Acta, 44 (19'~o) 17o. 14 V. N. I,EBARON, G. IIAUSER AND E. E. R u I z , Biochim. Biophys. Acta, 6o 11962) 338. a5 C. G. ltUGGINS, Nature, 184 11959) 1412. ts H . B. BURFORD AND C. G. I-IUGGINS, ,.Int. J. l~hysiol., 205 (I903) 23.5. 17 F. ANDRADE AND ('~. (;. HUGGINg, Federation Proc., 22 11963) 414 . xs F. ANDRADE AND ('. (;-. HUGGINg, Biochim. Biophys. Acta, 84 (1904) 98. 19 C. H. FISKE AND V. SUm*AROw, J. Biol. Chem., 66 (1925) 37.5. ~0 R. (;. BARTLETT, J. Biol. Chem., 234 (1959) 466. zl D. J. ]-IANAHAN AND J. N. OLLEY, J. Biol. Chem., 23I (1958) ~SI3. 22 R. J. BLOCK AND K. B. WVlSS, Amino Acid Handbook, T h o m a s , S p r i n g f i e h l , I950, p. 29.
Biochim. Biophys. ,4 cta, 84 (I964) 681 ( ; 9 3
KIDNEY PHOSPHOINOSITIDE COMPLEX
693
23 E. E. S.'~ELL, in P. GYORGYI, Vitamin Methods, Vol. I, Academic Press, New York, i95 o, p. 445,4 F. N. LEBARO.'% J. FOLCH AND E. ROTHLEDER, Federation Proc., I6 (r957) 2o9. t5 A. DESJOBERT AND F. PETEK, Bull. Soc. Chim. Biol., 38 (I956) 87I. ,2 R. S. BA,'~DURSKX AND B. AXELROD, J. Biol. Chem., 193 (I95 I) 4o5 • ,7 S. E. KERR AND G. A. KFOURY, Arch. Biochem. Biophys., 96 (I962) 347. 28 j . G. HAMILTON AND J. E. MULDREY, J. Amer. Oil Chemists" Soc., 38 (196x) 55z. 2, W. D. SKIT)MORE, AND C. ENTE~MAN, J. Lipid. Res., 3 (r962) 47I. ~0 R. M. C. DAWSON, Biochem. J., 75 (I96o) 43. s t H. WAGYER, A. LlSSAU, J. HOLZL AND L. HORnAMMER, J. Lipid. Res., 3 (I962) I77. s2 D. J. HA.'~AHAN, J. C. DITTMER, E. WARASmMA, J. Biol. Chem., 228 (1957) 685. s2 I). M. BROWN, G. E. HALL AND R. I,ETTERS, .]. Chem. Soc., (I959) 3547s4 j . GARBUS, I-t. F. I)ELucA, M. E. I.OOMANS AYD F. M. STROXG, J. Biol. Chem., 238 (1963) 59.
Biochim. Biophys. Acta, 84 (i964) 681-693