Gossypol inhibition of Ca++ uptake and Ca++-ATPase in human ejaculated spermatozoal plasma membrane vesicles

CONTRACEPTION GOSSYPOL INHIBITION HUMAN EJACULATED

U Kanwarl,

OF Ca++ UPTAKE AND Ca++-ATPase SPERMATOZOAL PLASMA MEMBRANE VESICLES

A.Batlal,

and A. 1 2

Department

Department

Institute

R.Minocha',

S.Sanyall,

of

S.Majumdar'

Rangal

Zoology,

Panjab

of Experimental

of Medical

IN

University

Medicine,

Education and India

and

Post-Graduate

Research,

Chandigarh,

ABSTRACT

plant-derived polyphenolic a actions in men, contraceptive

Gossypol, to exert

known

compound inhibits

Ca++ -transport ATPase in isolated plasma and Ca++ -activated membranes of ejaculated +h+uman sperm+ce$ls. It also inhibits ATPas+es, bound Mg - and Na +K -dependent the membrane systems. Ca and alkaline phosphatase 5'-nucleotidase It inhibition ATPase by gossypol is non-competitive. in Arrhenius expression of discontinuity abolishes the the and increaTes temperature dependence of Ca ++-ATPase Phosphatidyl choline and Na -deoxyof activation. energy inhibit Ca++-transport activity in the membrane cholate The apparent similarity of Ca++-transport inhibivesicles. indicate choline tion by gossypol and phosphatidyl may the possible capability of this compound to induce changes of the membranes, wherein in the lipid microenvironment Inhibitory effect of gossypol the integral proteins operate. that gossypol membrane Ca++-pump suggests OII the plasma may affect sperm motility by a mechanism which is related to the structure and functions of the plasma membrane. INTRODUCTION

Ca++ -transport against tial

a steep

pre-requisite

such

Acrosomal of

of

(capacitation)

Submitted Accepted

membrane

the

important

which

occurs to

overlying

for publication for publication

APRIL 1989 VOL. 39 NO. 4

the

gradient,

processes

and

sperm

involves

which

spermatozoa,

concentration

reactions

reaction,

exposure

plasma

for

acrosomal

as

in

inward

of

after

female

anterior

motility

(1).

a brief

period

reproductive

tract

Ca++ -dependent

the

place

in fertilization

control only

the

takes

is an essen-

half

fusion of

the

of

the

sperm

October 5, 1988 January 18, 1989

431

CONTRACEPTION

head it

with (2).

A

the

acrosomal

membrane

net

uptake

Ca++

identified confirm

(3) that

and

Ca++-influx

are,

involved

rence

been

have

acrosomal

membranes,

Mg ++-ATPase ATPase

in

rodent

sperm

plasma

membranes

is

a key

Ca ++-uptake Gossypol

the

the

in

Ca++ -transport

concentration

diffe-

plasma

membranes

Ca++-pump

the

or

membranes,

and and

in

and Ca+++

a

Ca++-

boar

The Ca++-binding

organising

function

the

of

several

abundance

in

been

A 23187

although

membranes

(4).

role

and

of

the

acrosomal

present

plays

occurrence

flagellar

sperm

has

the

sperm

including

ram

in

calmodulin,

in

beneath

ionophore

understood,

operating

sperm

probably with

in

identified

just

capacitation

kinetics

poorly

lies

Ca ++

with

induces

The

however,

mechanisms

during

experiments

reaction.

acrosomal process

bull

of

which

and

protein, and

spermatozoa events

associated

(5).

[l,l', 6,6',7,7'-hexa-hydroxy-5,5'-di-isopro-

pyl-3,3'-dimethyl-(2,2'-binaphathalene)-8,8'-dicarboxaldehyde]

isolated

species) male to

has

inhibit

and

enzyme

in

renal

non-competitive

shown

to

across

in

inhibit

the

red

examined

Ca++ -ATPase ejaculated

in

inhibitor

inhibition

was

C4

other

as

seeds

agent

Mg ++-ATPase

isozyme

competitive zing

the

reported

antifertility

genase

be

from been

(6).

in

This

compound

a variety

It

nature number

of

plasma

membrane

a

isolated

dehydro-

of

plasma

as

a

metaboli-

Na++K +-ATPase reported has

also

to been

systems

transport (11).

shown

function

is

Gossypol

inhibitor

potential in

been

nucleotide

hexose

a

has

although

gossypol

(10).

