LOCALIZATION, SUBSTRATE SPECIFICITY, AND THE EFFECT
. OF INHIBITORS ON ALKALINE PHOSPHATASES
OF TETRAHYMEEA GELEII W

By

Edward F. Degenhardt

AN ABSTRACT

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in Partial Fulfillment of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Zoology

Year 1955

Approved /£z47.JéE;"‘°zZ&:”“

 

THESIS

 

Edward F. Degenhardt

THESIS ABSTRACT

Organisms used for experimentation were cultured in
bacteria-free tryptone, tryptone-Mgel2, vitamin—enriched
tryptone, vitamin-enriched tryptone with added MgCl2, and
tryptone—citrate solutions with added MgCl2. Control tests
were utilized in all experiments. PhoSphatase was demon-
strated in various substrate solutions, e.g., glycerophos-
phate, muscle adenylic acid, yeast adenylic acid, adenosine
triphOSphoric acid, glucose—l-phosphate, and creatine phos—
phate with the calcium-cobalt procedure of Gomori (1952).
Results obtained with some substrate solutions were checked
against those obtained with sodium alpha—naphthyl acid phos—
phate substrate solution (azo-dye procedure).

(1) Validity of the calcium—cobalt procedure was estab-
lished; (2) results obtained with inhibitors and activators
and various substrate solutions suggest that multiple phos-
phomonoesterases exist in Tetrahymena; and (3) activity
values were established for phosphomonoesterases under var—
ious experimental conditions.

Type of culture medium and prolonged culture of organisms
in.3pecific media affected phOSphatase activity, i.e., in
'vitamin-enriched tryptone, citrate buffered (0.01M) tryptone
with added IvIgCl2, phosphatase activity (calcium-cobalt pro-

cedure) was inhibited. Increased concentration of citrate

2

Edward F. Degenhardt

(0.0hM) suppressed the calcium—cobalt reaction. Positive
azo-dye reactions were obtained in organisms for 0.02M and
0.0MM citrate buffered tryptone to which MgCl2 was added and
also in organisms from vitamin—enriched tryptone solutions.
Prolonged culture of organisms in tryptone solutions in-
creased the intensity and area of staining reaction with the
calcium-cobalt procedure. Organisms from tryptone solutions
which exhibited pronounced phosphatase activity and pre-
formed phosphates with the calcium-cobalt procedure were
negative for azo-dye phOSphatase.

Inhibitor experiments were carried out using semicar-
bazide, 0.002M; sodium arsenate, 0.001M; KCN, 0.0lH; sodium
glycocholate, 0.006M; HCl, 0.0lN; citrate buffered solutions
(pH 5.0), 0.2M; glycine, 0.25H; NaCl, 0.01F; saline, H202,
and distilled water (800 0.). Complete inhibition was ob—
tained with semicarbazide, sodium citrate, and HCl. Activa-
tion of ATPase was induced by KCN while enzymatic reactions
in all other substrate solutions were inhibited to a great-
er or lesser degree. Sodium arsenate and H202 caused no
apparent inhibition of G—l—Pase or Cr—Pase but did inhibit
glycerophOSphatase, A—S—Pase, A-3-Pase, ATPase. Sodium gly-
cocholate did not inhibit G—l—Pase while occasional inhibi-
tion of Cr—Pase occurred. Glycine inhibited glycerophOSpha-
tase, A—S—Pase, and Cr—Pase to a greater degree than ATPase
and G-l-Pase. Saline and NaCl induced slight or no inhibi-

tion.of glycerophosphatase, A—S—Pase, A—3-Pase, and ATPase.

3

Edward F. Degenhardt

Hot distilled water (80° c.) inhibited all enzymatic
reactions.

Reactions for phOSphatase were usually located in the
posterior ends of cells. Nuclear staining reactions (cal-
cium-cobalt procedure) were believed to be artifacts. How-
ever, occasional nuclear reactions with ATP substrate, under
certain conditions, appeared to represent true enzymatic

reactions.

LOCALIZATION, SUBSTRATE SPECIFICITY, AND TEE
OF INHIBITORS ON ALKALINE PHOSPHATASES
IN TETRAHYMEEA GELEII W

By
Edward F. Degenhardt

A THESIS

Submitted to the School of Graduate Studies of

EFFECT

Michigan

State College of Agriculture and Applied Sciences

in Partial Fulfillment of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Zoology

1955

ACKNOWLEDGEMENTS

The author wishes to express his sincere thanks to
Dr. R. A. Fennell for higusuggestion of the research problem
and also for his constant assistance, direction, and interest
during the course of this research.

Grateful thanks is also due Mr. Berley Winton, director
of the U. S. Regional Poultry Laboratory, East Lansing,
Michigan, for allowing the author use of the laboratory's
facilities during part of this investigation.

The author is also indebted to Dr. S. Lesher, recently
of the U. S. Regional Poultry Laboratory staff and now
associated with the Argonne National Laboratory, for valu-
able suggestions on technique.

Thanks is also due Mr. Joseph G. Engemann, graduate

assistant, zoology department, for the photomicrographs.

85.306431

Section

I
II
III

IV

TABLE OF CON ENTS

IIQTRODUCTIOIQ'. O O O O O O O O O O O O O O O O O O

I'AATELRIALS AND I‘ETHODS o o o o o o o o o o o o o o

RE‘JSULTS O O O O O O O O O 0 O O O O O O O O O O O

l.

A.

5.

6.

A Comparison of the Calcium-Cobalt and Azo-
dye Techniques for Localization of
Alkaline Phosphatase Activity. . . . . . .

A Comparison of GlycerOphosphatase and

Muscle Adenylic Acid Phosphatase
Activity in Specimens of T. geleii
Cultured in Tryptone Solution. . . . . . .

A Comparison of Yeast Adenylic Acid Phospha-
tase, Adenosine Triphosphatase, Glucose—
l-PhOSphatase, and Creatine PhOSphatase
Activity in Tryptone Solutions and in
Tryptone-Citrate Solutions with Added

MgC l2 0 O O O O O O O O O O O O O O O O O O

The Relation Between Concentration of
Citrate Buffer and PhOSphatase
ACtiVity O O O O O O O O O O O O O O O O O

PhOSphatase as Related to Inhibitor
Reactions, Substrate Specificity, and
Intracellular Localization . . . . . . . .

Nuclear Staining and Diffusion Artifacts
Observed in I, geleii. . . . . . . . . . .

DISCUSSION

1.
2.

3.

4.

l2

l2

16

3O

36

37

H2

Ca-Cobalt and Azo-dye Phosphatase. . . . . . 4h

The Relation of PhOSphatase Activity to
Variations in Culture Media. . . . . . . .

Enzyme Inhibition and Substrate Specificity
(multiple Phosphomonoesterases). . . . . .

Nuclear Enzymes. . . . . . . . . . . . . . .

52
56

TABLE OF CONTENTS (Cont.)

Section

VI

LITERATURE CITED. . . . . . . . . .

LIST OF TEXT-FIGURES

Text-figure

1 Relation between culture age, glycerophospha-
tase activity and A-SePase activity in
specimens of _. geleii w cultured in
tryptone media . . . . . . . . . . . . . . .

2 Relation between culture age, glycerophOSpha-
tase activity, and A-S-Pase activity in
specimens of T. geleii w cultured in
tryptone-MgCl2 media . . . . . . . . . . . .

3 Relation between culture age, glycerOphOSpha—
tase activity, and A-5;Pase activity in
specimens of T. geleii w cultured in
vitamin—enriched tryptone media. . . . . . .

4 Relation between culture age, glycerOphospha—
tase activity, and A-S-Pase activity in
specimens of 1. geleii w cultured in
vitamin-enriched tryptone media with added

'rigCl2oo00.000000000000000

5 Relation between culture age and enzymatic
activity in specimens of T. geleii w
cultured in tr tone—citrate solution
(0.01M, pH 5.5)pwith added Mg012 . . . . . .

6 Relation between culture age and enzymatic
activity in specimens of T. geleii w
cultured in tryptone media. . . . . . . . .

18

21

2h

27

31

33

LIST OF TABLES

Table

I The Effect of Inhibitors on the PhOSphomono-
esterases of T. geleii W. . . . . . . . . . . . 39

LIST OF PLATES

Plate

I Alkaline phOSphatase activity in specimens of
T. geleii. Figures 1,3, 5, and 6 stained
for glycerophosphatase using t‘;le Gomori Ca—
cobalt technique; figures 2 and A stained
for azo-dye phOSphatase using the Gomori
azo-dye technique . . . . . . . . . . . . . . . 68

Fig. l. 1hH-hour cells cultured in tryptone-
MgC12 solution;

Fig. 2. lHA—hour cells cultured in tryptone-
Mg012 solution;

Fig. 3. lhO-hour cells cultured in tryptone—
Mg012 solution;

Fig. A. lAO-hour cells cultured in tryptone-
MeCl2 solution;

Fig. 5. IMO-hour cells cultured in tryptone—
Mg012 solution; and

Fig. 6. IRA—hour cells cultured in tryptone-
Mg012 solution.

II Alkaline phOSphatase activity in specimens of
T. geleii. Figures 7, 8, and 9 stained for
glycerophosphatase, IO and 11 for A-S-Pase,
using the Gomori Ca-cobalt technique; fig-
ure l2 stained for azo-dye phOSphatase using
the Gomori azo-dye technique. . . . . . . . . . 70

Fig. 7. Ink-hour cells cultured in tryptone-
Mg012 solution;

Fig. 8. 792-hour cells cultured in tryptone
solution;

Fig. 9. IRA—hour cells subcultured in tryp-
tone solution for approximately
six months;

Fig. 10. 288—hour cells cultured in tryptone
solution;

LIST OF PLATES (Cont.)

Plate

Fig. 11. 216—hour cells cultured in tryptone-
MgC12 solution; and

Fig. 12. 26A—hour cells cultured in vitamin-
enriched tryptone solution.

