Demonstration of A DPNH: α-Ketoglutarate oxidoreductase activity in embryonic chick liver supernatant fraction

BIOCHEMICAL

Vol. 21, No. 6, 1965

DEMONSTRATION

OF A DPNH: EMBRYONIC

a-KETOGLUTARATE

CHICK

BERTRUM INSTITUTE

OF

COL~~MBIA

AND BIOPHYSICAL

LIVER

SHEID

CANCER

RESEARCH

UNIVERSITY,

November

IN OF

HIS

AND

WITH

WITH AN

THE

PH

ACID

TRACTABLE PROMPTED

A

I D IN

HI

THE

ACTIVITY

AT

WERE

CHICK ENZYME

WERE

OPTIMUM

CONDITIONS

ASSAY

SIDERED

A

* SERVICE NATIONAL

TO

GLUTAMIC

SUCROSE

MAMMALIAN

LIVER

DIGITONIN IN

ACTIVITY

FRACTION.

MITOCHONDRIA,

PH

A

(GDH)

GDH

ADDITION,

ONE

-ET

DEVELOPING

WITH

FRACTIONS

DEHYDROGENASE

(HIRSCHBERG

THE

BOTH

FRACTION, 5.2.

AND

EACH

AL -a*

CHICK

OPTIMUM

AND

THIS INVESTIGATION TRAINING GRANT

FRACTION

OF

EX-

1964), Two

LIVER.

7.5

8.2

-

ACTIVITIES AT

OF

VARIOUS

OF AND

THE

COMPONENT

(DE

DUVE

WAS CA

SUPPORTED AND

5011

INSTITUTE

601

IN

METHODS

FACT -ET

CONTAINED

GLUTAMIC-

A

IN

VIEW

THAT AL ,a*

BY UNITED RESEARCH

(GOT)

THREE THE

ENZYMES

FOR

SUBSTRATE

GDH

1962)

OF

ALL

TO THE

IS

AT

DEVELOPMENT

USED

AS

DPNH.

RESULTS,

ALL

POINTS

a-KETOGLUTARATE

OXIDATION

No.

ALSO

DEMONSTRATED

TRANSAMJNASE

SPECTROPHOTOMETRIC

THE

GDH

FOR

GLUTAMIC-OXALOACETIC

OTHER

EMPLOYED

MEASURED

ASSAYS

THIS

SPECIFIC

STANDARD

MITOCHONDRIAL

CANCER

THE

ENZYME

OBSERVED,

THE

DETERMINATIONS

THE

THIS

OBSERVED

SUPERNATANT

WITH

OF

PH.

THE

AND

SUBCELLULAR

IN

OTHER

SUPERNATANT

(GPT)

THIS

REACTIONS

THE

IN

IN

THE

IN

1965).

COMPARABLE

EMBRYO.

SURGEONS

MITOCHONDRIA.

ISOZYMES

OF

RSCHBERG,

PH

IN

REPORTED

GDH

AND

TRANSAMINASE

MEASURABLE

5.2

WATER

A

BIOCHEMISTRY,

AND

YORK

ENZYMES

(1959)

