394

Biochimica et Biophysica Acta, 385 ( 1 9 7 5 ) 3 9 4 - - 4 1 1 Q Elsevier S c i e n t i f i c P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 27630

SUBCELLULAR FRACTIONATION OF PIG PLATELETS

L E O N S A L G A N I C O F F * , P A T R I C I A A. H E B D A , J O H N Y A N D R A S I T Z a n d M I R I A M H. FUKAMI

Department of Pharmacology and Specialized Center of Throm bosis Research, Temple University School of Medicine, Philadelphia, PA 19140, (U.S.A.) (Received October 14th, 1974).

Summary Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule fraction (10 000 g) was subjected to a sucrose gradient fractionation. Primary fractions and the granule subfractions were studied electron microscopically and biochemically by following the distribution of markers of membranes, lysosomes or a-granules, mitochondria and dense granules. With this technique of platelet homogenization, 80% of the serotonin and 93% of the fl-N-acetylglucosaminidase were found to be particulate. In the gradient, mitochondria were sharply banded in a fraction {density 1.16--1.17) having a specific activity 10--100 times higher than the other fractions of the gradient. Serotonin-containing granules were found in a pellet of density greater than 1.27 and contained 60% of the serotonin and adenine nucleotides of the granule fraction. The lysosome markers that were monitored, acid phosphatase and ~-N-acetylglucosaminidase, exhibited different distribution patterns. Acid phosphatase showed the highest specific activity in the microsomal fraction with only 2.8% in the granule fraction, and this latter a m o u n t also appeared to be associated with membranes upon further fractionation, fl-N-Acetylglucosaminidase was present in both the granule fraction and in the microsomal fraction with nearly the same specific activity. However, that present in the granule fraction was clearly associated with granules that distributed over a wide range of densities on a sucrose gradient. The calcium distribution was followed to a t t e m p t to determine its subcellular location; 19% was found in the same subfraction as the serotonin-containing granules, but at least 50% of the particulate calcium was associated with granules distinctly separate from the storage granules.

* Address correspondence to: Temple University School of Medicine, Dept. of Pharmacology, 3420 N. Broad Street, Philadelphia, PA 19140, (U.S.A.)

395 Introduction

Although platelets lack nuclei, they contain numerous subceUular particles and organelles, some of which disappear during the platelet release reaction induced by ADP, thrombin, etc. (for reviews, see refs 1 and 2). These agents induce the specific release of serotonin, adenine nucleotides [3], calcium [4] and lysosomal enzymes [5,6] from intact platelets. Subcellular fractionation studies attempting to correlate the location of these released materials in specific organelles have been reported b y many laboratories [7--14]. For the most part, these studies resulted in a high degree of solubilization of serotonin and adenine nucleotides during platelet homogenization. The method of cell disruption appeared to be a critical determinant of whether or not a released substance was contained within discrete organelles or present in the cytosol. The highest yields of serotonin-containing granules reported in the literature were obtained by ultrasonication of platelets [7,8]. However, the latter investigations did not a t t e m p t to separate the different subcellular particles. A method for the mild disruption of intact platelets, using a modified French pressure cell, was developed in our laboratory. The mitochondrial fraction, isolated with this new procedure, demonstrated coupled oxidative phosphorylation, indicating that the procedure was mild enough to preserve delicate functions of subcellular organelles [15]. As the electron micrographs of the isolated mitochondrial fraction showed the presence of many granules other than mitochondria that appeared to be structurally intact, gradient fractionation with electron microscopic and biochemical characterization of the subcellular c o m p o n e n t s of this mitochondrial fraction were undertaken in a preliminary effort to obtain separated components for subsequent studies. Serotonin was monitored as a storage granule marker along with adenine nucleotides and calcium which are released similarly [3,4]. Acid hydrolases that were assayed as a-granule markers were ~-N-acetylglucosaminidase (EC 3.2.1.29) [9] and acid phosphatase (EC 3.1.3.2) although the latter enzyme seemed to be associated more with membranes and membrane fragments [16] than with agranules per se (see Results). The marker selected for mitochondria was a-glycerophosphate oxidase (EC 1.1.99.5) rather than succinate dehydrogenase (EC 1.3.99.1) because it had higher specific activity for the same subcellular location [ 1 5 ] . Lactate dehydrogenase (EC 1.1.1.27) was used to follow the breakage of platelets [ 1 5 ] . The results obtained show that French press homogenization of platelets, under conditions that prevent actomysin gel formation, not only yields coupled mitochondria but also a-granules and dense bodies while retaining 80% or more of the granule constituents of the preparation in a particulate form. An excellent separation on sucrose density gradients was obtained between the mitochondria and the dense storage granules, while the a-granules exhibited a wide range of specific densities. Methods

Isolation of platelets The platelets were isolated as described previously [15] from pig blood

396 that had been collected into acid-citrate-dextrose solution [17] at a ratio of 9 " 1 to give a final pH of 6.5--6.7.