III Alkaline phosphatase activity in specimens of
T. geleii. Figure 13 stained for azo-dye
phosphatase using the Gomori azo-dye tech—
nique; figures 14 and 19 stained for ATPase;
15, G-l-Pase; 16, Cr-Pase; l7 and 18, glycero-
phOSphatase using the Gomori Ca-cobalt
teemique O O O O O O O O O O O O O O O O O O O 72

Fig. 13. lhh—hour cells cultured in tryptone-
citrate solution with added MgCl2;

Fig. 1%. 72-hour cells cultured in tryptone
solution;

Fig. 15. #32 hour cells cultured in tryptone
solution;

Fig. 16. 216-hour cells cultured in tryptone
solution;

Fig. 17. INA—hour cells cultured in tryptone-
citrate solution with added MgCl2;

Fig. 18. 288-hour cells cultured in tryptone
solution. Organisms immersed in
0.2M citrate buffer for 15 min-
utes prior to incubation in
glycerOphosphate for 2h-hours; and

Fig. 19. 72-hour cells cultured in tryptone-
citrate solution with added MgClZ.

INTRODUCTION

The ciliated protozoa have been used to a limited extent
for histochemical studies. Alkaline phosphatase was first
localized in Specimens of Colpidium cannylum (T. geleii) by
Sullivan (1950). He maintained that enzymatic activity we
limited to the nucleus and that cytOplasmic reactions were
dependent on migration of the enzyme from the nucleus into
the cytoplasm. Elliott and Hunter (1951) demonstrated that
T. geleii contained an enzyme capable of Splitting phOSphate
from S-nucleotides. The enzyme involved was confined to the
cell and not released into the surrounding medium. Fennell
(1951) found positive phosphatase reactions in the cytOplasm
of T. geleii and attributed this activity to both preformed
phOSphate and phosphatase. Bouwman (1952) found phOSphatase
activity in both the nucleus and the cytOplasm of T. "eleii.
Hugard (1953) studied alkaline phosphatase activity in Spec-
imens of Ophryoglena and found that enzymatic activity was
absent in starved animals; upon ingestion of food, phospha-
tase activity became observable in the vicinity of the food
vacuoles. Thereafter, phosphatase activity appeared in the
macronucleus, micronucleus, and cytOplasm. Fennell and Marzke
(19bh) demonstrated that, in specimens of T. geleii cultured
in tryptone solution, cytoplasmic alkaline phOSphatase in—

creased from a minimum (24 hour cultures) to a maximum

(288 hour cultures) ana then rapidly decreased to zero
(360 hour cultures).

Alkaline phosphatase has been extensively studied in
vertebrate tissues. Danielli (19h6) made a critical study of
the calcium-cobalt technique of Gomori (1939) and Takamatsu
(1939) for the localization of alkaline phOSphatase. He

1.
,\

concluded that the positive reactions obtained with the
technique indicated the site of alkal'ne phosphatase activity.
Gomori (1952) found under certain experimental conditions
that both false—positive and false-negative reactions were
obtained with this technique. Danielli (1050) stated that the
Gomori technique suffered from a deceptive simplicity, and
one using the procedure should have a good knowledge of phy-
sics and chemistry.

Johansen and Linderstrom—Lang (1951, 1952) evaluated
the Ca—cobalt technique mentioned previously. They concluded
that the method was not dependable since (1) calcium phos-
phate has a tendency to form supersaturated solutions; (2)
the time required (0.1-2.5 seconds) for the phosphate to reach
a critical concentration was sufficient for diffusion of phos-
phate into adjacent cellular areas where precipitation oc-
curred on preformed crystal nuclei; and (3) the enzyme was
uniformly distributed in cells. Gomori (1952) and Gomori and
Benditt (1953), after a repetition of the Johansen-Linderstrom-
Lang experiments, concluded "there is no experimentally demon-

strable tendency to supersaturation under the conditions of

the correctly per erred histochemieal tests (calcium concen-
tration not less than 0.051, p: not less than 9.3), nor do

preformed crystal nuclei have any effect on the results".

U1

He concluded his di cussion with this statement: "Further—
more, the thesis that phosphatase is distributed evenly (or
at random) among all cells or even within one cell body
cannot be accepted."

Martia and Jaeoby (19e9) superimposed strongly alkaline
phosphatase—positive sections of liver tissue upon sections
in which phosphatase was not histochemically demonstrable.
Positive reactions were obtained in the underlying tissue.
They were unable, on the basis of their experiments, to say
whether or not positive reactions were due to diffusion of
the enzyme or to an enzyme activator. Yohoyama, Stowell, and
Kathews (1951), who superimposed enzyme active duodenum
sections on inactivated liver sections, believed that posi-
tive reactions in the nuclei of the latter were due to dif—
fusion of the enzyme fron enzyne active tissue into the in-
activated tissue sections. Danielli (1953) performed essen—
tially the same type of experiment by superimposition of
phosphatase-active kidney sections on inactive liver sec-
tions. he believed tlat positive reactions in the underlying
tissue were dependent on either diffusion of phosphatase
itself or an activator substance. Novikoff (1951) presented
evidence to show that Ca phOSphate was adsorbed on nuclei
and concluded that

the Ca—cobalt technique was not reliable

for localization of phosphatase. Gomori (1951) stated that

:‘ Jn‘ '—‘ .L. . «z 1—. .— [A arxu J- ’9 ~I . -, .—- r‘ ~ ‘5 J— . fl
and taut in Most cus-s ;_acle: r stainin. cas an artiiact.

consult Chevronont and lirket (19??) who hav extensivelv

'1

lienten, Ju1'15e, one Green (lQM-ia, 19121313) and 1La-n einer
and Selignan (19h?) enuloyod the azo—dye :ethod for deion-
stration of pho33hatase. rho Go 0; i (1932) - zo— dye method
was based on the supposition that naphthol liberated from
sodi‘: alpho- unantWV1 acid phosolzate would couple with an

azo—dye (Diazo Blue * salt) to for: an insoluble purple

Characterization and localization of various thSphor—

ylating en zynes was inve. Hti a.ted b

{<1
p—a
{J
C)
*1
i
c:
4
1
d
l
t;
w
‘4
a
1*
O
U]
0
m
'J
O

(1950,1952 . They used glycerOphosphate, 51L. cose-l—ahos ahate

fructose-G—ohos sphate elucos 6-ph03phate ntscle ad nVlic
- : o ? J

.5

acid, yeast adenylic acid, creatino phos phate, and adenos ine

triphosphate as substr a e

along with various enzyme activa-

U)

('1‘

OJ

tors and inni itors. Ihey concluded, on the basis oi their

research, that multiple onos3.on noostem~ se s could be ~.eztioa—

° (..x_.

i in fresh frozen rat tissues. Gomori (l9h9) utilized
nineteen different phOSphate—conm miiin' sub ostrates at

ferent hyclrogen ion concentrations. He was unable to cis-
tinsui sh phOSphatases other than th e corn n non-sgecific

‘Lb

alkaline and acid Tarieties with 18 of the substrates how-

so.

ever, one substrate (p-chloranilidOphOSphatc) did present
a different enzyratic distribution in the acid range.

ma
U. J.

Eewnan et a_. (1950) employed various suIs rate

U)

inhibitors and demonstrated three ma jor groups of phospha-
tase enzymes, i.e., cytoplasmic phosp’: a tases (groups I and II)
and nuclee r phosphatases (b roup III).

Studies on the role of phospha ase e- 1zynes in growth
and development have been made by numerous investigators.
IIoog (1940) found that alkaline ahosphatase increased in
Chicken embryos of 48 to 96 hours incubation, decreased be-
tween 96 and 144 hours, and then increased again between 144
and 240 hours incubation. Johnson and Bevelander (1947)
found that presence of a1 kaline ph03p11atase was correlated
with proliferation and differentiation of all cells that
were concerned with feat} er development. Bevelander and
Johnson (1949) demonstrated abundant pI osphatase activity in
aneloblasts during matrix formation and the subsequent ca 1-
cification of enamel. Karcznar and Berg (1951) observed dif-
ferences between alkaline phosphatase associated with the
larval liMb of Amblvstona and that functional in limb ontog-
eny and regeneration. Flavia and Pearson (1954) found phos-
phatase activity was reduced in rapidly growing tunors and
in tumor areas which were especially ana11astic. Clark et a1.
(1950) found that phosfl11atase activity in huiuans increased

between the sixth and ninth decades of life.

6
The function of phOSphatase in cell metabolism has been

treated by Krugelis (1946a), Kanen and Spiegelman (1948),

U

hstein and heier (1949), Bradfield (1950), Gold and Gould

d-

\O

f.

\O

l 51), Danielli (1951), £003 and Wenger (1951), and Lesher
l 2).

A A
\O
\J'o

.0

It is evident iron this review of the literature on
phOSphatases that the function of phosphatases, the action of
enzyme inhibitors, relative enzymatic activity in various
substrate solutions, and the existence of multiple phospha-
tases is still open to question. Questions have also arisen
concerning the reliability of the Gomori technique for demon—
stration of alkaline phosphatase in cell nuclei.

It is the purpose of this study to: (1) test the relia-
bility of the Gomori technique for demonstration of alk1line
phOSphatase in cell nuclei; (2) ascertain whether or not, on
the basis of enzyme inhibitors, localization, and staining
intensity, multiple phosghatases exist in 1. geleii; and (3)

show the activity value for phOSphatase in specimens of

1. geleii from various ages and types of culture media.

MATERIALS AND KETHODS

Specimens of 2. geleii (strain U) used in the follow;
ing eXperiments were cultured in bacteria-free (l) tryptone
solution; (2) tryptone-M3012 solution; (3) vitamin-enriched
tryptone solution; (4) vitanin—enriched tryptone solution
with ac dded hg 019; and (5) tryptone—citrate solution with
added LgClQ.

