The distribution of polyol:NADP oxidoreductase in mammalian tissues

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

THE DISTRIBUTION

OF POLYOL:NADP OXIDOREDUCTASE IN MAMMALIAN TISSUES

James P. Weaver and Albert I. Winegrad Rex S. Clements, Jr., George S. Cox Institute, Department of Medicine University of Pennsylvania, Philadelphia, Pennsylvania, Pa. 19104

Received August 21, 1969 SUMMARY Polyol:NADP oxidoreductase and L-gulonate:NADP oxidoreductase in the soluble fraction of rabbit and rat tissues can be separated and identified by a The validity of the method was vertical starch-gel electrophoretic technique. established by comparing the electrophoretic mobilities of partially purified polyol:NADP oxidoreductases and L-gulonate:NADP oxidoreductases with the bands of enzyme activity detected in the soluble fraction of the tissues from which they were isolated. Polyol:NADP oxidoreductase was found to be widely distributed in the tissues of the rabbit and rat; this suggests that polyol synthesis from aldoses may occur in many mammalian tissues. INTRODUCTION Polyol:NADP restricted seminal

oxidoreductase

(E.C.

1.1.1.21)

has been thought

to have a

It was known to be present in distribution in mammalian tissues. 1 4 2,3 but was thought to be absent from vesicles, lens, and placenta,

other

tissues.

lated

from brain,

However , polyol:NADP oxidoreductases have recently 6 7 5 7 Interest aorta, sciatic nerve and pancreas.

tribution

of this

enzyme derives

reduction

of glucose

and galactose

in part

from evidence

to their

respective

been isoin the dis-

suggesting polyols

that is

increased

related

to

the development diabetes

of pathological lesions in the lens and neural tissues in 8,9,10,11 and galactosemia. Cells containing polyol:NADP oxidoreduct-

ase (and in which rate

limiting)

abnormally

the

intracellular

are thought

certainty

soluble

to be a.t risk

if

of an aldose the plasma

substrate

level

is not

of the aldose

is

elevated. The distribution

with

transport

fraction

can contribute

of polyol:NADP

by demonstrating of tissues significantly

oxidoreductase

the NADPH dependent

since

L-gulonate:NADP

to the observed

347

cannot

reduction

be determined of aldoses

oxidoreductase

activity.

However,

(E.C.

in

the

1.1.1.19)

the --in vivo

Vol. 37, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

significance

of polyol

is uncertain

since

from

brain

the Km's

in

for

hexoses

are significantly 596 same tissues.

the

the reduction

of aldoses

this

activity

and pentoses

to determine

soluble

(Mano et.

of these

from rat

liver,

but

concentrations.)

of rabbit

the distribution

of the enzymes isolated

oxidoreductase

technique

oxidoreductase

fraction

oxidoreductase

than those of polyol:NADP oxido12 were unable to demonstrate al

electrophoretic

of polyol:NADP

in the

greater

at low substrate

starch-gel

and identification

by L-gulonate:NADP

by L-gulonate:NADP

only

A vertical

ductase

catalyzed

and aorta

reductase

assayed

formation

permits

the

and L-gulonate:NADP

and rat

tissues;

it

separation oxidore-

has been employed

two enzymes. METHODS

Adult, tissues

male,

to be studied

containing

weight

1:l.

The homogenates

Aliquots

was:

of the

sodium

employed.

The electrode

electrophoresis

nitroblue

detection that

buffer

(0.1

L-gulonolactone

the enzyme isolated

pared

from

the

Identical lactone

Polyol:NADP

liver

staining

obtained

gel

volume other

would patterns

length

reduced

(0.13 for

M),

four

348

tissues

prepared

pH 6.3

gel

(0.5

Ver-

at 4'C.

En-

in carbonate(70 mM), NADP (2 mM),

(0.13

mu) at 37'~

as substrate to permit 12 Mano et. al had reported only

mM).

to 22 cm was

hours

of the

oxidize

L-hexonic

were observed

was prepared

to

at 4'C.

was chosen since

pH 7.3

2-mercaptoethanol

methosulfate

from Nutritional

oxidoreductase

all

the

M),

30 minutes

L-gulonolactone

oxidoreductase

from rat

of buffer

to a 15% starch-gel

slices

containing

(0.05

and for

phosphate

thin

buffer

x g for

at 9 Volts/cm

(5 mM), and phenazine

of L-gulonate:NADP

lactones.

pH 9.6

lO:l,

containing

was sodium

by incubating

M),

lens

the

were decapitated;

The ratio

were applied

out

rats

phosphate

at 100,000

form with

buffer

in the dark.

their

5:l;

(33 mM), pH 6.3

was carried

tetrazolium

90 minutes

kidney

Buchler

was detected

tris

mM) at O'C.