Preparation of subcellular fractions 1--4 The procedure used to disrupt the platelets was essentially the same as described earlier [15] except for the following modifications. The isolated platelets were washed once with a modified Tyrode's solution containing 0.2 mM EDTA and no calcium [18] at 10--15°C. After centrifugation, the washed platelets were suspended in an ice-cold ionic medium (referred to as "incubation m e d i u m " in the previously published Method) consisting of 100 mM KC1, 50 mM Tris chloride, 5 mM MgCl2 and 1 mM EDTA, pH 6.5, at a ratio of 10--15 g wet weight of platelets per 16--20 ml of medium. 2 ml of the incubation medium containing 10 mg of the proteolytic enzyme nagarse (EC 3.4.4.16) and 5 mg of ATP was added to the platelet suspension and the mixture incubated for 5 min at room temperature. The platelets were then isolated by centrifugation at 1 0 0 0 g X 10 min at 4°C and resuspended in ice-cold incubation medium containing 1 mM ATP and 0.35% bovine serum albumin (Sigma, fraction V, fatty acid poor), pH 7.4, (isolation medium) at an approximate wet weight to final volume ratio of 15 g per 30--40 ml. The platelets were then lysed in a French pressure cell (4°C) and fractionated as previously described. A summary of this procedure is shown in Fig. 1. Fraction 2 was washed twice with isolation medium and suspended in 0.25 M sucrose mid 10 mM Tris chloride, pH 7.4, at a protein concentration of approximately 10 mg/ml for sucrose gradient fractionation. (For adenine nucleotide determinations, fraction 2 was washed twice with isolation medium containing no ATP.)

Preparation of sucrose gradient fractions A--F Sucrose

SUSPENSION

OF

gradients

PELLET

+

x

formed

as

follows:

solutions

of 0.8, 1.0,

PLATELETS

BROKEN

1,000 g

were

i0 min

S UPE RNATANT

(fl) i0,000 g PELLET (f2)

+

x

i0 min

sLrpERNATANT i00,000 g PELLET (f3)

SUCROSE STEP GRADIENT (0.8 - 2.0 M)

lO0,O00 g

5 BANDS fA-E

+

x

+

x

30 rain

SUPERNATANT (f4)

60 m i n

PELLET fF

Fig. 1. F r a c t i o n a t i o n S c h e m e . f = F r a c t i o n , f ( 1 - - 4 ) d e s i g n a t e d p r i m a r y f r a c t i o n s a n d F ( A - - F ) the s u b f r a c t i o n s o f f2.

397 1.2...2.0 M sucrose were prepared in 5 mM Tris chloride and 0.1 mM EDTA, pH 7.4. Steps of 1.5 ml each were layered into three 13-ml cellulose nitrate centrifuge tubes (SW40 rotor rubes, Spinco, Palo Alto, Calif.). These were incubated at 37°C for one hour to soften the interfaces and cooled to 0°C. Approximately 1.5 ml of the twice-washed fraction 2 was carefully layered onto each tube and fractionated by centrifugation at 100 000 g for 60 min (rotor SW40, Centrifuge L2-65B Beckmann). The fractions were then removed from the top of the tubes band by band, using standard techniques. For the e n z ~ n e assays, each fraction was diluted with an equal volume of 0.25 M sucrose buffered with 10 mM Tris chloride, pH 7.4, centrifuged at 150 000 g for 30 min and the particles resuspended in a minimal volume of the 0.25 M sucrose with Tris chloride; supernatants of these fractions were checked for enzyme activity. Serotonin, adenine nucleotides and calcium were assayed using the gradient fractions directly.