Tryptone solution (Bacto-tryptone, Difco Laboratories,
15 gm;; KH2P04, 1 gm.; and 1 liter glass-distilled water)

‘wa 3 use as an experimental medium and also for the mainte-
nance of stock cultures. Repetitions of experiments were made
with organisms that were maintained in vita min-enriched sol—
utions prior to inoculation of experimental cultures (tryp-
tone, tryptone—M3012, etc.). Tryptone—Ngc 2 solutions were
made by the addition of 1.07 gm. KgolZ to the basic medium.
Vitamin—enriched tryptone solutions were made by the addition
of 1 mg. riboflavin, 1 mg. thiamine hydrochloride, 1 micro-
gram nicotinic acid, and 0.5 nicrogran biotin to the basic
medium. Vitamin—enriched tryptone solution with added Mg012
was made in the same manner as vitamin-en nrich ed tryptone
medium except that 1.07 gm. of Mg312 was added. The final
culture medium (tryptone-citrate solution with added MgCl2)
was varied slightly by replacing the KH2P04 buffer present

in the basic: medium with a O. 01" sodium citrate buff e-r (2.06

gm. sodium citrate and 0.63 gm. citric acid per liter of
culture solution). Iagnesium chloride was then added to this
solution in the amount of 1.07 gm. In other experiments,
either 4.72 gm. of sodium citrate and 0.84 gm. of citric
acid, or 8.44 gm. sodium citrate and 1.68 gm. of citric acid
was substituted, i.e., 0.02M and 0.04M citrate buffer, re—
spectively, for the 0.01H buffer. The final pH before steri-
lization was set at 5.5. ’

Organisms used in all experiments were cultured in 125
m1. Erlenmeyer flasks. Eacteria-free cultures of T. geleii
were established by the transfer of 1 ml. of bacteria-free
culture solution, in which organisms were abundant, to each
of fifteen 125 ml. Erlenmeyer flasks which contained 75 m1.
of sterile culture solution.

For localization of alkaline phosphatase in T. geleii
from various culture solutions (tryptone solution, tryptone-
MgC12 solution, etc.), tests were made 72 hours subsequent to
inoculation and every 72 hours thereafter until organisms
disappeared from the culture solutions.

Test organisms were obtained from the cultures by trans-
fer of approximately 12 m1. of the culture solution (cultures
were then discarded) to a 15 m1. centrifuge tube. The tube
was centrifuged at 1500 r.p.m. for not longer than five
minutes. Eleven milliliters of the supernatant were removed
with a pipette. The orga1isms were left suspended in 1 ml.

of culture medium. The cellular concentrate was withdrawn

from the centrifuge tube using a 1 m1. pipette. A drop
(0.1 ml.) of this cellular concentrate was then placed on
each clean albumen or gelatin coated slide and allowed to
dry in air. The slides were transferred to 50 m1. of either
cold C.P. or U.S.P. acetone (50 C.) or 85 per cent alcohol
in a coplin jar and left for 3 to 36 hours (usually 24 hours)
for fixation. The slides were removed from the ceplin jar,
hydrated to water, and stained, using one of the Gomori
Ca—cobalt tests for alkaline phOSphatase (Gomori, 1952).
They were then dehydrated, cleared in xylene, and mounted
in balsamr

The test for glycerOphOSphatase was run as described
by Gomori (1952). Tests for 5-nuc1eotidase were run as des—
cribed by Gomori (1952) using 20 mg. of muscle adenylic acid
as a substrate. This technique was also utilized when yeast
adenylic acid and adenosine triphosphoric acid were used as
substrates. In tests using creatine phOSphate (calcium salt)
and glucose-l—phosphate (potassium salt), the same technique
was used except the pH of the substrate mixture was adjusted
to 9.4 instead of 8.3. The Ca-cobalt technique was checked
in most instances by substitution of sodium alpha-naphthyl
acid phosphate (azo-dye method of Gomori, 1952) for the
Gomori substrate solution.

Slides were incubated in the following substrates for
two hours: muscle adenylic acid, yeast adenylic acid, adeno-

sine triphOSphoric acid, creatine phosphate, and glucose-l-

lO

phOSphate; they were incubated for one hour in sodium glycero-
phOSphate. When sodium alpha-naphthyl acid phosphate was used
as a substrate, the incubation time was five to ten minutes.
The incubation temperature in all cases, except when sodium
alpha-naphthyl acid phOSphate was used, was held at 370 C;i
l0 C. The incubation temperature for the latter was 200 Cfit2o C.

In inhibition experiments the slides were exposed for
15 minutes to an inhibitor mixture prior to immersion in the
substrate solution. Final concentration of inhibitors was:
sodium arsenate (0.001M and 0.01M), semicarbazide (0.002M
and 0.02M), citrate buffer (0.2M, 0.01M, and 0.000lM,pH 4.5-
5.0), H01 (0.1N), KCN (0.01M), H2O2 (1 ml. 30 per cent H202
made up to 50 ml. with distilled water), HaCl (0.01h),
saline (0.14M), sodium glycocholate (0.006K), and glycine
(0.25M). Water used for inactivation of enzymes was heated
to 800 C.; slides on which organisms were mounted were im-
mersed in it and left 15 minutes.

In other experiments MgCl2 was left out of the substrate
mixture in order to ascertain the enzyme activating effect
of this salt.

In control tests either glycerOphosphate was omitted
from the incubation mixture or distilled water was used as
a replacement for the substrate salts.

Estimation of enzymatic activity was made using a sub-
jective rating scale. Reactions were given values ranging
from 0-6: zero, no reaction; 1, very weak reaction (few

positive cells, etc.); 2, light reaction; 3, moderate reac-

11
tion; h, heavy reaction; 5, very heavy reaction; and 6,
extremely heavy reaction (reactive area large, heavy black
precipitates,all cells positive). Criteria used for ascer—
tainment of reaction values were: (1) the intensity of the
staining reaction; (2) the relative concentration of the
reaction granules; (3) the area of the reaction; and (M) the
relative number of positive cells (few, most, all). This
estimate also involved all the cells on the slide rather
than individual cells or groups of cells. The study involved
approximately 1,000 slides; more than one reading per slide
was made, and an average was taken.

It should be noted that this estimate of phOSphatase
activity is in no sense a quantitative value. It does, how—
ever, indicate trends in enzymatic activity and does provide
a means of qualitative comparison. Subjective rating scales
for the estimation of phosphatase activity similar to this
were used by Kaengwyn-Davies at al. (1950, 1952) and Wachstein
and Meisel (195%).

Organisms used for experimentation were obtained from
various ages of cultures subsequent to seeding. The age of
cultures ranged from 72 to 1,080 hours. In most experiments
culture age varied between 72 and 360 hours.

1. §,eomparison g: the Ca—cobalt and azo-dye techniques

 

for localization of alkaline phosphatase activity. Doyle

at al. (1951) described the chemical aSpects of the staining
reaction in the Ca-cobalt procedure of Gomori (1939). Reac-
tion products obtained with the azo-dye method were described
by Novikoff (1952). The azo—dye method of Gomori (1952) was
used to check the validity of localization of phosphatase

wi h the Ca-cobalt technique. Both procedures are indirect
methods for localization of enzymatic activity.

In the Ca-cobalt method, inorganic phOSphate is liber-
ated from a phOSphate ester by the enzyme, and it is pre-
cipitated as Ca phosphate within the cell. Subsequent treat-
ment of the precipitate with cobalt and ammonium sulfide
transforms the colorless Ca phosphate into a dark brown or
black precipitate at the sites of enzymatic activity.

In the azo-dye method, the organic portion of the mole-
cule is utilized for demonstration of enzyratic activity.
Phenol is liberated from a phOSphate ester (sodium alpha-

naphthyl acid phosphate) by enzyme action. A cOIpling of the

l3
phenol with the azo—dye produces an insoluble purple precip-
itate in enzyme active areas of the cell.

Three generalized types of precipitates were obtained
in Specimens of l. geleii with the Ca-cobalt technique, i.e.,
(1) fine granular black precipitates; (2) an intermediate
precipitate (fine granules plus a variable nuwber of larger
black granules); and (3) a large vesicular precipitate.

1. Fine granular precipitates were usually encountered
in Specimens from young cultures of organisms. Figure 1 shows
Specimens from a 144 hour tryptone—MgCl2 culture. Individual
granules which were stained bluish-black were barely resolv—
able with a high power lens system (X400). They were usually
arranged to form a narrow oblique band at the posterior end
of the cells. In other individuals the fine granules were
randomly or uniformly distributed in the cytOplasm.

Organisms from this same 144 hour culture when stained
with the azo—dye technique exhibited a precipitate which
differed in several respects from the precipitate des—
cribed in the preceding paragraph. The azo-dye precipitate
was dark purple in color. A concentration of precipitate at
the posterior end of the cell was the exception rather than
the rule. In some specimens the anterior end of the precipi-
tate was adjacent to the nucleus, but the posterior end
terminated several microns anterior to the posterior tip of
the cell (fig. 2). In other specimens the precipitate was

restricted to one or more longitudinal bands. Precipitates

14
concentrated po 8 terior to the nucleus, including the longi—
tudinal band-preciip nit at tes, —xhibited a variable number of
large purple granules which were invested by fine purple
granules. The larger cytoplnsmic granules (two or more) in
other individuals were randomly distributed in the cytOpla sm.

2. The internediate type of precipitate, obtained with
the Ca—cobalt procedure, is illustrated in Mi gure 3. Speci-
mens were obtained from a 140 hour tryptone-KgT12 solution.
It is evident that the precipitate consisted of a compacted
mass of large and 81811 black granules. In most specimens the
enzyme active area was restricted to the posterior end of
the cells, but in some organisms precipitates were either
uni iformly or ra-rolly dist -ri buted in the cytoplasm. Conpacted

recipitates were sufficiently extensive to cover the pos-
terior one —fourth of the cytOplasn.

A co parison of the Ca—cobalt reaction (fig 3) with
the azo—dye reaction (fig. 4) in specimens from the sane lhO
hour culture, shows that the intracellular phosphatase active
area was located in essentially the same area of the cell,
i.e., in the majority of organisms both Ca—cobalt and azo-
dye precipitates were concentrated in the posterior portion
of the cell. The co parison also shows that th e extent of
the phOSphatase active area was essentially the same with
both procedures. It is also evident in figure 4 that a few
Specimens exhibit a precipitate that has a tendency to be

arranged in longitudinal bands

3. Large granular Ca—cob lt precipitates were identified

in specinezis cultured in tryptone, tryptone-IgClg, and vita-
min-enriched tryptone with added Mg312 solutions. Spherical
vesicles, principal components of the precipitate, had an
average diameter of three microns. They occurred singly or
in clumps ranging in number from three to SilC or more (fig.
There we S a ter de ency for vesicle to be concentrated at the
posterior end of the cells, but in some specimens they were
arranged in a linear series. In other specimens they were
uniformly or randomly distributed in the cytOplasm. Staining
reactions were variable with the Ca-cobalt technique. Range

of color varied from light gray to dark black. Gray vesicles
were homogeneous in appearance, and the darker vesicles ex-
hibited variable amounts of blacn particulate matter uni-
formly distributed within the vesicle. A conparison of vesi-
cular particulate materials within vesicles with the fine
particulate matter in the posterior ends of cell S showed
that from a morphological point of view they were indistin-
giishable.

