Histochemical studies of phosphatases separated by starch gel electrophoresis

174 HISTOCHEMICAL BY

STUDIES STARCH

OF PHOSPHATASES

GEL

M. SANDLER’ Department

of Anatomy,

SEPARATED

ELECTROPHORESIS’ and G. H. BOURNE

Emory Received

University, March

Atlanta,

Ga.,

U.S.A.

27. 1961

T HE

question of specificity of enzyme activity of the alkaline phosphatases has been the subject of many papers [i, 3, 71. The histochemical distribution of enzymes dephosphorylating /I-glycerophosphate, adenosine triphosphate, thiamine pyrophosphate, pyridoxal phosphate, glucose-6-phosphate and numerous other high and low energy phosphate esters have been reported from this and other laboratories [2, 8, 91. Heretofore the question of specificity has been confined to studies of histochemical preparations or biochemical determinations on whole homogenates. The comparison of histochemical results with biochemical results has in the past been difficult in that there was no simple way of studying the multiple enzyme systems or the effect of fixation procedures, embedding, etc.; the latter have been studied with regard to the percentage of activity lost, disregarding the question of the isozymes (term suggestedby Markert and Moller [6] to describe the different molecular forms in which proteins may exist with the same enzymatic specificity). Which of the isozymes is losing activity in what procedure is a question that to date remains unanswered. The use of starch gel to separate protein mixtures has been described [lo] and the demonstration of non-specific alkaline phosphatases using naphthol salts has been describedby Hunter and Burstone [4]. To our knowledge the demonstration of specific phosphataseson starch gel has not been described in the literature. The use of sodium alizarin sulphonate (alizarin red S) as a stain for the calcium deposited in the Gomori alkaline phosphatasetechnique was first proposed by Bourne 111.He added alizarin to the incubation mixture. This forms a “lake” with the calcium ions in the mixture and the calcium deposited as the phosphate salt as a result of phosphatase activity was then pink in colour and could be localized under the usual optical microscope. As a histochemical technique this was not very satisfactory, as there was a great deal of background staining and the intracellular localization was not very precise. In the demonstration of specific phosphatases on starch gel, after trying many techniques, we are of the opinion that the use of alizarin red S in very small amounts (10 mg/lOO ml of incubation mixture) is the best method available at the present time for the demonstration of specific phosphatases. The results of preliminary trials with human serum showed that with sodium B-glycerophosphate the bands of enzyme activity differed from those obtained with 1 Supported by grants from the National Heart Institute Health (B-2038) and the Muscular Dystrophy Associations 2 U.S.P.H. Fellow in Cardiovascular Stud-ies. Experimental

Cell Research

24

(H-1553), of America

the National tnc.

Institutes

of

Historhemistry

Fig. I.-7’iswe: tl, calf intestinal

(I, Human serum; alkaline phosphatasc.

Suhstrule: Sodium a-naphthol acid

phosphate:

and sodium

h, human

acid phosphate:

by olecirophorosis

serum:

c. calf

intestinal

li5

alkaline

sodiutu S-glyceroptlosphite:

phosphatase:

ant1

xotliurn *-naphlhol

,!-glycerophosphate.

sodium a-naphthol acid phosphate used as the substrate. These techniques have also been applied to the investigation of the phosphatases present in various tissues. Results indicate the presence of at least five separate alkaline glgcerophosphatases in the rat liver. \\‘e have also differentiated adenosine triphosphatase and &glycerophosphatase in rat muscle with the aid of starch gel electrophoresis. It is of interest that a commercial preparation of calf alkaline phosphatasel showed an identity between the bands of enzyme activity obtained with the alizarin technique and the azo dye technique. \\‘e intend to extend our investigations to study the effects of fixation, hot paraffin ’ (California

Riochemicals

Corp.