7.6

AT WAS

TWO

SUCROSE

WITH

PYRUVIC

OPTIMUM

ISOZYMES

AND

OF

PHYSICIANS

OF

SOLOMON

RE-EXAMINATION

MITOCHONDRIAL

HIRSCHBERG

NEW

DISTRIBUTION

OPTIMUM OF

WITH

THE

LIVER,

PH

A

RECOGNITION

(SHE

OF

CHICK

ACTIVITY

YORK,

IN

FRACTION*

DEPARTMENT

OF

ACTIVITY

17, 1965

STUDY

EMBRYONIC

ERICH

COLLEGE

NEW

Received

OXIDOREDUCTASE

SUPERNATANT

AND

RESEARCH COMMUNICATIONS

STATES PUELIC GRANT No. CA

OF

THE

THREE

INITIATE

SIMILARITY

GENERALLY

MOST

PH

ACTIVE

OF CONAT

HEALTH FROM

02332

THE

PH

THE

Vol. 21, No. 6, 1965

VALUES

ABOVE

FURTHER

NEUTRALITY,

THE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NATURE

OF

THIS

ENZYME

ACTIVITY

APPEARED

WORTHY

THIS

COMMUNICATION,

DEHYDROGENATION

MATE

IN

THE

DIRECT PRESENCE

EVIDENCE

OR

CHICK

IS

TRANSAMINATION

LIVER

ENZYMATIC

OF

AGAINST

PATHWAYS

THE

FROM

THE

PARTICIPATION

a-KETOGLUTARATE

5.2

FRArTION

AT

PH

SUBSTRATE

TO

a-HVDROXYGLUTARATE

SUPERNATANT

CONVERSION

PRESENTED

BUT,

OF

TO

INSTEAD,

GLUTA-

FOR IN

THE

THE

DPNH.

OF

METHODS SPECTROPHOTOMETRIC FROM

POOLED

0.25M

1950).

MEASUREMENTS:

EMBRYONIC

A

WATER

BY

GDH

NH&*,

ENZYME,

DATION FIRST

FEW

THEN

MINUTES.

GOT

KARMEN

(1955)

METHODS

OF

GOT

WAS

ALSO

GPT

ACCORDING

ASSAYS

WAS

ESTIMATED To

WAS

A

AND

PHOSPHATE

TO

THE

PH,

WERE

ESTABLISHED

BY

WAS

ASSAYED

WROBLEWSKI

1952)

CONSISTED

AFTER

10

ADDED

WITH

(1956)~

LADUE

(1953).

THE

OBTAINED

FROM

DPNH,

OF

MIN.

AT

ACCORDING

VARYING

HOGEBOOM,

INCUBATION, MIXING.

310

THE Mp

FOR

SPECTROPHOTOMETRICALLV

AND

MCHENRV

IN

AND WERE

SPECTROPHOTOMETRICALLY

GPT

AND

(MD)

PREPARED

1965).

BUFFER.

COLORIMETRICALLY

CALDWELL

DEVELOPMENT

EXTRACT

ANFINSEN,

PROPER

FOLLOWED

AND

DURING (SCHNEIDER

HIRSCHBERG,

OR

AND

WERE

STAGES

DIGITONIN

AND

(OLSON

ADJUSTED

DPNH

OF

(SHEID

ACETATE

a-KETOGLUTARATE,

VARIOUS

FRACTIONS

CENTRIFUGATION

AND

MIXTURE

AND

AT

DIFFERENTIAL

PELLET ASSAY

SUBCELLULAR

LIVER

(Mw)

EXTRACT

MITOCHONDRIAL THE

CHICK

(1:lO)

SUCROSE

THE

TO THE

AMOUNTS

OF

SHEID

LINEARITY ENZYME

OXITHE

BY

THE

RESPECT

I VELY.

ET

(1965)

AL. OF

THE

EXTRACTS

AND

ENZYME

IN

INITIAL

EXPERIMENTS. CHROMATOGRAPHIC IN

0.1!

AND

METRIC

MANNER

105,000

CENTRIFUGED

OXIDATION

OF AS

PH 5.2.

BUFFER, AT

ASSAYS

DPNi-i

X

AT

THAT PH

THE

G

BRIEFLY

SHOWED

SUCROSE

EMBRYONIC

DETERMINATIONS:

ACETATE

CENTRIFUGATION ICE,

OF

INVESTIGATION. iN

THE

BIOCHEMICAL

WAS TO

THE 5.2

SUPERNATANT

SUPERNATANT

HEATED REMOVE

RESULTING IN

THE

CHICK

FOR

FRACTION

5

MINUTES

COAGULATED

FRACTIONS.

OF THE

602

WAS

AT

FRACTION

65Oc,

EXTRACT

BY COOLED

IN

SPECTROPHOTOCATALYZED

a-KETOGLUTARATE ENZYME

HOMOGENIZED

OBTAINED

PROTEIN.

SUPERNATANT

PRESENCE

LIVER

IN WAS

THE DILUTED

THE SAME

Vol. 21, No. 6, 1965

I:10

0.2

AND

BIOCHEMICAL

ML

PH 5.2,

BUFFER,

TABLE

1.