Large granular precipitates were produced by experi-
mental procedures. Specimens of T. cclcii cultured in tryp—

—.h——

tone-12g012 solution were concentrated at the bottom of a

5).

15 n1. centrifuge tube by centrifugation at about 1500 r.p.m.

All but 1 ml. of solution (in which Specimens of Tetrahynena
were concentrated) was removed witha a pipette. Organi ns

1

tested for alkaline phOSpnatase at this stage in the proced-

16
ure exhibited positive reactions (fig. 6). The centrifuge
tube was left for five minutes subsequent to the addition of
14 m1. of 0.01H citrate buffer (pH 5.1). Organisms were
again concentrated by centrifugation, and then 14 m1. of the
citrate buffer was removed with a pipette. Organisms that
were left in the centrifuge tube were vigorously shaken, and
then they were transferred along with 1 ml. of citrate sol—
ution to 75 ml. of bacteria-free tryptone-MgCl2 solution in
a 125 ml. Erlenmeyer flask. Specimens of T. geleii prior to
transfer to tryptone-K0012 solution did not show either vesi—
cles or phOSphatase activity.

After two hours incubation in the Erlenmeyer flask,
Specimens of T. geleii were removed from the tryptone—MgC12
solution, affixed to slides, transferred to cobalt nitrate
solution, and left for five minutes prior to immersion in
ammonium sulfide. An examination of the preparation Showed
an abundance of intracellular and extracellular vesicles
(fig. 7). Phosphatase activity ranged from a weak (gray ves-
icles) to a strongly positive reaction (black vesicles). It
is evident from these observations that positive phosphatase
reactions can be obtained in Tetrahymcna when incubation in

substrate solution is omitted.

U)

'1:

2. §_conparison g: glycerOphosphata e anr muscle adenylic

f 2. geleii cultured

acid phOSphatase activity in Specimens
in tryptone solution. Control slides were utilized in all

Xperiments. Careful consideration was given to positive

17
reactions that were in part dependent on preformed phos-
phates. Thus, the results given in the following paragraphs
actually represent trends in phOSphatase activity.

It is evident in text-figure 1 that phOSphatase activity
in glycerOphOSphate (pH9 4) increased from about 1.5 (72
hour cultures) to a maximum of 3.0 (144 hours), and then
decreased to zero (360 hours). Phosphatase activity increased
again to reach a second maximum of about 2. (504 hours), and
then it decreased to about zero at 648 hours. A third maxi-
mum (3.0) was observed in specirens from 792 hour cultures
(fig. 8). PhOSphatase activity then gradually decreased to
nearly zero in 1,080 hour cultures.

It was apparent late in these experiments that cells
aintained in tryptone solutions for approximately six months
exhibited increased anounts of preformed phosphates (fig 9)

with an average value of 5.0-6.0 (text—fig. 1). Under these
conditions enzyme inhibitors were utilized for making a dis—
tinction between false-positive reactions and true phospha—
tase reactions.

It is also evident in text—figure 1 that phosphatase
activity in A—S—P (pH 8.3) was similar to enzymatic activity
in glycerOphosphate, i.e., maximum and mininum activities
were evident. A comparison of enzy Watic activity in the two
substrate solutions S lows that succeeding maxima in A— —P,
subsequent to 288 hours, beca :1e progressively lower, 3- 5 at

288 hours, 2.0 at 432 hours, and 1.0 at 792 hours. :urtb er-

TEXT -FIGm n3 1

Relation between culture a.e, ‘lycerOphosphatase activity,
mid A—S-Pase activity in Specimens of T. geleii W cu tured
in tryptone media. Activity value ascerta :ed as described

under :aterials and methods.

(), T. geleii W cultured in tryptone media for
31x nontn s;

. , 2;.gClC11 x! c1-lturec 1n tryptone‘media and
stained for a-,—Pase act1v1ty oy the
calcium—cobalt method of Gomori; and

C) ’.T. “eleii N cultuer in tryptone “e118. an
stained for "lvcer0p.os)“atase activity
by the calcium-cot alt net: Mo of Gonori.

 

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20
more, enzymatic activity decreased to about zero in A-b-P at
360, 576, and 93 hour cultures. 0n the other hand, glycero-
pnosphatase activity decreased to zero at 360 and 1,080 hours
A comparison of figure 8 with figure 10 shows that maximum
phosphatase activity in A-S-P substrate solution (288 hours)
was higher than it was in glycerOphosphate.

Tex t— fi 1g1 1T8 2 summarizes the results obtained with
organ isns cultured in tryptone—IgClg solutions. A comparison
of text-figure l with text—figure 2 shows that phosphatase
act1v1ty 1n tryptone-I g012 solution was
tryptone solution. In tLe former, glycerOpiosphatase activity
increa ased fro: about 3.0 in 72 hour cultures (the maximum
activity in tryptone solution) to a maximum of about 5.0 in
360-432 hour cultures. Enzymatic activity then decreased to
a minimum 1 about 1. 0 as the age of the culture increased
from 43 2 to 720 hours. Phosphatase activity increase d a ain
to reach a second maxiuum of 3.5 (792 hours), and then grad-
ually decreased to 2.0 as the age of the culture increased to
1,08011ours.

Text -fig ure 2 also shows that A-S—Pase activity was more
erratic than that of glycerOphosphatase in specimens cultured
in tryptone—thlg solution. In the former, enzynatic activity
was at a minimum of 0.5 at 72 hours. A—b-Pase activity then
increased to a maxiium of 5.0 at 216 hours (fig. 11), de-
creased to a second rininun of 1.5 at 360 hours, and then
acreased again to reach a second uaxinun of 5.0 at 432 hours.

Tiniua of zero again occurr e0 at 576 ancl 720 hours

I

21
EXT-FIGURE 2
Relation between
and A-fi—Pase act

culture age, glyceroohosphatese activity,
1vity in specimens of :. geleii U cultured

in tryptone—Kg012 ncdia.
C), Glyceronhosohatase activity, and

., .1-5-Pase activity.

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23

Results obtained with organisms cultured in vitamin-
enriched tryptone solutions are summarized in text-figure 3.
PhOSphatase activity in glycerophosphate increased from 1.0
at 72 hours to a maximum of 3.0 at 288 hours, and then de—
creased to a minimum of 0.5 at 648 hours. It is evident in
text-figure 3 that enzymatic activity in A—S—P tended to
parallel enzymatic activity in glycerophOSphate with the
exception that in the former activity was lower. a-b_Pase
activity in 1-5-? increased from zero at 72 hours to a max—
imum of 2.5 at 144 hours, and then decreased to almost zero
at 360 and 432 hours.

A comparison of text—figure l with text-figure 3 shows
that, in general, total enzymatic activity was lower in or-
ganisms grown in vitamin-enriched tryptone solutions than it
was in organisms fron tryptone media. It also shows that in
the former (vitamin-enriched tryptone solutions) enzynatic
activity increased more uniformly to reach a maximum, and
then it decreased to a mininum, whereas in tryptone solutions
numerous naxina and minina were observed. A comparison of
text-figure 2 with text-figure 3 shows that enzymatic acti—
vity vas lover in cells grown in vitamin—enriched tryptone
solutions than it was in organisms cultured in tryotone—
Mg012 solutions.

These results are in general agrcefient with those of
Fennell and harzke (1954) who found that strongly positive

1

reactions (Ca-cobalt), waich charact rizee organisms fron

(D

24
TEXT—FIGURE
Relation between culture age, glyceroohosohatase activity,
1 ,4 '0 o o o .0
and A-p-Pase act1v1ty in specimens of 2. geleii w cultured
in vitamin—enricned tryptone ned'a.
C), GlyceroahOSphatase activity; and

., :1- S—Pase activity.

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Relation between culture a~e, glycerOphc sphao se activity,
and A—f—‘ase activity in saecimens of 2. fieleii U cultured
in VitLHLu-C rie111 d trvfia 01-0 media with aueed ;

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A comparison of text—figure 4 with text-figures l, 2,
and 3 shows that enzymatic activity in both $13 cerovhos-

:1i11— enriched

::
"J:-

phate and 3-5-? drOps much more sharply in v
tryptone solutions with add. 0,6 LgCl2 than in the other e: :pe ri-
nen tal culture mec‘1ia. It is also ev vilel 1t ( text- fig. L:) that
enzvuatic activity wa initially higher in vitamin—enriched
tryptone with added L3012 than it was in tryptone, tryptone—
LgCle, and vitamin-enriched tryptone media.

Results obtained with organisms from tryptone-citrate
solutions ~ith added Lg012 are summarized in text-figure 5.
Envvratic activity in blvceroonosmete substra be solution
rose from a “inr“un of 1.0 at 72 hours to a razinum of 2.0
at lbrh hours, de\. creasing to a second minimum of 1.5 at 21o
hours. Enzvmatic activity reached a second maximum of 2.0 at
238 hours. The activity of the enZTne in A—S— P w as al: ost
zero in organisms from all ages of culture. Enzymatic acti—
vity was absent at 72 hours. It increased to about 0.1 at

144 hours and 216 h urs, and then it decreased again to zero

hours.

CO

at 23

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comparison of text—figure 5 with tex —figures 1, 2,

h shows that n ximun glycerOphosphatase activity was

Lo
.5
9.:

, ani

lower and total A_5_Pase activity was lowest in tryptone-
MgClp solution in which the concentration of citrate buffer

xra.s 0. 01M.