Assays fJ-N-Acetylglucosaminidase activity was assyed by measuring the a m o u n t of p-nitrophenol (400 nm) released by the hydrolysis of p-nitrophenyl-~-Nacetylglucosaminide [9] after acid deproteinization and alkalinization of samples that had been incubated for 30 min at 25°C in a citrate buffer, pH 4.5, in the presence of 0.5% Triton X-100. Acid phosphatase was measured at pH 4.8 in fresh samples with p-nitrophenyl phosphate as the substrate as described by Linhardt and Walter [19] with the following modifications: 0.2% Triton X-100 was included in the assay mixture and the reaction was incubated at 0°C for 30 min to avoid an apparent inactivation of the enzyme at higher temperatures. The reaction was not linear with enzyme activities less than 0.15 pmol per 30 min. a-Glycerophosphate oxidase was measured polarographically, either via the mitochondrial electron transport chain or in the presence of cyanide with phenazine methosulfate as an artificial carrier [2]. It was necessary to assay the nagarse-treated platelets in the presence of 0.5% Triton X-100 although the oxidase activity in fraction 2 was inhibited about 50% with the detergent. Fractions 1--4 were also treated with Triton to obtain consistent recovery data, but the sucrose gradient fractions A--F were assayed w i t h o u t detergent. Adenine nucleotides (and guanine nucleotides) were estimated as the difference in absorbance at 260 and 310 n m (max = 15.7 mM -~ cm -~) on neutralized extracts of samples that had been deproteinized by 10% perchloric acid [21,22]. Calcium was analyzed by atomic absorption s p e c t r o p h o t o m e t r y on a Perkin-Elmer 308 spectrophotometer. The samples were extracted by 0.1 M HNO3 (final concentration) and insoluble material was removed by centrifugation. Calcium levels were determined by comparison with standard calcium solutions. All measurements were made in the presence of 1% LaC13. When calcium was analyzed in solutions of high sucrose concentrations, the samples were diluted to a final concentration of 0.3 M sucrose and read against calcium standards prepared in 0.3 M sucrose. Serotonin was assayed directly as total 5-hydvoxyindoles by the fluores-

398 cence emitted at 535 nm u p o n excitation at 295 nm in 3M HC1 [23,24] *. A sample volume o f 0.2--0.5 ml was homogenized in 2--2.5 ml of cold 0.1 M HC1 with a teflon pestle in a glass vessel and the protein was precipitated by the addition of 1 ml of 10% ZnSO4 and 0.5 ml o f 1 M NaOH. After centrifugation of the samples, the supernatant solutions were acidified with 0.5 ml of 12 M HC1 and the fluorescence was quantitated by comparison with standard concentrations of serotonin creatinine sulfate treated in the same manner as the samples.

Electron microscopy The samples were suspended in 1% glutaraldehyde in cacodylate buffer (pH 7.4, 350 mosM) and fixed in the cold for 30 min. The subcellular material was sedimented by centrifugation at 12 000 g for 10 min. The pellet was rinsed briefly with cacodylate buffer and fixed with buffered 1% osmium t et roxi de in the cold for 1 h. The osmium was rinsed out with buffer, the sample dehydrated with ethanol, and e m b e d d e d in Epon 812. Sections were double-stained with uranyl acetate followed by lead citrate.

Materials Nagarse was obtained from the E n z y m e Development Corporation, New York. The sucrose used in the gradient was from Mallinckrodt, Analytical Reagent grade. Serotonin creatinine sulfate and p-nitrophenyl-fl-N-acetylglucosaminide were f r o m Sigma Chem. Co. All o t h e r chemicals were commercial preparations o f the highest purity available. Pig blood was generously provided by the Penn Packing Co. of Philadelphia.

Results In our earlier work on platelet m i t o c h o n d r i a [ 1 5 ] , occasional platelet preparations aggregated prior to French pressure cell t r e a t m e n t and had to be resuspended by light hand homogenization in a glass vessel with a teflon pestle. The occurrence of specific release of nucleotides or o t h e r released materials was n o t mo n ito r ed , because the coupling and functions of the isolated mitochondria did n o t seem to be affected by the release reaction. However, it was necessary for the present study to establish that the m e t h o d of homogenization being used was n o t accompanied by specific release or aggregation so that the contents o f the isolated subcellular granules would a p p r o x i m a t e as nearly as possible the in situ levels. The release reaction was m o n i t o r e d by measuring the absorbance at 260 nm in the deproteinized platelet supernatant solutions after washing and after nagarse-treatment. When ATP was used in the medium, the added adenine nucleotide had to be subtracted from the total a m o u n t f o u n d in the medium to

* The possible presence of significant quantities of 5-hydroxyindoles other than serotonin was c h e c k e d b y t h e e x t r a c t i o n m e t h o d d e s c r i b e d b y W e i s s b a c h e t al. [ 2 5 ] . S e r o t o n i n v a l u e s i n u n treated and nagarse-treated platelets were 101--107% of the total 5-hydroxyindoles (4 determinations).