AND BIOPHYSICAL RESEARCH CO~VWJN~CATIONS

WAS

INCUBATED

WITH

AND

9 pM

FOR

DPNH

THE EFFECT OF GENASE ACTIVITY

AMMONIUM IN THE

TONIN

EXTRACT

(MD))

CHICK

LIVER.*

PH

FRACTION

OF

90 6

FM

a-KETOGLUTARATE,

HOURS

IN

A

ION ON TRUE MITOCHONDRIAL

AND

VOLUME

/J,M

IV-DAY

MITOCHONDRIA

(Mw) (Mw)

MITOCHONDRIA

(Mw)

MITOCHONDRIA

(Mw)

MITOCHONDRIA

(MD) (MD) (MD) (MD)

MITOCHONDRIA MITOCHONDRIA MITOCHONDRIA

5.2

** t

THE SAMPLE

AT

EASTMAN

WATER

TWO

THIS

THE

50%

(12:3:5)

IN

ASCENDING

BY

BROMCRESOL

OF

OR

300 320 0 0

DUPLICATE

CHICK

NH4+,

I~~/MIN/MG

LIVER

ADDED

GEL

SHEET

SPOTS

ADDED,

ONS

T RIC

WAS

APPLIED PAPER,

THE

THE TO

WHATMAN

TYPE

K301R.

P-DIMETHYLAMINOBENZALDEHYDE.

603

IDENTIFIED

0.15M. RESULTS ON

REACTION

WERE

I&DAY HATCHING.

ARE

WAS

1

STOPPED AND

FILTER

BUTANOL-ACETIC

EMPLOYED WITH

12

DAYS.

ESSENTIALLY

CENTRIFUGED, NO.

AN

SEPARATE

INDICATED

MIXTURE

(16:1:3) WERE

THE

ASSAY

OF AFTER

PERFORMED

COENZYME. ACID,

FRACTION

DAY

NI TROGEN.

SPECTROPHOTOME THE

ASSAYS ONE

WHERE

DETERMINAT

ETHANOL-AMMONIA-WATER

CHROMATOGRAPHY. GREEN

x

TRICHLORACETIC

SILICA

OR

OF E

IN

OF

DUPLICATE

SUPERNATANT

ACTIVATED

OBTAINED AND

TIME,

DISAPPEARANCE

OF

WERE LIVER

ACTIVITY: OF

2950

+

CONCENTRATION

FINAL

OF

0 0

-I-

THE

ADDITION

+

5.2 5.2 7.6 7.6

CHICK

COMPLETE

540 0

0

RESULTS

TEMPERATURE.

+

+

EMBRYONIC

AVERAGE

0

7.6 7.6

ANALOGOUS

SPECIFIC

+

;:;

SUPERNATANT

*

ACTIVITY

0

7.6 7.6

SUPERNATANT SUPERNATANT

ROOM

(AVERAGE)t

5.2

SUPERNATANT

AT

EMBRYONIC

SPECIFIC

MIXTURE

MITOCHONDRIA

ML.

GLUTAMI~ DEHYDRO (Mw) AND DIGI-

OF

ADDITION**

ACETATE

3

OF

ARTIFACTUAL (WATER EXTRACT

FRACTIONS

NH 4 +

ASSAY

FINAL

AND

SUPERNATANT

100

As VAPOR,

BY A

PAPER ACID-

SOLVENTS FOLLOWED

OR

BIOCHEMICAL

Vol. 21, No. 6, 1965

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS TABLE SERVED

1

SHOWS

ACTlVfTY

IN

FRACTIONS

WERE

DIFFUSION

TECHNIQUE

OR

THAT

GDH

STIMULATED

GDH

ACTIVITY

EFF-ECT

ON

CONFIRMED BUT

IN

WAS

THE

NOT

RIBOSOMAL SUPERNATANT LIVER,

RAT

HEPATOMAS.

FRACTIONS BUT

PHOTOMETRIC ISONIAZID,

THE

OF

AN

THE

HAD

ENZYME

OF

RAT

DEMONSTRABLE

7.6

BY

EITHER

EASED

ON

OF

ANTAGONIST

GPT

PYRIDOXAL

AND

ON

THE

(TOMKINS

SUPERAND

BUT HAD

A

ONE-EIGHTH

NOT

IN

THE

STIMULATORV

EMBRYONIC

NOT

(1957)

ASSAY

NOT

LIVER,

AND

THE

MITOCHONDRIAL

THE

PHOSPHATE

AT

WAS

CONDITIONS

(BRAUNSTEIN,

FOUND

AND

THE OR

NOVIKOFF

SUPERNATANT METHODS;

OXIDATION

THE

IN

MOUSE

COLORIMETRIC

FURTHER, 5.2.