Ho

3. A conpar son f .aast adem rlic acid eLosseatase aieno-

— —- 4——

K1
r")

sine trinhosohatase, glucose-l-ohosuhatase, and creatine
nhosnhatase activity in tryptone solutions and in trypton ne—
citrate solutions with add ed 330 2. It is clearly evident
from a co parison of text-figure 5 with to: :t- f15ure 6 hat
phosphatase activity was Mi 5he er in all substrate solutions
(A_3—P, etc.) in organisms obtained from try stone media. It
can be seen that enzyratic activity in A—3-P was alrtost ao-
sent in or5 anisns from tryptone-citrate solution with added

L5C12 (te: {t— fi5' .5), while in tryptone (text-fig. 6) A-3—Pase
activity was at a maximum of 3.5 in 72 hour cultures. This
was followed by a minimum of 1.5 at 144 hours, a second
maximum Of 3.0 at 216 and 288 hours, and a subsequent de—
crease to zero at 360 hours

Ens; Inatic ac tivity in ATP substrate solution unifor 1y
increased in or 5‘anisms from 72 hours to 288 hours in tryptone-
citrate solutions with adden L5012. Activity at 72 hours was
at a minimum of 0.5, gradually increased to 1.5 at 14% and
216 hours, and then increased to a maximum of 2.0 at 268
hours. The Opposite reaction was evident in cells grown in
tryptone solutiw . The activity was at a maximum of H.5 at
72 hours (fig. 14); this was followed by a gradual decrease
to zero at 330 hours.

Enzymatic activity in G- —P substrate solution is shown
in text- figure 5 by ma} :ima of 2.5 at 72 and 288 hours, and

minima of 2.0 at 144 and 216 hours. The Opposite effect is

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31

TEXT- TI IGURE 5
culture age and rz'ratic activity in succi-
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G—l—Pase activi‘y;

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Cr—Pase activity;

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A—3—Iase activity.

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Relat1onmoetx'reen.culture age and enzymatic activity in soeci-
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o , G—l—Pase activity;
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35
shown in cells grown in tryptone where the results, when
illustrated graphically, form an inverted "V". A minimum of
5.0 occurred at 72 hours followed by a maximum of 6.0 at 216
hours, succeeded by a second minimum of 3.0 at #32 hours (fig. 15).

It is evident in text-figure 5 that enzymatic activity
in Cr—P substrate solution increased from 2. 0 at 72 hours to
a ma.xi:1un of? .5 at 1A4 hours, and then it decreased to a
minimum of 2.0 at 216 hours. This was succeeded by a second
naxinun of 3.0 at 288 hours. It is also shown in text-figure
6 that Cr—Pase activity in tryptone rose from an activity
value of 3.0 at 72 hours to a maximum of 6.0 at 21611ours
(fig. 16), succeeded by a minimum of zero at 360 hours.

A conparison of teth-figures 5 an 6 also shows that
enzyMatic activity in trypton ne (text- fig .6) was either
represented by a curve that increased to a peak and then
declined to a low level (G—l-P and Cr-P) or one in which
there was essentially a continued lowering of enzymatic
activity with culture age (ATP and A—3—P). Enzyuatic activity
in cells grown in tryptone—citrate so1ution with added K5012
(text-fig. 5) is e: {hibited by curves essentially the reverse
of those text-figure 6, that is, there is a gra ual rise
in enzymatic activity in ATP and Cr-P substrate solutions,
or there is a fall in enzymatic activity to minimal level
which is followed by a rise in activity (G-l—P). The activity

of A— -Pase is here repr (sented by zinina activity.

 

and onosohatase activity. Organisms from Vitamin-enriched
tryptone solutions with aeded IgClg in which the concen-
tration of citrate buffer was increased to 0.04M were used
in this series of ex oerL lents.

rigure 13 shows that strongly positive alkaline phos-
phatase reactior IS were obtained with the azo—dye procedure
in orgar1isms from 144 hour cultures (216 hours subsequent to
establishment of citrate buffered cultures). 0n the other
hand, algaline nhosyhatase activity was not deronstrated in
organisms fron this save culture with the Ca-cobalt proced—
ure (fig. 17). Lssentially the sane results were Obb ained
1 organisms from 96 hour cultures (2’8 hours subsequent
to establislment of citrate buffered cultures). The enjeri—
ent was renea ed again with organisms from a 96 hour cul-
ture (total tine in<fltrate buffered solutions, 384 hours).
Positive reactions were a ain Ootained with the azo-dy pro—
cedure, aLd no reaction was ostained with the Ca—cobalt
procedure although both A—J-P and ATP were substituted for
glycerOphosnhate in the susstrete solutions. Specirens incu-

bated in the latter exhibited nuclei which were stained

citrate erffered solutions by subcul uriitg for #9 days, and
then sr1cultures were established in which the concent tion

a :1- 1. wuno‘ m ,1 . .1. , . 11.1 4-? 1‘
01 c1t1ate eh11cr wws lflCTCeSCd 60 0. who hihety- s x hours

37
subsequent to seeding, negative reactions for alkaline phos-
pha tase were obtained witl1 both the azo— lye and Ca— cobalt

procedures.

 

 

specificity, aLd L tr acellular localization. Six phosphate-
containing su1strates were utilized at different hydrogen ion

(3.:

concentrations, e.g., so iu1n glycerOphoSphate, glucose-1-
phOSphate, potassiu21 salt (G—l-P), and creatine phOSphate,

alcium salt (Cr—P), pH 9.4; muscle adenylic acid (A—fi-P),
yeast adenylic acid (A-3_P), and adenosine triphOSphoric
acid (ATP), pH 8.3.

In general, all substrates tested gave positive reactions
which were usually restricted to the posterior ends of the
cells. Part of this posterior activity was due to preformed
phosphates as was wiovn by control slides. Hydrogen ion
concentration of the substrate solution was an ifiportant
factor in demonstration of preformed phOSphates, i,e, he

concentration of pre1orned phOSphate was greater at pH 9. 4

than at pH 8.3. ho method was devised for removal of phos-
phate without destroying all phos aha ase reactions alon ng

11th the preforted phOSphatos. In all cases critical com—
parisons of control and substrate slides were me do, and only
those results are reported which demonstrated distinct dif—
ferences between experimental and control orga1isas.

Various conpoundsl1enown to cause inhibition or activa—

tion of phos atase enzyznes, haengwyn-Davies et a1. (1950

38
1952) and Newman gt al. (1950), were used in conjunction
with t‘11e substrates. 0rganisz3 were left in the various
inhibitor sol*Mt ons for 15 rainutes prior to immersion in
substrate solution. Results obtained are presented in Table I
which summarizes he effect of inhibitors and activators on
the phosphononoesterases of T. geleii. It is apparent that

DH 5, and 0.1K H01 com—

J.

0. 002 II se:1icarbazide, 0.2K citrate,
pletely suppressed all phOSphatase activity. These inhibitors
did not distinguish between false—positive and true enzyme
reactions. This was not the case Mi1the other inhibitors
used. Potassium cyanide, 0. 01K, ter ded to activate QTPase.
It induced moderate inhibition of A-5;Pase, and caused
slight or no inhibition of glycerOphOSphatase, G-l-Pase, and
Cr—Pase. Its action on A—3—Pase was erratic. Sodium arsenate,
0.001M, caused less inhibitiono glycerofi mo phatase, ATPase,
G—l-Pase, and Cr—Pase than of A—S_Pase or A—3—Pase. Glycine,
0.25M, caused little reduction of G—l—Pase activity, slight-
ly more inhibition of ATPase, and induced moderate to heavy
suppression of glyc cerOphosphatase, A—S -Pase, A-3- Pase, and
Cr—Pase activity. Saline and 0. 0111 h Cl caused little or no
reduction of enzymatic activity. Distilled water at 800
alway51indr1ced inhibition of enz; matic activity but seldom
completely suppressed positive phosphate reactions. H202
caused no inhibition of G—l-Pase and Cr—Pase; glycerOphos-

Q

p1_atase and 3—3- Pase were inhibited toa agreater degree than

either ATPase or A—S—Pase. Sodium glycocholate, 0.0063,

39
TABLE I

THE EFFECT OF IEEIBITOH? AKD ACTIVATORS ON THE
PHOSPEOKONOESTERASES OF 1. ggisll w

 

Glycero- A-S—P A-3-P ATP G-l-P Cr-P

 

phOSphatase ase ase ase ase ase
pH of
substrate 9.% 8.3 8.3 8.3 9.% 9.H
solution
Sodium 0.00lM 0-1 1-2 0-2 O-l O O
arsenate 0.01M 2 % --- --- _-_ _-_
Semicar- 0.002H 4 h 4 H H #
bazide 0.02M 4 H h h h u
KCN 0.01M 1 2 o—l-// %/ 0-1 0-1
Sodium
glyoo- 0.006M --- --- --- --- 0 0-1
cholate
Citrate 0.2M h 4 H 4 fl 4
buffer 0.01M 3 --- --- --- --- _-_
pH 5.5 0.000lM l --— -_- -—- _-- ---
HCl 0.1N 4 h H M H H
Glycine 0.25H 2-3 2-3 2—3 1-2 O-l 2-3
neroent in 1-2 1 1-2 0-1 0 O
h9 ml. H20)
Saline 0-1 0-1 O—l —-- —-- ---
NaCl 0.01M 0 0-1 O-l O-l --- -——
M3012 #0 #0 /O /O /O /O
Distilled

water 800 C. 3 3 3 3 3 3

 

TABLE I (Cont.)

Specimens of I, geleii mounted on slides which served as con-
trols were normally positive for preformed phOSphates with
the Gomori Ca-cobalt technioue. Organisms incubated in sub-
strate solutions without prior immersion in inhibitor or
activator substances also exhibited positive staining with

this technique.

Explanation of Table number values is as follows: ---, no
data available; 0, no inhibition; 1, slight inhibition; 2,
moderate inhibition; 3, heavy inhibition; 4, total inhibi-
tion; //, activation; #0, no activation or inhibition.

Ml

caused no inhibition of G—l-Pase and little suppression of
Cr-Pase activity. The addition of thl2 to the various sub-
strate solutions did not seem to inhibit or stimulate phos—
phomonoesterase activity. This was in contrast to the usually
accepted fact that Hg012 is an activator of phOSphatase
enzymes.