(Gossypium nonsteroidal

L-lactate may

of

(9), by

slices

plants

(7) and (8).

cell

system

This compound ++ Ca -uptake and

membranes

from

human

spermatozoa.

Preparation

432

a potential

systems

MATERIALS

samples

cotton

sperm

testis of

of be

to

were

of

Sperm

collected

Plasma from

AND

METHODS

Membrane healthy

Vesicles

human

donors

:

Semen through

APRIL 1999 VOL. 39 NO. 4

CONTRACEPTION

self-masturbation spermatozoa. rated

Sperm

0.2

MgC12

(MOPS), same by

pH

an

high

first

in

at

the medium

tion.

of

10,000

g

in

same.

9-fold

for

30 The

of

and The

estimated

by

Lees

and

standard method

Paxman

(13). of

The

APRIL 1989 VOL. 39 NO. 4

the

dodecyl

using

was

sucrose

four

was

whole

cell

homo-

enzyme membrane

membrane

sulphate-Lowry

was

than

Na+I?+-ATPase

ensuring

bovine

at

suspended

plasma the

40 of

times

greater

enzymes,

preparation

for

centrifuged

showed

of

g

solu-

interface

pellet

and

superna-

sucrose gradient

and

the

pellet

resuspended

diluted

isolated

crystalline

membrane

resultant

35,000

purity

concentration

sodium

Rufo -et al.

to

marker

This

the

the

and

the

centrifuged

at

MOPS

resultant

of

and

at

preparation

confirming

protein the

mM

comparison

integrity

vesicles.

10

membrane

5'-nucleotidase,

activity

located

in

a microtip

was

(d20=1.05)

removed

The

in

the

centrifuged

membrane

the plasma

min

pellet

15%

acid

thoroughly

with

a discontinuous

and

and

min.

enrichment

genates

on

was

sucrose

then

M sucrose,

environment.

10

The

by were

sulfonic

resuspended

suspension

and

fraction

layer

35,000 the

was

for

min.

layered

gradient

M

30

(d20=1.17)

membrane

0.25

g

10 min

0.25

Instruments)

The

motile

cells

disrupted

ice-water

an

cent

the

propane were

were

pelleted

and

and

80% motile

haemocytometer

containing

EDTA

medium.

for

and

d20=1.17/1.05

of

at

g

40%

The The

with

and

in

per

was

min

cells

1 mM

g for

35,000

composed

min.

washed

as

semen 10

(B.Braun

the

at 6,000

tant

The for

morpholino

speed

centrifuged

resuspended

in

The

containing

than

progression

a Neubayuer's

buffer

mM

ultra-sonifier at

g

with

more

forward

temperature in

(12).

10

contain

and

room

1,500

and

7.4.

buffer

probe was

at

repetitively

mM

to

counted

earlier

centrifugation washed

at

were

described

found

motility

subjectively

cells. as

and

Sperm

was

procedure

serum

as

the

based

on

the

(14).

433

CONTRACEPTION

Measurement plasma us

of

Ca++-uptake

membrane

vesicles

earlier

for

(15),

adopting

Assay

of

as

ATPase

described 30

mM

120

mM

NaCl

at

37OC, and

Rufo

the

the

acid.

and

dent

preparations

were

corrected

of

plasma the

activity

was from

activity of

Pi

the

release

initial (AEa)

enzyme were

1.5

the

presence

affinity

and

Bergmeyer For

were

freshly

and

addition

or

434

enzyme

the

All

10% from

Fiske

two

and out

indepen-

ATPase

values

the

absence

was

measured

Ca++-dependent ATPase ++ the Ca -independent

the

The

Na+K+-

ouabain-sensitive the

difference

absence

of

0.1

mM

(Km)

maximum

the

energy

earlier

alkaline

methods

Na-

as

and

described and

by

and

(Vmax)

as

as

of of

carried

in

calculated

constant

velocity

measured

measured

was

ml

of

(Mg++-dependent).

measured and

MgC12,

liberated

were

subtracting

ATPase

mM

1

method

and

contain-

addition with

repeated

EGTA

by

5'-Nucleotidase were

the

membranes.

measured

pre-incubation

phosphorus

were

mM

total was

Substrate

was

7.5

of

37OC

the

vesicles

medium

salt),

determinations

plasma

AIPase in

of

measured in release ++ Ca -independent ATPase

of

the

at

by

(16).