176

31. Sander

Fig. 2.-Tisstre: f, rat liver. Substrate:

n, Rat

Xdenosine

heart:

triphosphate;

6, rat

muscle;

adenosine

cm1 G. H. Bourne

C. rat triphosphate;

heart;

d? rat sodium

muscle;

c, rat kidney;

p-glycerophosphate;

and sodium

j3-glycerophosphate: odiurn B-glycerophosphate; and sodium P-glycerophosphate. wax and various temperatures on the intensity and distribution of enzymes visualized in our zymograms [5]. In the meantime the technique has already demonstrated the following: 1. That there are a multiplicity of glycerophosphates. 2. That some glycerophosphatases are identical with the enzymes demonstrated by the azo dye technique. 3. That other glycerophosphatases are not demonstrated by the azo dye technique. 4. That the technique will permit the differentiation of substrate specific phosphatases, e.g. adenosine triphosphatase and /l-glycerophosphatase in rat muscle. In conclusion it becomes obvious that as with esterases there is a multiplicity of phosphatases and further investigations with the starch gel electrophoresis technique will not doubt demonstrate the degree to which they overlap with substrate specificity and pH optima. Experimentnl

Cell Research

24

Nistochemistry

17;

by electrophorosis

REFERENCES t.

2. 3. 4. .j. (i.

i. X. 9. 111.

~~OUKNE. BOUKNE, I:REIMAN,

c;.,

@Uli”f.

.J. i%Tpff.

Ph7JSiOf.

32,

1 (1943).

G. H. and GOLARZ, hI. N., Nnfwe 183, 1741 (1959). D. G. and KAPLAN, N., J. Hisfochem. Cytochem. 8. 159 HVNTER. R. L. and BURSTONE. 31. S., .I. ffistochem. ~&tochern. 8. HUNTER. R. L. and MARKERT, C. I,., Science 125, 13-94 (1957). ~IAKKERT, CL. L. and MOLLER, F., Proc. N&l. dead. Sci. L’..S. 45. PE.~RSE, X. G. E.. Histochcrnistry. Theoretical and Applied. Boston, 1960. SANDLER, 31. and BOURNE, G. H., J. Gerontol. 15. 32 (19Ml). --Circ. RPS. VIII, 1274 (1960). S~rrrrtr~s. 0.. f~inchem. J. 61. 629 (1955).

THE

EFFECT

OF DILUTION

METABOLIC

PROPERTIES

and

CARBON

L MOUSE

F. WHITFIELD

and

Health

Physics Chalk

Division River,

Received

753

R.

, Atomic Ontario,

March

(19.59).

Little.

l~rown

DIOXIDE

OF SUSPENSION

OF STRAIN J. Biology

AND

(1960). 38 (1960).

K: Company.

ON THE

CULTURES

CELLS H.

RIION

Energy

of Canada

Limited.

Canada

27. 1964

IT has been generally observed that the metabolism of tuulour cells has two main characteristics; the respiraton rate is depressed by glucose (Crabtree effect [S]) and the cells produce large amounts of lactic acid. In this communication we will show that simple dilution with fresh medium enhances both of these characteristics of L mouse cells in suspension cultures. \\‘e will also present evidence which suggests that this dilution effect is due to a decrease in the CO,content of the culture medium. Two strains of L mouse cells Ill], Ll and R3, have been used in this study and their origins have been described previously (15, 151. Since the responses of these two strains to dilution were identical, the results were pooled. The methods of handling and maintaining suspension cultures, and of determining total cell concentrations, have been described elsewhere (11, 161. \?able cell numbers were indicated by the inability of live cells to stain with Erythrosin I3 [9]. In these experin:ents. portions of 200 ml suspension cultures containing 5 to 7 105 cells per ml \I (w diluted with fresh medium (80 per cent CMRL-1066 and 20 per cent horse serum) to contain 1 1W cells per ml. The diluted and undiluted portions of each culture were then incubated for 3 hours. The same number of cells (c. 1 10’) from undiluted and diluted portions were resuspended in 2.4 ml of Webs- Ringer phosphate solution 1131. To this was added 0.3 ml of horse serum, which prevented cell death during incubation in the respirometer flasks. The respiration rate of these suspensions was measured by the direct method of \\‘arburg 1131; the gas phase in the flasks was air. Glucose (I< rng iu 0.3 ml) was added at .iO uiin and the oxygen consumption