(30 ul)

buffer

and Wistar

in

were centrifuged

constructed

bicarbonate

2:l;

supernatant

phosphate

zyme activity

(0.5

liver

A specially

tical

rabbits

were homogenized

2-mercaptoethanol

tissue

with

New Zealand

Biochemicals from rabbit

with

acids

L-gulonate

for the

and pre-

Corporation. lens,

sciatic

nerve,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

pancreas

and aorta

.was prepared

as previously

from rabbit

described.

kidney

L-gulonate:NADP

and pancreas,

and from rat

oxidoreductase

kidney

by the pro-

6 cedure these

reported

for

the rabbit

enzymes were:

8.0 mM; and rat

rabbit

kidney

aortic

enzyme.

kidney

The Km L-gulonolactone

30 mM; rabbit

pancreas

of

2.0 mM; rabbit

aorta

5.3 mM. RESULTS

Two distinct, oxidizing

activity

the soluble served

the

of rabbit

in the absence

1:

(Figure

anodal

electrophoretograms

I)

Neither

or L-gulonate.

as polyol:NADP

band (Band II)

L-gulonolactone

band was ob-

The slowly

oxidoreductase,

as L-gulonate:NADP

of

migrating

and the more

oxidoreductase

from

observations. oxidoreductase

prepared

had the same electrophoretic

Band I demonstrated

Figure

in the starch-gel

of L-gulonolactone

Polyol:NADP and pancreas

bands of NADP linked

tissues.

has been identified

migrating

following

migrating

were demonstrated

fraction

band (Band I) rapidly

anodally

in the soluble

from

mobility

fraction

rabbit

lens,

in the starch-gel

of the

same tissue.

Diagram of vertical starch-gel electrophoretograms NADP linked L-gulonolactone oxidizing activity in fraction of rabbit tissues.

(A) (B) cc> CD) (El

Polyol:NADP oxidoreductase from Polyol:NADP oxidoreductase from Polyol:NADP oxidoreductase from L-gulonate:NADP oxidoreductase L-gulonate:NADP oxidoreductase

The cathodally migrating not been characterized, of substrate.

it

activity is not

349

sciatic

observed observed

nerve, system

(Figure

as

1)

of the the soluble

rabbit lens. rabbit sciatic nerve. rabbit pancreas. from rabbit kidney. from rabbit pancreas. in rabbit lens in the absence

has

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

L-gulonate:NADP ney had the fraction

same electrophoretic

of rabbit

pancreas

Polyol:NADP separated the

only

fraction

1, only nerve.

There

was also

the starch-gel from

the

nerve

Figure

IO

a good correlation

from

in

2:

60

steps

the

Both 2),

can be

employed

in

ammonium

on DEAE-cellulose,

the column.

oxidoreductase

(Figure

the soluble

oxidoreductase

the starch-gel

between

and by enzyme isolation.

20 30 40 50 Fraction Number

in

and kid-

1).

was chromatographed

I

0

demonstrated

- one of the

was recovered

by enzyme isolation

pancreas

When the 40 to 80% saturated

and L-gulonate:NADP

method

tissue

sciatic

rabbit

and L-gulonate:NADP

Band I was present

sciatic

NADP oxidoreductase

(Figure

5,6,7

oxidoreductase

shown in Figure of rabbit

enzymes.

from

as Band II

on DEAE-cellulose

of rabbit

polyol:NADP

mobility

oxidoreductase

of these

prepared

and kidney.

by chromatography

isolation

sulfate

oxidoreductase

(Figure

2)

As

electrophoretogram

identification in rabbit

of polyol pancreas

by

enzymes were recovered

and both

Band I and Band II

Rabbit Nerve

I

70 Fraction Number

The desalted 40 to 80% saturated (NH4)2 SO4 fractions (prepared as in reference 7) were pumped onto 2 x 25 cm DEAE-cellulose columns previously equilibrated with potassium phosphate buffer (5 mM) pH 6.8; the columns were washed with 200 ml of the same buffer, and developed with a linear gradient (700 ml) of the Ten ml fractions were collected. same buffer (5 m M to 25 mM). Enzyme activity was assayed spectrophotometrically at 340 w in 1.0 ml of phosphate buffer (67 mM) pH 6.2 containing NADPH (0.1 mM) D-xylose (0.3 M) plus Li2 SO4 (0.4 M) indicated by and either: or D-glucuronate (20 mM) indicated by closed circles. open circles, (The polyol:NADP oxidoreductase Assays were carried out at 30°C. peaks were subsequently chromatographed on Sephadex G-1OO).7 350

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

were present

in the

Arsenis reductase chondria

and Touster

(E.C.