Pi

calculated

portion in

of

membrane

min by

min

by

enzyme

for

ATPase

5

Inorganic

membranes.

activity

(Na

started 20

spermatozoa1 described

activity 1 ml

ATP

experiments

addition

in

After

determined

The

duplicate

by

was

border

ATPase

mM

by as

technique

(14)

KCl.

after

was

Subbarow(17). in

0.05

reaction

reaction

al.

mM

terminated

brush

: The

et --

20

trichloroacetic

measured

filtration

Tris-HCl, and

Ca++-uptake

intestinal

rapid Activity

by

ing

ATP

rat a

: was

of

of of

ouabain. apparent

activation

(18).

phosphatase Huang

activities

and

Keenan

(19)

(20),respectively. -in

vitro

prepared of assay

it

addition by did

medium.

of

dissolving not

alter

Gossypol

gossYPol, in

10%

the was

the

solutions

alcoholic pH

of

received

the

saline uptake through

APRIL 1989 VOL. 39 NO. 4

CONTRACEPTION

the

courtesy

of W.H.O.

Gossypol

Research

Programme,

Geneva,

Switzerland. Phosphatidyl were

added

persions

to

results have

6-8

on

been

analyzed

Ca

earlier

semen

membrane

(PC)

based

samples

in

were

in

a

two

deoxycholate sonicated

pooled

single

Ca ++-ATPase

-uptake, on

sodium

assay

dis-

(18).

preparation ++

and

Ca++-transport

as described About

plasma

choline

the

together

other

and

independent

for

experiment.

membrane

The

enzymes

preparation,

in duplicate. RESULTS The

plasma is

a

course

time

membrane

vesicles

two-component

followed state.

by The

bation.

a

by

is at

the

about

and

PC

and 1 uM deoxycholate ++ of Ca -uptake to 40 min thereafter. uptake 30%

At

by

and

The

ATP

for mM

inhibits systems,

maximal Vmax

the and

effects

the

linear

inhibits the

amounts

to

incu-

inhibits

uptake.

point,

PC

ATPase,

of

Na-

5%

Na-

portion

the process

reduction

in

greater

activities typical

kinetics.

than

4.54

activities the kinetic

APRIL 1989 VOL. 39 NO. 4

ATP

activity

of

shown

is

found

type

of

the

I. sub-

required

to be

proteinlmin. and

and

Table

concentration

Ca ++-ATPase

analysis

in

Michaelis-Menten

umolelg

ATPase,

5'-nucleotidase are

The

Ca++-ATPase is

Ca++-activated

Na++K+-ATPase,

follows

saturation

the

reduce

time and

Mg++-dependent

phosphatase

half and

completely

of

nM

is

steady

respectively.

hydrolysis

strate

PC

min

10

the

of

of

40-min

insensitive

alkaline

30-40

shows

uptake rate

rapid

of

kinetics

The

achieving

to about

and

Na-deoxycholate

60%,

ouabain

the

initial

process,

Fig.2

the

spermatozoa1

Fig.1.

concentration

25%. on

up

by

in

An

slower

linear

-uptake

shown

process.

uptake

deoxycholate

++

Ca

is

relatively

Gossypol

Ca++ -uptake

of

13.33

Gossypol other

substrate

enzyme require-

435

CONTRACEPTION

cd

90.

3

no:

d_

d.

103.

.d_

10

20

30

40

50

60

70

min Fig.1

436

The time spermatozoa1 or absence

course of Ca++-uptake by plasma membrane vesicles of gossypol.

human ejaculated in the presence

APRIL 1969 VOL. 39 NO. 4

CONTRACEPTION

Na-

DEOXYCHOLATE

PHOSPHATIDYL

I

10

20

40

30

50

WOUNE

(5%)

(1JJM)

60

min Fig.2

Effects of N a +-deoxycholate and g+hosphatidyl (5%) choline (1 PM) on the kinetics of Ca -uptake by human ejaculated spermatozoa1 plasma membrane vesicles.

APRIL 1999 VOL. 39 NO. 4

437

?