MITOCHONDRIAL,

WAS

7795

INVOLVING

PH

ACTIVITY

ACTIVITY

EMBRYONIC

AND

PH 5.2.

ACTIVITY

OF

OR

REACTION

THE IT

MORRIS

SPECTROPHOTOMETRIC

AT

IN

LIVER.

BUT

THE

METHOD. APPEAR

CHICK

LIVER,

604

THIS

(1952) DID

OF

7.6.

PH

AT

SOLOMON’S

5.2

ACTIVITY

INDICATED DF

PH

A COUPLING

GOT

OF

ADENOSINEDIPHOSPHATE

ABOUT

FRACTION

IN THE

EFFECT

(EDTA)

ACTIVITY,

MOUSE

THAN

EXTRACT.

DUCK

OR

LESS

FRACTIONS,

OLSON-ANFINSEN AT

SUPERNATANT

ACTIVATORS

377oC

AT

OB-

CONWAY

MITOCHONDRIAL

NO

MITOCHONDRIAL

UNDER

EMBRYONIC

MEASURABLE ASSAY

GDH

THE

AND

THE

THE

I.E.

ACTIVITY.

SUPERNATANT

FRACTIONS

FORMER,

INDICATED

WITH

EXPERIMENTS

CHICK,

PH

THE

THE

MEASURABLE

READILY

AT

ONLY

DPNH,

WAS

IN

(DES)

BOTH

LOW

WITH

NUCLEAR

ADULT

ACTIVITY

MITOCHONDRIAL

PRESENT

FRACTION

RAT

GOT

IN

ACTIVITY OR

IN

REPORTED

MEASURABLE

ENZYME

THE

WITH

NEGLIGIBLE,

INHIBITORS

MITOCHONDRIAL

DIGITONIN

4.5-4.8,

PH

AT

ON

KNOWN

IN

FOR

MlTOCHONDRfAL

ASSAY

NH3

OF

WERE

BOTH

AN

ETHYLENEDIAMINETETRAACETATE

(1959)

SOLOMON FOUND

THE

OF

PREINCUBATED

INHIBITED

FRACTION.

ONLY

WHEN

BUT

SUPERNATANT

7.6

OR

EFFECT

1961)

AMOUNTS

NH,I++

NO

CONTRAST,

NH&+.

OF

THE

PH 5.2

REQUIRED IN

ADDITION

DIETHYLSTILBESTROL

EVEN

1961).

YIELDING,

5.2.

PH

THAT

THE

FRACTIONS.

ACTIVITY

THE

AT

YIELDING,

FRACTION

AT

ESTABLISHED

AND

SUPERNATANT

(ADP)

UPON

ILLUSTRATES

(TOMKINS

NATANT

ASSAY

FRACTIONS

2

DISCUSSION

SUPERNATANT

GDH

THE

DEPENDENT

MITOCHONDRIAL

TABLE

THE

AND

ONLY

OF THE

ADDITION

1960)

SPECTROOF

AT

A

FINAL

BIOCHEMICAL

Vol. 21, No. 6, 1965

CONCENTRATION

OF

SUPERNATANT

1

&-~,

FRACTION

ADDITIONAL IS

X

RESULTED

AT

PH

EVIDENCE

PRESENTED

IN

7.6,

3.

LIVER

WAS

INCUBATED

AT

PH

TABLE

2.

RESPONSE

OF

TRUE

ACTIVITY

TO

KNOWN

THE

5.2

HAD

THE

WHEN

NO

EFFECT

THE

GDH

OF

FRACTION

a-KETDGLUTARATE

ART~FACTUAL

INHIBITORS

AND

GLUTAMIC AND

GOT

OF ON

INVOLVEMENT

PH

FRACTION

RESEARCH COMMUNICATIONS

INHIBITION

SUPERNATANT

WITH

AND

95%

IN

BUT

AGAINST

TABLE

AND BIOPHYSICAL

ACTIVITY

ACTIVITY

IN

AT

OR

THE

OF

EMBRYONIC

DPNH,

NO

PH

THE

5.2.