1.1.1.10)

in guinea

contributes

starch-gel

pig

is liver.

to the activity

reductase

prepared

electrophoretogram. have recently

(Figure

reported

that

1)

xylitol:NADP

oxido-

present in the cytosol as well as the mito13 However, it is unlikely that this enzyme of Band I or Band II,

from acetone

powder of whole

since guinea

xylitol:NADP oxido14 pig liver (which is

13 largely gel

cytoplasmic

was found

in origin)

to migrate

cathodally

in

the starch

system. Table

tissues

I summarizes

as determined Table

II

starch-gel

by starch-gel

summarizes

and L-gulonate:NADP

the distribution

electrophoresis

the distribution

oxidoreductase

electrophoresis.

in rat

and L-Gulonate:NADP

Tissue Lens Kidney Liver Brain Sciatic Nerve Pancreas Adrenal Jejunum Adipose Tissue Diaphragm Heart Aorta

enzymes in rabbit and enzyme isolation.

of polyol:NADP tissues

The electrophoretic

Table Distribution

of these

as determined mobilities

oxidoreductase by vertical of Band I and

I

of Polyol:NADP Oxidoreductase

Polyol:NADP Oxidoreductase + + + + + + + + + + + +’

Oxidoreductase in Rabbit

Tissues

L-Gulonate:NADP Oxidoreductase

* + + + * *

*

*

+ * + + + + + +’ *

* The observation of a definite band of enzyme activity in the starch-gel electrophoretogram of a tissue is indicated by +; an asterisk (*) indicates that the enzyme has also been isolated from the tissue as indicated in the Neither enzyme could be demonstrated text and references 6 and 7. (+'I in the unconcentrated soluble fraction of aortic homogenates, but polyol: NADP oxidoreductase and L-gulonate:NADP oxidoreductase isolated from rabbit aorta6 had the appropriate electrophoretic mobilities. 351

Vol. 37, No. 2, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Distribution

of Polyol:NADP

and L-Gulonate:NADP

See Table

I for

extensive

explanation

soluble

fraction It

every

tissue

will

rat

and rabbit

tissues

tissues

run in

the

same gels.

bands observed

in

has not been undertaken,

was shown to have the

of rat

*

+ 4 -I-I+ +

of the anodal

of rat

oxidoreductase

+ + +

of symbols.

identification

electrophoretograms

L-Gulonate:NADP Oxidoreductase

+ + + + + + + + + + + +

were the same in

ate:NADP

in Rat Tissues

Polyol:NADP Oxidoreductase

Lens Kidney Liver Brain Sciatic Nerve Pancreas Adrenal Jejunum Adipose Tissue Diaphragm Heart Seminal Vesicles

though

Oxidoreductase

Oxidoreductase

Tissue

Band II

II

rat

same mobility

the

Alstarch-gel

kidney

L-gulon-

as Band II

in

the

was demonstrated

in

kidney.

be noted

that

:NADP oxidoreductase

polyol

studied. DISCUSSION

These studies tributed it

in mammalian

has previously

this

8 the

lens,

that Recent

glucose

that

tissues

other

polyol:NADP

and is not

been observed.

enzyme in tissues

uncertain.

suggest

than

oxidoreductase

restricted

The physiologic the

seminal

to the

is widely few tissues

substrate

vesicles

(where

concentrations However, the presence of significant 6 10,ll 15 10 kidney, and aorta of normal brain, nerve, may be an important

observations

suggest

that

substrate the

sorbitol 352

for

in which

or substrates it

is

glucose)

of sorbitol animals

the enzyme in these content

dis-

of the aorta

is in

suggests

tissues. is

for

subject

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 37, No. 2, 1969

6 to hormonal rabbit

lens,

properties

sciatic

from aldoses,

may be a normal process

nerve,

of a regulatory

formation

of substrate

in

and pancreas 7 It enzyme.

and in particular

metabolic

may be subject

, polyol:NADP

In addition

regulation.

activity

oxidoreductases

have been found is

therefore

sorbitol

to regulation

by factors

to have some of the

possible formation

in many mammalian other

from

that

polyol

from glucose,

tissues,

and that

this

than

the concentration

the plasma. ACKNOWLEDGEMENTS --

This work was supported in part by USPHS Grants AM 04722, AM 05556, We thank Mrs. M. A. Fletcher and Mrs. J. C. Feuer forand GM 06405. expert assistance.

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