& g i= $ 8

I

are based on within 5%.

phosphatase

The results which agreed

Alkaline

two

independent

membrane

10.00

1.54

3.33

3.33 10.00

11.11

11.11

25.00

Na++K+-ATPase

5'-Nucleotidase

16.66

20.00

25.00

Mg++ -ATPase

preparations,

4.54

4.54

13.33

(pmole/g

Control

Vmax

13.33

Km(mM)

Ca++ -ATPase

Enzymes Gossypol

Membrane Control

Plasma

VESICLES

-analyzed

at 37OC)

MEMBRANE

PARAMETERS

in

duplicate

2.77

1.19

5.00

12.50

2.56

Gossypol

protein/min

RESULTS OF -in vitro ADDITION OF GOSSYPOL (10 PM) ON THE MICHAELIS-MENTEN KINETIC OF Ca++ -ATPase AND OTHER MEMBRANE BOUND ENZYMES IN HUMAN EJACULATED SPERM PLASMA

TABLE ---

CONTRACEPTION

ment

shows

that

Ca++ -ATPase,

although

the

Km

is

the non-competitive The studied. and

2Z°C

activity

in

was

showing

decreased,

of

was

Ca++-ATPase temperature

expression

the

with

a

was

lines

point

break

point

a lowered

were

parallel,

lost.

was

also

sensitive

of gossypol,

both

break

of

indicating

of

although

and

Vmax

(Fig.3).

to be

In the presence

obtained,

proximity

the

thereby

inhibition

found

Arrhenius

(Fig.4).

decreases

dependence

activity

nonlinear

around

not

nature

temperature

The

gossypol

Gossypol

resulted in a continuous AEa. The AEa was 0.45 -1 in control and 0.61 in the presence of gossypol.

treatment kJoules.mol

DISCUSSION

The system

which

is

antifertility

plasma

the

Ca++ -transport

in

an

evaluated.

was sperm

plasma

lymphocytes, all

of

ATPase

are

a high

inhibited the

by

of the

supports

same

be

order

to

for

net

presumably

membrane, the

Ca++ -ATPase

been

that

and

al.

the hypothesis

flavonoid

they

and

neutrophils, systems. to ouabain,

quercetin,

ATPase

that

(21)

erythrocytes,

and

Ca++-transport

the

acti-

characterized

et -of

for Ca++,insensitive

between

and

(21)

and are

and

transport expressions

enzyme. In

inhibitor

to

thought

erythrocytes,

In

has

to

male

affinity

responsible

macrophages

affinity

similarity

in

Breitbart

properties

Ca++-ionophore

apparent

activities

ATPase

adipocytes,

which has

plasma

by

High

(15).

system

gradient,

This

potential

membranes

vesicles

the

membrane

similar

possesses

described

transduction across

a

described.

synaptic

electrochemical

Ca ++-transport

gossypol,

also

membrane

energy

membrane

been

lymphocytes,

intestinal

vity

by

are

systems

macrophages,

against

inhibited

compound,has

Ca++ -transport

study

plasma

spermatozoa1

of

our

both

APRIL 1989 VOL. 39 NO. 4

assay

system,

Ca++-transport

gossypol and

is

an

Ca++-ATPase

effective activity.

439

CONTRACEPTION

1.8

GOSSYPOL /

1.6 1.4 1.2 f.0 5.8 0.6

0.4

02/ // 0.1

0.2

0.3 4

Fig.3

440

substrate Effect of spermatozoa1 ejaculated presence ATPase in the

O'l,

0:s

(mM)

the human concentration on membrane bound Ca'+plasma or absence of gossypol.

APRIL 1989 VOL. 39 NO. 4

CONTRACEPTION

eTEMPERATURE 49O

3

3-Y

3.2

3.1 ;

Arrhenius ejaculated ATPase in

x10?

(Co, 3o"

20"

1P

4"

3.3

3.4

3.5

3.6

;

temperature dependence of plots of bound spermatozoa1 plasma membrane the presence or absence of gossypol.

APRIL 1989 VOL. 39 NO. 4

huygn Ca -

441

CONTRACEPTION

The

ATPase

fashion.

Also,

direct

Gossypol which

is

been

bound

a

also

ram

and

with

bull

existing

lipid

from

may

shown

the

membrane

(9),

although

the

partially

to

10

!JM as

inhibit

Ca++-

activities

although

effect

the

compounds,

the

affect reducing may

plasma the

have

on

at

in

higher

Ca++-uptake

was

may

the

but

vesicles likely the

not

safely

apparent

right to

is of

concluded vesicles similarity

their that

is of

the

and

PC.

the

a

the

of

half where

a

number

system

proteins,

and

PC

lipids thereby

sites.