Substrate Specificity was demonstrated utilizing stain-
ing intensities and the presence or absence of enzymatic
reactions. In general A—S—Pase and A-3—Pase gave lighter and
less intense reactions than either glycerOphosphatase or
ATPase. It can be seen in text-figures 1—5 that if the reac-
tion for glycerOphosphatase was absent, the reaction for
A-S—Pase was also absent. On the other hand, the reaction
for glycerophosphatase did not seem to depend on he pre—
sence or absence of A-S—Pase. The behavior of A—3—Pase in
this respect was essentially the sane as A—5.Pase. ATP
usually gave the best results of any substrate used, i,e,

a good distinction between control slides and substrate
slides could usually be made. ATPase activity was in most
cases demonstrable even if the A_S_Pase and A—B—Pase reac-
tions were absent (text-fig. 5). The reverse was not ob-
served. When A—S—Pase and A-3-Pase were present, their acti-
vity was less than that of ATPase where direct comparisons
were made (text-fig. 5—6). The ATPase reaction usually was
less intense than the reaction for glycerOphosphatase in

tryptone-citrate solutions with added HgCl2 (text-fir. 5).

Q

The reactions for -l—Pase

and Cr— Pase tended to be

higher than those obtained for ATPase and glycerOphOSphatase,

and always higher than the

(text—figs. 5—6). In general there was

Cr—Pase to give a stronger reaction than

cells were cultured in tryptone-citrate

14.2012 (text—fig. 5). I-v'hen on 3’ tI'Y‘fi‘COHQ

culture medium, the reverse seemed true

A differential location of enzymes

f.

T geleii was not

4-.
—

monoesters in demonstra

eaction for A-S-Pase and A-3—Pase

slight tendency for
G-l-Pase when the

solution with added

was present in the

(text-fig. 6).

attac1m1 bh s

‘

o pho-

,1.
O

ble in 0 5a nisns

from tryptone, tryptone—thlQ, vita min-enriched tryitone, or

vitanin-enriched tryptone with added MgC

presented a different pattern of enzvna

12. However, ATPase

tic activity in cells

grown in tryptone—citrate solutions with added MgClg

(O. 01H, ph 5. 5),i .e., both nuclear and

was deronstrated.

J.

1 L

6. Euclear s aining

y-

gelei .

Q)

Iucle

T.

was observed

cytonlasnic activity

facts observed in

most frequently

with glycerophospmi te, G-l- P, Cr— P, and ATP, less frequent-

ly with A-5—P and A—3-P, and never with sodium

,
a 11

cid p cephate (azo-dye).

With few exceptions (noted below),

in control solutions and in the various

tions also exhibited nuclear sta ini ing.

0.2:]:

leCGTODhOSphate for 24 hours 5 ave only nuclear rca ctions (fig

0

i:

citrate buffer (ph 5) for 15 “lflUbCo and incuoa

alpha-n hth~l

ganisns incubated

LhiL itor re ra

"d

P3

Cells subjected to

9

ln

1

80.

.18).

43
The nuclear reactions observed with ATP in cells cul-
tured in tryptone—citrate solution with ac ml d LgClg (ph 5.5)
alape ared to be of a different ty and intensity than the
ther nuclear reactions (fig. 19). 1ne nuclei were very da ”
and hie ghly discernible. Cells from the same cultures incup
baa te d in A—5—P ,G-l-P, and Cr—P substrate solutions (93 8.3)

d not show this sane type of nuclear reaction. The nuclear

O;
H.

reaction in ATP we 5 de21mo strated only in cells ra.ised in
culture media where the 0.01M citrate buffer (pH 5.5) was
present. However, the reaction was not always observed. It
was seen when cytOplasmic reactions were either positive or

negative. Nuclear reactions other than the one just described

seem to indi cate the presence of di ffu on artifacts.
It has already beenr n.entioned th at nost specimens of

T. geleii exhibited preformed phOSphates. It should be noted,

however, that in dividing cells preformed phosphates and
enzymatic reactions were either entirely absent or a few
the nu—

-.~v

faint— st: ining reaction granules were located near
cleus. Posterior cvtoalaS“1c rte ctions were never observed

A diffuse g-ra" staining in the cytoal sn

Ho
.5
n.
4
E—J.
{34
In].
Us

0'}
O
O
H

Q
.

. . - ‘ an 1 ~ ~ . .w 1 an o 3"! n a s"-
1mned1atel¢ hen mtl the pellicle 1“ all ozganisns was assa ed

l. Ca—cozelt :c ago—eja o;oso;atase. G llama and Jackson
'1 ‘10 7pm,“; .~r\ «n:— -.1.‘ V1 2' A .A - . .
(i93oa) 3-1“ 312:5. Lat bots pLObpflOLleothaSES an“ ohos:
_ [a 0" r- o J. '0 ‘ J_ h n on“ O
{Jeu 1h a variety Oi plant end ¢nlmfll tis-

sues. Ph s;io”1esucrrses lioerated phenol but did not swlit

- 1‘ *1. . r - 1' . 1' ”v ~ ‘2‘ 1' ‘ ‘ "I
once 3110 l aCiu frog ilpdeflglpuos;flate. 1neseautn018 con-

-

'2:

O

cluded that polynucleoti6ases, which Split nucleotides from

nucleic aCies, were oiosohoiiesterases.

eat on two leaetions: (l) phospiocicstereses split nucleo-

J-

tides from nucleic acid; and (2) phosohomonoesterases hydro-
-formed phosohetes in the tissues of
higher verteorate animals was renortee by :OL 1rne (19+h).
Elliott and Punter (1951), who imi'es ti; ated phosvhetase

(S-uueleotidase) in 2, f leii, dc: castrated the during he

.4

Kamen and Spiegelman (19%8)e°.intamc that if "inorganic
phosphate" was stored in or anisrs, it was probably erived
from the breakdown of organic oucsvuwt . hill er (19h?)
believed that the accumulation of Ca pies ohete in iixed cells

was an ar tif (wet.

u

\J'l‘

iis strcly s; o"ec1 that the position
of preformed ohosphate ("inorganic phosphate”) in l. geleii

was in essentially the same position as the purple precisi-

tate which was obtainefi with sodium alpha -ncp 1thyl acid.

d“

phos) late substrate solution. lhis 31::

('50
Vt.)

s that if phosphate

'3

storage occurred its location was in essertially th: sane
osition as enzymes hydrolyzin; organic onogvnate co pOllndS,
i.e., in the posterior end of the cells.

Other 0 ser vations made in this study sugges t that only
Ca—cobalt piosonatase (glyceroohos hatase) was functional
Turthermore, Ca-cobalt reaction were inhibi
from citrate buffered (0.01M, pH 9.5) tryoto
and they were co pletely suppressed when the concen ration
of citrate was increased from 0.0lI to 0.04K. 0n the other
hand, organisus fro: the 0.0%? citra'e-tryptone solutions
exhibited strong jositive reactions with the azo-dye tech-

nique. nepetitions of these tests auproxinately 384 hours

tryptone were oositive
teanique.

A discussion of the fuiction of phosahatases can be

established on the basis of presence or absence of enzyratic
activity in organisms fro: various culture solutions (tryp—

f1

tone, t yjtone—Igo 9, vitamin-—nriched tryptone). Fennell

.1.

and Karzke (1954) also founfl that svecimens of Totrahvrcna

/
40

‘ f
C.)
H
:7
p.

156 in vitagin—en riches tryotone for several months

did not exhioit phosdhatase activity (Ca-cobalt technique).

Ho

Observations wade in th 3 Study showed bunt organisms
Twita‘in—enrichefl cultlres exhi hited abundant nh037hatcse

0 O I a -\I .f‘ _: fly.“ I I \
activ1ty Vlth the w,o—d_3 e technique. JlJCC it 18 well esta

lisheu tiat grovt d1 of TC trahvrena was cooleiated bv aflditio

 

 

of Vitanins to tryotone solutions, ib see 8 rec sontole to
assunc that Vitaxins su7ply some ssecific unit, i.e., pos-
sioly corn rte", v ich hates it possible for ago—dye on s-

.1..:. '- 4-- - Ya . . . . ..
ase to exert a role in ne synthesis or plot07laSA.

(D
t
.3
d
‘9
d
J
L24
.7
I

7 n .7 f7- ‘ a“. .. ”J—

neyernoi anu ureen (1950) dc oastr_1
- P‘fi . 1. - 1 1,‘ A ~ - r0 7- . 1— ~ I
‘ase reacts Wltfl pnosonate acceptors as »ell as wits pnos—

phate donors; i1 no acceptor is present, the phosaha e be-

. l4. ' . ., - - A -‘-~nJ.- ...- . ' .L I”. .- ..
CCCGJbOT 13 present, tie phosj ate ;“OLp is at ilrst trans—

‘

fer ed iron the higner to tne lavcr encr"y levels oc

;ni Ca-cohalt p os-

o L‘

‘rnei v: n

SJ
0
O
0
:j
3

‘nn-lv‘ . -"‘,“’:' 4‘1 ‘.-“-‘— --~(—v
j7<atas—s 1413c l ti autism, .nie latter .1;r
'-. ~ 1" ~. . V '7 (N - '. '. or‘ ' \ ,- .0 . - ‘ - 3 ~-
tr nsnnos7lorjlation, i.e., ph037 e was tran.ifrrea from

,

a higher en<r3y co 7ounc

J

b0 a level level co 7ounfi. Further—
:ore, both La—cohalt arfl are—five 7h037hat7se fay cooperate
in biological synthesis. lovcver, Ca-coialt uhosuhatise ma?
iunction in ,le TC?C?S~ manner in cells *sixtsinec in tr"7-
tone sol finals: for scvrrffl_;xxrfl1s, i.e., ifi3:33 co:c:rned.