399 obtain the figure for the a m o u n t released; as a result, the estimations in these cases may n o t have been as accurate as when no ATP was added. However, the results always correlated well with t he a m o u n t of material retained by the platelets, and the total recoveries after corrections for added ATP was nearly 100%, just as in the preparations with no added ATP. Serotonin estimations provided an additional check on t he release reactions and confi rm ed the validity of the adenine nucleotide measurements. The degree of aggregation was checked for by visual exam i na t i on of the platelet suspension and occasional phase contrast microscopy. Occasional preparations u n d e r w e n t bot h specific release and aggregation with the previously described m e t h o d , and 40--60% of the total platelet adenine nucleotides appeared in the supernatant of the nagarse-treated platelets with less than 5% retained in fraction 2. When no release or aggregation occurred, 0--5% of the total adenine nucleotides was f o u n d in the supernatant of the nagarse-treated platelets, while the rest was particulate. Sometimes aggregation was observed when little or no adenine nucleotides were released extracellularly. In these instances, the total recovery of adenine nucleotides in all the fractions com pared to the starting material was only 40--60%, perhaps due to f o r m a t i o n of h y p o x a n t h i n e [ 2 6 ] , for which the ratio of e~ 50/e~ 60 is 1.32 at pH 7 and e~ 60 is only 8.1 mM -1 cm -1 [ 2 7 ] . Elimination of the nagarse t r e a t m e n t f rom the procedure was found to be undesirable, because only a poor separation of bands and no pellet was obtained upon sucrose gradient fraction w i t h o u t it. A single washing of the platelets with a modified T y r o d e s solution w i t h o u t calcium and with EDTA at pH 6.5 (see Methods) prior to t r e a t m e n t with nagarse and the use of a nagarse incubation medium with a pH of 6.5 instead of 7.4 was f o u n d to prevent the release reaction and aggregatiom The aggregation that was unaccom pani ed by release was avoided by including ATP in t he protease incubation step. One o th er modification of the fractionation procedure t hat was a t t e m p t e d was the exclusion of ATP from the isolation medium. ATP was originally included in the preparation to p r o t e c t t he m i t o c h o n d r i a from swelling in the ionic medium. However, since coupled m i t o c h o n d r i a were n o t essential in these fractionation studies, ATP was deleted to facilitate d e t e r m i n a t i o n of adenine nucleotide distribution. When this was done, almost all of the serotonin, calcium and much of the adenine nucleotides were f o u n d to be solubilized.

Primary fractionation The distribution of the assayed c o m p o n e n t s in t he four fractions isolated by differential centrifugation f r om the French press h o m o g e n a t e is shown in Table I. The nagarse-treated platelets were taken as 100%, since the variation in specific activities for a given assay was less from one preparation to the next with nagarse-treated platelets than with washed platelets. Differences in removal of plasma proteins from the platelets by small variations in the daily washing and draining techniques were p r o b a b l y responsible for the variability in specific activities with the once-washed platelets. The protein lost with nagarse t r e a t m e n t a m o u n t e d to 15--20% of the washed platelets, while less than 1% of the lactate dehydrogenase was lost. It was established in preliminary studies th at when no release or aggregation occurred, t he loss of the measured markers due to the nagarse-treatment was low. The levels of serotonin, for

I

are expressed

* The

amount

recovery

platelets

Total

4

3

2

1

OF

ASSAYED

COMPONENTS

8.8

4.7

* 5.6)

* 6.3)

* 1.2)

* 0.3)

+ 4.0)

* S.E.

of nagarse-treated

(92.8

(43.8

(

(

(35.5

(loo%)*

Protein

as the mean

6.8

0.8

platelets

(78.6

(

(21.8

14.5

(10.6

2.8)

varied

+ 2.6)

f

* 0.2

+ 3.5)

* 1.6

* 1.5)

f 1.8

13.0

from

+ 2.5

23.9 3.7

700-3000

(78.7

(20.2 f

t 5.1

56.5

* 5.6)

f 0.6)

+_ 0.5

depending

n.m. -

9.0

(78.4

(

5.2

+ 1.7)

f 5.8)

(39.5

f 2.4

15.4 (29.9

(100%)

on the

+

amount

99.2

(

i

34.1

f

t

?

6.7

+

34.7

f r

15.9

1.7

of platelets

5.7)

4.6)

10.0

0.3

0.9

1.7)

8.9

+_ 0.8)

20.0

(

(

(

173.2

56.6

used

1.2)

for a given

f 3.4)

f 3.4)

* 1.4

6.8 14.5

f 0.5

+ 0.6

f 1.9)

f 2.3

i

f 0.8

3.2

4.2

22.0

51.7

63.6

(100.6

(

(

(

(

preparation.