lipid-sensitive

and

understood

surrounding

Cat+-transport

gossypol

outer

Phospholipids

cell

binding

The

saturated

vesicles,

is poorly the

opening

limits.

transport

in

that

out

operate.

transporter

of

that

by

have

of

side

the

calcium,

possibility

to

the phospho-

critical

of PC may

The mechanism

accessibility

membrane

uptake

microenvironment

expected

in

removing

the

influence

(23).

the

and

beyond

conformation

the be

is

systems,

inhibited

the

add

Na-deoxycholate

dispersion

of

to

paper

by

structure

lipid

bilayer

shown

present

Ca++-transport

membrane

protein

been

membrane

442

Gossypol

of

Ca ++-ATPase

inhibited

vesicular

the

lipid

transport have

and

molecule

(9).

with testis

membranes,

the

of a sonicated

modified the

of

greatly

closed

addition

it

complex.

subunit

been

inhibitory

of

knowledge

Na-deoxycholate

and

penetrate

concentration,

and

plasma

the

in

similar has

to

polyphenolic

bind

catalytic

a

sperm

results

is

up the

of

the

At

to

the

substrate

molecules

activity

from

components.

(22).

uptake

and

shown

result

appears

and

protein

study, gossypol ++ Ca +Mg +:ATPase

The

of

with

non-competitive

membrane

enzyme

a polyaldehyde

cyclase

concentrations lost

molecule the

with

a to

with

bind

enzyme.

uptake,

appear

gossypol

conclusively

competitively,

our

may

in

lipophilic

readily

adenylate

purified in

of

and

bind

inhibited

inhibition

being

membrane

has

was

interaction

Gossypol the

activity

However, in

sperm

process inhibition

APRIL 1999 VOL. 39 NO. 4

CONTRACEPTION

of

ca++ -transport

the

drug

to

may

the microenvironment The is

more

The

of

associated

The

bulk

two

lines

also the

of

the

energy

involvement

(25).

Being

of gossypol viscosity active

rotational in

possibly a

in

lipid by

bulkier the

could

protein

lead

is

a

and

lipid out.

thereby

binding the

reduced

in the hydrocarbon

of

by

in

the

solubilization core

AEa

as

reflect implying

altering

an increase

in the

mobility

the

in

gossypol

the

increase

bilayer Such

loss

essentially

transmembrane

the

(24).

although

in the membrane

structure,

to

operate

However,

may

be

which

microenviron-

increasing

which

plane

be ruled

seemsto

non-linearity

there

proximity.

or

limiting

membrane

cannot

the

activation, slope,

lipid

and

intrinsic

phase,

transition,

show

of the molecule

on

protein

viscosity

of

by a steep

restriction

activity

and

and

proteins

that

thermal

phase

the

abolishes

implying

are parallel

evident

enzyme

gossypol

lipid

of

Ca++-

sensitive

lipid

integral

of

is the

enzymes of

of

modify

lipids

plot

temperature

bound

and

membrane

Arrhenius

changes

ability

limit.

expression

fluidity

expression,

the

affects

membrane

the membrane

presence

Arrhenius

is

linear

subtle

the membrane

in which

the

the

with

gossypol in

activity

most

to

components

a reasonable

of

in the

with

influences ment

beyond

enzyme

back

membrane

visible

the discontinuity property

traced

with

interaction

clearly

ATPase.

be

interact

of its

substrate in

micro-

presence in microof

the

(26).

REFERENCES 1.

2.

3.

Yanagimachi, R. In : Fertilization and embryonic development in vitro. Mastroianni,L.,Jr. and Biggers, J.D. (eds.),ppm Plenum Press, New York, 1981. Russel, L., Peterson, R. and Freud, M. Direct evidence for formation of hybrid vesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction in boar spermatozoa. J. Exp. Zool. 208: 41, 1979. ,SS:gh,iz,P., BabcoclFs, “dFco~p~n~nl;“ybfH.~,p~~~:~~~~~ influx of spermatozoa. Biochem. J. 172 : 549, 1978.

APRIL 1999 VOL. 39 NO. 4

443

CONTRACEPTION 4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

444

Paterson,

R.N.,

Ashraf,

M.

and

Russel,

L.D.