V " O Q
;conosu;o;;lati a. as a cens.fiue1cr, 7 os.;t

accufulates in the coste770r an? 0“ tie cell.

q _ o -q o 1“! _ 1 - o- .“ I ’1‘: _
Coserrations cescribel in the 7Cecezl33 713:3 lialc ted
4-- J- ‘A 4-. _ o _. 1 “L '_o J_ r 0 r1 N _o _o
two 1 tJCallv occurr Ln sr7strttes existed in i. Cleii.
—

anisns were culturea in tryptone—ICC 3 solution and then
were rashed with 0.0ht citrate buffer (7; 5.1) for removal
of preformed fl71037hates. Stron ’ly posi tive 7ho37natase re-
act'ons were obtainec in the posterior region of cells after

.1.

resuspension in tryptone- _K3Clp solution for tvo hours sub—

sequent to in ersion in cobalt nitrate and an oniun sulfide.

It .ight be Cr ued that nositive reactions vlich vCre ob—

c+

ained with this procedure .ere d pendent on diffusion of
preforned p11037hates fron the culture solution into the cell.
Other observations do not supjort t is views Or-anis:7s tZ1Ct
were washed in citrate and transferred to Gomori glycero-
p11ospe7 te solution exhibited the sai7e tV7e of reaction but
to a lesser d.3ree.

It was shown in he preceding pages that Mp ci ens of
2. seleii e::hibited, after pro13n3ed culture in tryptone
solutions, an abundance of cyt07lasnic vesicles. The Ca—

tive reactions in ves-cles

H.

cobalt procedure revealed that pos
were dependent on glyceroplosphatas- since preformed phos-
phates rere removed Lit} citrate buffer prior to the stain-
in3 p1rOCCaure. The question arises as to the relationsi1ip
between 7aosnrstase-7ositive vesicles aLd

positive a eCs in th- posterior end of the cells. The latter

C)

reaction was found to be lepenient on both preformed phos—

phate and alkaline phosphatase. A co 7arison of the r action

,4
(D
*‘3
(D
p.
{3‘
04
H
(.0
rt
.5
D

that the heavy conCCn tration of pre foraed 7hQS7hate in the
posterior end of cells was de7endent on alkaline 7h087ha—
It is still o7en to question as to use taer or not pre-
.7ate is utilized by dividing cells. Observations
made in tais study lend su77ort to this View. It was found
that preferred phosphates ni3rated from the posterior end of
the cell to a 7erinuclear position and ultimately disa77eared

entirely after transfer of or3anisus to free;

 

 

he 7recedin3 7C 3es

(7+

he results described in
for localization of 3lvcer07ho.s 7tha in various culture

7-1
r1
uL

media are in general Coreenent witll ties e of ennell an

Iarzke (1954). The use of A—5_P as a substrate further in-
creased the validity of the evidence presented by these

authors as it was now possible to compare phosghatase acti-
vity in organisms from various culture media in two differ-

ent substrate solutions. The activity of n-5—Pase, a though

mnerally lower, tended to 7: rallel that of gly30207h037ha-

tase. Decreased activity of n- _PCse ni3nt be eruleined on
the premise that CaClq when adeezl to the in uoation 1 Ciui1

acted as an nhibitor. hep7el and hilnoe (1931) found this

49
lyOphilized snal {e venom .They found that in the presence of
M3312 the reaction for A-b_ Pase was inhibited by CaClZ, and

his inhibition could be overcome by the ac ddition of more
NgClQ. Kovikoff (1932) also observed that calcium ions strong—
ly inhibited the action of A—b-Pase. In these experiments
both HsClg and CaCl2 were present in the substrate mixture.
Assuming that the results obtained by t1 ese workers are
correct, the conditions for A-S—Pase inhibition were satis—
fied in these experiments. The possibility that hydrogen ion
concentration was the controlling factor in lowered A-S-Pase
activity cannot be r1 11ed out since pEI values used in these
eXperimcnts were at optima for maximum enzymatic activity in
mammalian tissues, i.e., pH 8.3 (A—b-Pase) and ph 9.4 (gly-
ceroghOSphatase), Gomori (1952). Optizm uhydrogen ion con-

entration for enzynetic activity in Tetrahygena may differ
from that found in vertebrate issues.

The conditions causing erratic A_5-Pase reactions when
magnesium we added to the basic culture mediurn was not sat-
isf actorily explain M on the basis of Ca012 inhibition or
unfavorable pH outima. It was evident that addition of mag—

nesiuu ions to the basic culture medium tryptone) did not

~4.)

‘40

have the sane mtivating effect on A—5_Pase activity as t
did on glyceroohosohatase activity, i.e., glyceronr osphitase
activity reached a h 3her total level and was sustained for

a longer period of ti:. :e than was A-7_P:se act1v1tv. These

results su33cst, but do not demonstrtte, that :30 2 is not

I.“

an efficient activator of A_R_Pase. 'ac
and Iclanus ,t al. (19b2) found that na3nesium was not essen-

'a1 * auxin}: A- - as- a, ' 't wn_. I— ‘- an“ I' moe
t1sl f01 P1 1 11 5 P1 0 ct1v1ty, 1ile eppcl d 11

slvcine buffer 1M uced

{I‘M uh‘ a.

(1991) found that addition of £3319 to
aronounced activation of A—b—Pase.

It was obs e1‘ ved by Fennell and Larzke (1954) that lit-ClO
L.

:1
{1)
1*
{:5
o
"3
0

‘5
m
rI)
:1

added to the trvdtone culture :met glvceronhos—
phatasc activ1ty. hesults obta'nel in this st‘dy are in gen—
cral agreerent with these findinfis.

It was found during the course of this study that the
addition of vitamins to tryptone—I3 Cl2 solution increased
A-S-Pase activity to a greater de3ree thaz1 glycerOphoSpha-
tase activity, whereas the a lr?.ition of vitsrlns alone do—
crsased A—b-Pase activity.

Fennell and Iarzh e (195M-) fou11d that cells cultured in
Vitfii'fifl- enric} _ trywtone solutions exhibited decreased and
1

ultiuate inhibition of .1 cerov oss*st se activit" and that
t

ic activity was erratic after the addition 01 I331

bear out their observations in tiis respect. It would seem

-- : ..,,_. ° -1 .L .1. .3- ' . 4.1- ' 1,, .° 4..., 1.1:
fr01 oaser.e ions nude 11 t1ls study that in Vitanin—enriched
fl -. ,-.. 1 4“.“\ '-1 v-v- .z—‘r‘ - .1.
a lesser cc.rce than . cerop M 1A1 ese, alt

p M - a. ~" Ln ‘0 A...‘ ‘An 1" _o ”‘0
01 3lyceros osn at se. inis su33csts that perdaps Vitau1ns

O
H-
} 3
O
H
C
{J
U)
(D
C1-
Fwd
,4
O
F
0
C1.
H.
1:;
F'.
ct
K:

along with L301? onerate in SOTO way t

of A-g-Pase more t 11an that 01

s1

1

The lowered activity of A—S—Pase and A_3_Pase after sue-

M1
0
\n
V
Pb
o
H
V
b
C)
:1
E3
(+
m
c‘
g
7”

s titution of 0.013 citrate buffer (31
fer in t“"otone-n Clq solution was attributed to tne citrate
buffer. Furthe1: re, activity of A- _Pase and n_3_P1se in
rate buffered solutions was alieccea to a
than rlveuro11osesotrse activity, e.g., A_S-Pase ana A—B-Pasc
did not snow the gradial increase in activity as was tne case
with other phOSphononoesterases (31"cer031o 1hetase, etc.).
Increasing the concentrzztio n of citr:.te buffer from 0.01K to
0. 8K corp etely innioited glycerephosphatase reactions. It
seens reasonable to assure that 1017 concentrations of citrate
(0.01H) 1ad a tendency to arour Ce a were wt.ble acid culture
neCiun than phosphate buffer. As a consequence, growth rate

of or 3anisns was naiLtained bu at a lower level than it we

U)

i tryptone solution. Furthernore, omission of the phOSphate

ru
y.)

be ffe or decreased phosm1 ate eo;1centration in the culture sol-
utions. Under these conditions enzvtatic activitv was de—
creased to a greater degree in A—5-P and A—3_P substrate

solutions than it was in the other susscrate solutions used

Results presented in the preeeCing pages suggest that
phosunatase reactions in g. eeleii are directly correlated
or ‘ne culture of organis13. It
seems reasonable to assume nat tryptone solutions contained
suboptinun conce11t1ations 1 accessor1r growth-promoting
substances. In citrate buffered (O. 0“} ) vita: 11H -eLriched

J-

tfrp cone solutions transfer 01pROS1has1 from Ligh energy

C: ’3
(—

co pounds to low ener5y co pounds was inhibited an 5rowth
rate of organis's was retarded. Kann (199%) shoved that in
the mold A's1er5 illus :1i5er the type 0 5rovth ncdiun marked-
ly affected such thin5s as the rate oi ohosghatc utilization,
phosphate content, and growth, etc.

3. lnzvre inhiaition aLi substrate specificity (nUltiu le

1e utilization of inhibitor ubstances

Specific enzymes hydrolyzing multiple phosyhononoesterascs

in l. rfeleii. It is sisnificant to note that all eno,-es

s)

were aci and heat labile. Iaen“""1—D°Viese al. (1970

OHJ' _...___.
%72) and ev"1n ct al. (1973 nede exhaustive studies on
phos.ha tase localiz1tion and reaction to inhibitors and

activators. On the basis of their findings, they concluded

)

e functional in mammal-

"3

that nu] tiplc 1n031neronoe° rases a
ian tissues. En;cl (l9k6, 1950) denonstratci that the alka—
line pnOSphatases of both the mammalian kidner and intestine
differed in their sensitiveness to KCE and HCl inhibition.
Fennell and Lerzke (19's), using only glycerOphOSphate as a
substrate and senicarbazide (0.023), sodium arsenate (0.01H),
and reduced an. oxidized 5 utathione (0.025H) as inhibitors,
were unable to reach a conclusion on the existence of several

enzyme systems in l. releii.