TRANSAMINASES CHICK

GLUTAMATE

DEHYDRDGENASE

ACTIVATORS.

ADDITION

SPECIFIC

ACTIVITY

(AVERAGE)*

MITOCHONDRIA

(Mw)

MITOCHONDRIA MITOCHONDRIA

(Mw) (Mw) (Mw)

MITOCHONDRIA

MITOCHONDRIA

(MD)

MITOCHONDRIA

(MD)

MITOCHONDRIA

(MD) (MD)

MITOCHONDRIA

7.6 7.6 7.6 7.6

540 5 x 1 x 5 x

7.6 7.6 7.6 7.6

1 x 5 x

7.6

::2 5.2 ::: 5.2

SUPERNATANT

AT

37%

SUPERNATANT SUPERNATANT

*

AVERAGE RESULTS LIVER

OF DUPLICATE WERE OF THE FROM

CHICKS

SUPERNATANT

WAS

FORMED.

INDICATED

BY

SOLVENTS.

GLUTARATE IS

ENZYMATIC.

REVERSE AND

AFTER

DPN-,

WERE

REACTION PHENAZ

DAY IN

PRODUCT

METHOSULFATE,

NHL;t

HATCHING.

THESE

DETERMINATIONS

ON

SHORTER IN

THE

BOILED

TIME

WAS

NOT

ADDED

AND

TO

THE

ENZYME NO

1937),

US I NG METHYLENE

605

a-HYDROXYGLUTARATE,

TWO

SUBSTRATA

PERIODS,

BOTH

MIXTURE.

EXPERIMENTS,

OR

WAS

THE LIVER

EXPERIMENTS.

REACTION

WITH

(WEIL-MALHERBE, I NE

330 320 320 330 300

DES DES DES EDTA ADP

THE

PRESENT

PRELIMINARY

AFTER

OF

FOR

INCUBATION IN

11000

VALUES FROM TWO SEPARATE EXPERIMENTS. SAME ORDER FOR l&DAY EMBRYONIC CHICK

INCUBATION

a-HYDROXYGLUTARATE

IO-4i

1 x IO-3i

CHROMATOGRAPHIC

AFTER

0

7000

EDTA ADP

5 x IO-4"

ONE

THE

10-3;

5 x IO-5M 1 x IO-4i 1 x IO-4i

FRACTIONS

RATHER,

5; 1060

2450

SUPERNATANT SUPERNATANT

DES EDTA ADP

5 x IO-5M DES

SUPERNATANT SUPERNATANT

10-5M IO-3ii 10-4"

THE

AND

WITH

ABSENCE

OF THAT

EVIDENCE

WAS

OBTAINED

a-HYDROXYGLUTARATE ELECTRON

AND

a-HYDROXY-

SHOWS

AS

TWO

a-KETOGLUTARATE

EXTRACT

BLUE

AS

THE

REACTION FOR

AS

THE

SUBSTRATE

ACCEPTORS

AT

Vol. 21, No. 6, 1965

PH

5.2

7.6.

OR

INVOLVED

IN

TABLE

3.

THE

THE

RF

BIOCHEMICAL

EXACT

PRESENT

VALUES

LIVER

NATURE

AFTER

GLUTAMATE GLUTARATE

OR

MIXTURES

SILICA

GEL

0.36, 0.21, 0.79,

0.37 0.22 0.82 0.13 0.79 0.40

0.10,

IS~CITRATE

0.71, 0.43, 0.54, 0.32, 0.36,

LACTATE

0.64,

PY RUVATE

A) B)

MALATE

MIXTURE

0.36, 0.21.

A:

BUTANOL-ACETIC

BLANK

SOLVENT

TO

PAPER

AND

OF

BE

OXIDOREDUCTASE

ESTABLISHED.

CHROMATOGRAPHY

STANDARD

OF

SINGLE

EXPERIMENT

MORE

OF

PYRUVATE

AND,

AND

LACTATE

THE

ORIGIN

0

0.21: 0.22,

0.27 0.22

0

ARE

(12:3:5);

SOLVENT

WITH

THE

0

A.