* 0.4

k 2.7)

(90.9

+ 1.3

2 0.5) t 7.4)

3.3

3.8 (20.3

(

2.7

2 2.5)

f 2.8 (22.0

+ 6.6) 30.7

* 0.6

+ 0.5

(46.0

8.9

(

21.4

7.4

73.1

_+ 0.3

(100%)

l&9

Serotonin

(100%)

1.6

Calcium

nucleotides. in parentheses.

Adenim

is shown

Methods).

(100%)

+

nucleotides

oxidase

58.5

adenine

8.9 f 0.1

Total

phosphate

(see

or compound

of protein

activity

per mg

enzyme

per min

of recovered

consumed

ol-Glycero-

percentage

of oxygen

mg of protein

5.4)

+ 1.9)

+ 0.4

+ 2.5)

* 0.6)

* 1.8

* 3.5)

? 0.3

+ 0.4

2.8

(26.0

(

6.1

(29.7

8.6

+ 5.5)

? 1.0

(39.5

6.7

9.4 (100%)

* 0.9

(100%)

5.4

Acid

The

or natoms

protein.

phosphatase

of

glucosaminidase

n = 5.

mg

1-4

/3-N-Acetyl-

with

per

produced

IN FRACTIONS

activities are expressed as nmol of p-nitrophenol and serotonin concentrations are given as nmol

Nagme-treated

Fraction

results

calcium

Enzyme

DISTRIBUTION

TABLE

All

401

example, were 7.4 -+ 0.5 nmol/mg of protein in nagarse-treated platelets and 5.8 +- 0.3 nmol/mg of protein in untreated platelets with a recovery of 92.2 + 3.3% of the total serotonin in the platelet pellet centrifuged from the diluted nagarse solution. The recoveries of added serotonin in platelet rich plasma were 100%, indicating that no destruction of 5-hydroxyindoles was occurring. A typical electron micrograph of the resuspended nagarse-treated platelets is shown in

-

IP~-"

"~IP.

'Ib

.t -

N

Fig. 2. (A), Electron micrograph of nagarse-treated platelets (X13 320)~ (B), Electron micrograph of fraction 1 (X 13 320).

402 Fig. 2A. Some of the platelets appear to have undergone release at this stage, but most of the platelets, although altered in appearance from untreated platelets, seemed to contain intact granules. Fraction 1 (see Fig. 1), contained unbroken platelets, large fragments of platelets and granules (Fig. 2B). The protein recovered in fraction 1 ranged from 30--40%, (Table I) and the assayed enzymes contained in this pellet were in a comparable range. Approximately 15--25% of the total platelet lactate dehydrogenase, a cytosol marker, was recovered in fraction 1 and the rest in fraction 4; less than 0.5% was measured in fraction 2 [15]. Adenine nucleotides, calcium and serotonin, were present in fraction I at a higher recovery level than the marker enzymes or total protein, indicating that there was an enrichment of storage granules in this fraction. A large increase relative to nagarse-treated platelets in the specific activity of a-glycerophosphate oxidase and in the amounts per mg of protein of adenine nucleotides, calcium and serotonin was seen in the 10 000 g pellet, fraction 2. Numerous mitochondria, a-granules and dense bodies were apparent in the electron micrographs (Fig. 3A). Except for acid phosphatase, the relative specific activities of all the marker components increased 2- to 6-fold in fraction 2 relative to nagarse-treated platelets (Fig. 4). a-Glycerophosphate oxidase, adenine nucleotides, calcium and serotonin levels peaked in fraction 2, but the activities of acid phosphatase and ~-N-acetylglucosaminidase were somewhat higher in fraction 3 than in fraction 2. Fraction 3 contained few intact granules

Fig. 3. ( A ) , E l e c t r o n m i c r o g r a p h o f f r a c t i o n 2 ( X 3 5 3 0 0 ) ; (B), F r a c t i o n 3 ( X 3 5 6 0 0 ) ; (C), S u c r o s e grad i e n t s u b f r a c t i o n A (X 3 5 4 0 0 ) ; (D), S u c r o s e g r a d i e n t s u b f r a c t i o n B (X 3 5 4 0 0 ) .

403 Protein

I F, 0 Acid

~

.

11

100%

Phosphatase

2

Adenine Nucleotides

2

I--U

u u_ u

4-

~-N-Acetylglucos aminidase

2

Calcium 4

2

,

Subcellular fractionation of pig platelets.

Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule frac...
3MB Sizes 0 Downloads 0 Views