Effect

of Calmodulin antagonists on Ca++ uptake by boar spermatozoa. Biochem. Biophys. Res. comm. 114 : 28, 1983. Jones, H.P., Lenz, R.W., Palewitz, B.A and Cormier, M.J. Calmodulin localization in mammalian spermatozoa. Proc. Nat. Acad. Sci. (USA) 77: 2772, 1980. Chang, M.C.,Gu, Z. and Sakesena, S.K. Effects of gossypol on the fertility of male rats, hamsters Contraception 21 : 461, 1980. and rabbits. Kalla, N.R. and Vasudev, M. Effect of gossypol acetic acid on the motility and ATPase activity of human spermatozoa. Andrologia 13 : 95, 1981. Montamat, E.E., Burgos, C., Burgos, N.M.G., Rovai, L.E., Blanco, A. and Segura, E.L. Inhibitory action of gossypol on enzymes and growth of Trypanosoma 218 : 288, 1982. cruzi. Science Katherine, L., D.G.T., William, M.A. and Olgiati, William, A.T., Jr. Gossypol inhibition of adenylate cyclase. Arch. Biochem. Biophys. 231 : 411, 1984. Xiopeng, B., Yunjuan, Z., Huifen, Y. and Zhirong, Z. The on ATPase activity of effect of gossypol the kidney. Scientia Sinica 24 : 573, 1981. Haspal, H.C., Corin, R.E. and Sonenberg, M. Effect of gossypol on erythrocyte membrane function:Specific and interaction inhibition of inorganic anion exchange 234 : 575, with band 3. J. Pharmacol. Expt. Therap. 1985. S.N. Kanwar, U., Batla, A., Ranga, A. and Sanyal, Effect of Solasodine (C27H4302N) on the morphology, buffalo bull motility glycolytic enzymes of and spermatozoa. Ind. J. Expt. Biol. (In Press). of the Lowery Lees, M. and Paxman, S. Modification of proteolipid protein. procedure for the analysis 47 : 184, 1972. Anal. Biochem. Rufo, +$.A., Schoff, P.K. and Lardy, H.A. Regulation of Ca spermatozoa. J. Biol. -transport in bovine Chem. 259 : 2547, 1984. K. Minocha, R., Sanyal, S.N., Wali, A., Gumbhir, S. Effect of cholesterol and temperature and Majumdar, pertu;tations on membrane hydrolyses and transport and glucose in guinea pig brush border membrane of Ca Intern. 16 : 1019, 1988. vesicles. Biochem. Hopfer, U., Nelson, K., Penotto, J. and Isselbarker, isolated border K.T. Glucose transport in brush membranes from rat small intestine. J. Biol. Chem. 248 : 25, 1973. determiFiske, C.H. and Subbarow, Y. The calorimetric Chem. 66 : 375, J. Biol. nation of phosphorus. 1925.

APRIL 1999 VOL. 39 NO. 4

CONTRACEPTION

18.

19.

20.

21.

22.

23.

24. 25.

26.

Minocha, R., Wali, A., Sanyal, S., Sadana, T. and Majumdar, S. Phospholipid dependence of rat brain microsomal glucose-6-phosphate phosphohydrolase. Biochem. Intern. 14 : 483 1987. Huang, C.M. and Keenan, T.W. Preparation and properties of 5'-nucleotidase from bovine milk fat globule membranes. Biochim. Biophys. Acta 274 : 246, 1974. Bergmeyer, H.V. Determination in serum withp-nitroof Enzymatic Analysis, phenyl phosphate. In : Methods Press, New York, 1963. PP 783, Academic Breitbart, H., Stern, B. and Rubinstein, S. Calcium ++ -ATPase activity in ram spermatozoa transport and Ca Biochim. Acta plasma membrane vesicles. Biophys. 728 : 349, 1983. Breitbart, H., Arden, L. Rubinstein, S. and Nass Effect of acetic acid on Ca ++-transport gossypol and ATPase activity in plasma membranes from ram 7 : 439, 1984. and bull spermatozoa. 1nt. J. Androl. Spector, A.A. and Yorek, M.A. Membrane lipid composition and cellular function. J. Lipid Res. 25 : 1015, 1985. Sandermann, HY. Regulation of membrane enzymes by lipids. Biochim. Biophys. Acta 515 : 209, 1978. M. Glucose Yuli, I., Wilbrandt, W. and Shinitzky, cell membranes of modified lipid transport through fluidity. Biochemistry 20 : 4250, 1981. parameters Shinitzky, M. and Inbar, M. Microviscosity and protein mobility in biological membranes. Biochim. Biophys. Acta 433 : 133, 1976.

APRIL 1989 VOL. 39 NO. 4

445