0.49

THE

SOLVENT.

ETHANOLSILICA PATTERNS

(1960).

BOILED

THE

ENZYME

SEPARATE

0

SPOTS

INDICATES

GEL

AND

SEPARATIONS

SMITH

BY WITH

OF

B: ON

CHROMATOGRAPHIC

TWO

VALUE

0.32 0.13

SOLVENT

PRESENTED

YIELDED

RF

0.61 0.72

EXPERIMENTS EACH

MIXTURE

LACTATE

AN

TWO FOR

DATA

INCUBATION

B.

OF

GIVEN

WITH

0.30 0.30

0 0.62

0.59: 6) 0.50, A)

ACID-WATER PAPER

0.30 0.12 0.41

0.16

0.20

RESULTS

PAPER

0

0.10

SOLVENT I NG

0

0.80 0.52 0.61 0.40 0.48 0.73

0.11,

ENTIRE

FOLLOW

0.10

0.19,

PROBABLY, IN

0.22 0.08 0.29

0.28

ON

THE

0.19, 0.09, 0.25,

0.44

AGREEMENT

CONSISTED

GEL

0.36

SATISFACTORY

ESSENTIAL

SILICA

0.24.

THE

B

SOLVENT

0.45 0.27 0.88

0.35 0.36 0.68

CHICK

COMPOUNDS.

PAPER

0.51

(16:1:3).

AMMONIA-WATER

IN

REMAINS

a-KETOGLUTARATE

SOLVENT A ------------------------------------------------------

a-HYDROXYGLUTARATE a-HYDROXYBUTYRATE

WERE

DPNH:

THE

THIN-LAYER

INCUBATION

a-KETOGLUTARATE

A

OF

EXPERIMENTS

SAMPLE

INCUBATED

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

ARE

BLANK

EXTRACT. IN

SOLVENT

SPOT

A

REMAINING

AT

CHROMATOGRAPHY.

REFERENCES

A.

BRAUNSTEIN, (EDITORS), DEDUVE, HIRSCHBERG,

C.,

IN:

NEW

E.

CALDWELL,

E.,

F.,

“THE

YORK:

AND

ENZYMES”,

P.

ACADEMIC

MCHENRY,

E.

W.,

ARCH.

R., D.,

AND BAUDHUIN, AND OSNOS,

M.,

WATTIAUX, E., SNIDER

D.

H.

BOYER,

1960,

PRESS,

BIOCHEM.

P.,

BIOPHYS.

ADVAN. ADVAN.

K.

AND

PART

P.

Q,

ENZYMOL,. &, ENZYME REGULATION

SHEID,

J. CLIN. INVEST. 2, 131 (1955) A., AND ANFINSEN, C. B., J. BIOL. CHEM. m, W. C., AND HOGEBOOM, G. H., J. BIOL. CHEM. B., AND HI RSCHBERG, E., SUBMITTED FOR PUBLICATION B., MORRIS, H. P., AND ROTH, J. S., J. BIOL.

SMITH,

I.,

MYRBAECK

150 97 (1953) 291 (1962) 2, 301 (1964)

A,

A.,

KARMEN,

67 (1952)

J.

OLSON, SCHNEIDER,

SHEID,

LARDY,

2,

VOL.

“CHROMATOGRAPHIC

INTERSCIENCE SOLOMON,

J.

SOLOMON, TOMKINS,

J. G.

WEIL~ALHERBE, WROBLEWSKI,

B., B., M.,

AND

PUBLISHERS, BIOCHEM.

J.

66,

DEVELOP. BIOL. AND YIELDING,

H., F.,

BIOCHEM. AND LADUE,

CHEM.

123

(1950)

(1965) 2&, 3016 NEW

TECHNIQUES”,

(1965)

YORK:

1960, VOL. I 261, (1957) 1, 182 (1959) K.

J. J.

ELECTROPHORETIC

m,

L.,

21, S.,

COLD

2080 PROC.

SPRING Sot.

606

HARBOR

SYMP.

26,

331 (1961)

(1937) EXPTL.

BIOL.

MED.

a,

569 (1956)