 

r—"~ ° w- 1 '. ?\
.LAAO 1.1.1411. 5.}

tion or activation 0: phosphatase enzymes

H0

in 2. geleii was the princi oal :et;od 1805 for making a

distinction between them. However, intensity of the enzymat-

ic reaction in specific substrates was also uti ized. In

the posterior ends of cells were Obtained wita .cll sai)tiat
alutions. The activation of ATPase by KCN (0.001h) was re-

:ortcd bv Stern et 5;. (l9bl). The ability of KC; (0.0lh) to

activate only ATPase in 2. scleii sup1orts the view that this
enzyne is a distinct entity. ATPase activity was easily do-

monstrated, i.e., preformed sheephates did not occlude en—
zynatic activity anc in many tests preformed phosphates were
lackin51ltoge her. On the basis of these observations, it
appeared that either the concentration of ATPase was nirn,
or Specific activators of anase existed in ietrahynena. Add-

itiono £5012 to the culture Hol-tion neither increased nor

d creased A”Pase activity. Daile ey (1942) and Dulois g_

(D

(1943a, 1993b) found that calcium was a Specific activator of

‘hlS enzyme in mammalian tissues. Since CaC19 was adwed to

H 0
0.1

the substrate solution in these experiments, it s su55este

x)

hat the Ca ion may have es crti lly the sane ctiV1tin

effect on the ATPase of Te raavncna. The presence of AEPase

in Tetrahvnena mav have in vivo si: 011 icance in that it re—

‘4_7 ‘1 h

 

leases nhosghate from nEP, a suosta1ce cont- inin5 hi5h energy
bones. Thus, ener5y is race available for netabolic needs.
It may also funC‘ion as an activator of the substrates in—
volved in glycolytic reactions. 13? has also been found to

be identical with or intinetelr bouni to nyosin, the con-

tra tile element in rannalian muscle (Dulois, 1993b). Its

€17? T3 03. INC-7.1108 in

div'sion

I

c:ezj_l

t1". .1

LhAax

54

SSOCZLE'.

"1:11,? b0 f‘
\J

J- ‘ . - s» V -. 7.
e “my tea 111 sc>1e 1r:;’ ruxtn

d body rcr1ool1s

saialnstrate solutions, and the differential effects of inhibi—
“tcsjrs, iidicatcd the esesence of two differert enzymes. Iow-
Cfircar, on the basis 01 Karrirents performed dur’ng nis
iatxrasti5ation, no data was obtai11ed that secned conclusive
enuxau h to swranort this vie: 1x10s ure of or5anisvs to sodium
zirxsenste (a Olh) an. ISI (0.011) prior to innersion in
£1—Q3-P induced erratic st1in ':15 reactions in Specitens of

2;. geleii, i.e., results were seldon duplicat-d. O. the other
lrsiid, or5anisn incufiated in 1.5-? due!1 to 1nrersion in

C‘
L.)

‘.
K,
‘W

-glv’Ll

inc te-
and

8'91 it 11-5

f1 r-L‘.’\ J
“ K43401” 31171101’5
'5 'L A ~~ ° I"!
sit ”“ses 1n -.
’1'" 1 - , -n .1- ,-
‘ “S 5101, 812-100 6:7.

-.f--o:~rever ,

1 ‘If

"def.

" '3
t.‘.l can

(«r1

. 11
lsbvfig

Kennel

P "110'. n-

H.

-« ca

(0.25I), ano sodium

.1. 4.1- -_: J. -_- . ' -
to the e_ 18 tence of ~ ...l Liple __1-. o" 1.0 -:ono—
,‘ . '. —/\ P's—s 1 J- . (8 L: v -: J‘V '. -»- r. _" a ‘
«8101 . 11nu :1 1c aetithwr.u1 l— .111 Jr-P
‘ 7v . J—. '\ w ' n A-~.- .1-
;J‘;. G]— L)‘ (71.1 5301—L11 \j-Z‘IS‘. .1.\,-UG (KM) . _.'\’Il: :) or I120? .

1‘1 '3‘ _ _ 'V _‘ ' ‘7 .L. r‘ ,t. _I _O _ - a' 1
lvce°0JMQSJ1eease ECulVl v fies oo-

.5.
'L QHU ..‘ ._ v

Kowever, ifiAioition o

n _ 37‘ "’n"“ 3' o 1“ '3" A 0“? o a o _ I3 r‘1'v _ _ ~‘
nervel. Mel (O.ulh) inelccd More lmfllDl 103 o; O_Jee°OJMOC-

‘ '\ II"r. -» . “.1 A. _~ Um C 'r J' \ A l‘ " , 1
iijase £433 e1 Mcr M-l-Plsc or Jr—Lese. u; b - 0 -Cr ewe,
~ vv 1’ [fl fur-I:- r ‘ (71- .‘*‘\:".:'L: * ’- V'A/K-v \ I" r1~ ‘ J- A
81; 1-6 \u..- ) <3M1~C ore 1J4h-plblon ol .rn MUOPAOQJIQbOS“

- , -.-‘\ ‘ .I‘ § -.'~ R .A. -f-. 7’1 r ‘ 1 '2 . H < ‘ ‘l' - -- I. 1 '. 1“."
JnoMon0u£ueheses o. ;. Jelc-1 aQQCdf Lo De Que eluMer to a
-~1 471”": J ’9 “Hobo «a - cw “a fix (“th o "tn '3 4.5,- n
' CO; ;D-L.--r.- k;-LOL1 0-; C:.L¢..JL.J 0.1. 1.0. .3 O. .C’ .4»: penile.) .1 <.. ‘ .ngblg,
- - ,3 ° ,.- ° - ‘I . . - J... ,..
cleurly ulSCnfnlblG Jr cese. LomJlete puo.thor once. ergo
o _ 1‘ o 1" _9 J__ 9 _. a _o '1 . ‘. ,' r '\ __o -| .7 .. r‘ .1... _. _'_1 _
lgllJlglon by aCLwS an» .m. lclrJ Zifle la SqubLOfl sceuee b
1 - ‘ ~r--~ (‘3 I“ F‘ (N J‘ 'I r7 . 7. r 1"* r1 '
be eue t ean-,;e . ell «£131- etloM Dy ;.J‘*.,_ro'|vs 5'; . Pre-
“‘ "\ 'L VA A, o 1 f‘ . f1. - . ‘. ' J ' .\ . :1
p-053Mabe we soluble 13 Hell solubloa and se 1corb-z1ue,

_ ' J- .- , fl, -' .1. .. . _ _
resulcin; 1n comslebelg negative conurol and substra e

?

amLo, pertiel reduction in stain1n7

. . J""P -. 4" . 1 ‘3‘ “"\ A V n 1" ~-~« . fl “‘\ - ’. .
uCtlvlbj ajjOaTCQ b0 be tue rhs7lt I 04em1cel coMllnatlon
-. -7-“ ° 7-°1-'- T134.“ 1 v. A ,.:.fi,. ' -.. .: °|,. ° .
of tlu 1anglLor , u“ tde enzyme, result Lo 1n ibelpltut101

or inactivation o; 610 en~y;e involved. In owner cases, a

cowklhrinxx: of the vEZITKTIIL. tion prthxf‘ (3 OQW£“*F) I’i' m1

A . '| 1’ . 1 '3 '1' A f‘ . ‘ ‘ ‘ ' ‘1 ‘- : "" . 'fi .f‘ ‘, r-v'vw-q r\ J‘ .
tha lflfllQleOT resulted 1n an app; Cut TCuuC 1 M Ol enoJM.b1c
’3- _’ o T _o tVI n "‘1 __i {31,1 f...‘ ’79 f—_ 'J,‘ 4,1. .0 1‘-..7 ”1: (""1C‘ 1" a .10 ;—:t J.
MC bl ii J o it -.;O..L 6 “013011ng ,._.._J_ _-l.LAb OJ. 0]. u' .l..-LQ. .u $1. u.) be 1 b0
- M. . n 4.1., ,. '. .Lx 1. 6.x--. .1. '1 ...L.: ,‘H, ,. 'v .1 .-
1anr810n o: “e cello lfl ble sung CDC SOLMVLOH TCLMC:Q tAlS

tYDe of intioi+ion. Hot water (8 O C.) inhi%ition seemed to

work by COJ“W7¢J101 of the en: rxe protein, tlus reducing
activity to that of the prcl rxed p 08M .Iate prescnu in bne

cell. 'ther anzfime in: Jitions WJJ”“*CC to Eesult lr m a

7" . h J‘ . '0 J‘Q'.’ . t ‘ -.‘J‘. ‘ ‘ ’. ‘ r3 " 'J ‘
COUblnublOD ol ble anove MGHLLOJCJ c.lec s.

The ability 0? the phos Jho: enorstor ses to rosiSt these
r reactions apparently was the reason for the Speci-
ficitr of the inl1ibitor--e1m"m reaction. Active tion of

'v seems to be the result of the activator
M.ter1al squJl in 13 a needed metallic ion to t1-e enz:,711e.

.h
J.

nescrele o .e of action of these inhio Jitors,
the Specific inhibitory effect prteuced 03 then on certain

~ . vb v 1 f‘ I) - r". hJ'.~‘\ o J-‘n n -.
enzYnes 13 a Very e: :cellent *C~e oi scfluret1n’ tne verious

U
if
Q
'3
p.
}
Q

.3
O
53
(L
(’1
rt
(3
l J
r)
U]
i”)
{2)
pro
0
1...)
.L"
O
H.
(.1.
Ho
O
}.
r
"x
H
J
(“A
U]
< t
:3
’4.
:1
i“
D-x‘
3..)
C W
H.
:3:-
Cr
0
1’3
r3

I

sity are of slight or no use. Eovevcr, it was roune not
unless prOJer cent :15 ene bCCHHLOhCQ were maintained, the
use of inhibitors with the Ce-c obalt proceoure yieldeL c n-

flicting results.

‘3

4. H JcleJr ensv“os. ine Speciiicity of the Gonori tech-

-

Ilique for den nstretion of nuclear JDOSJhet ses has been

questiomx d by various int est istors; an ng then were Gomori

0‘...
(1951), Lcduc anf Dempsey (1951), K8 ti“ and Jacoby (1949):

ane Zovikoi f (1932). Tnese invcs t1; o.tors believed that nuclei

F7
Q)
4
O
p.)
H
E) *3
,..)
’4.
C}-
< 1
{-5
0
FS
0
C.)
6

10591836. rurthcrnore, enzyme

csc*1csl means, v”1le De“sce (1943) use? er'lyticol procedures
1n ‘sc ce‘onstiction of J ospnatcsc—posit’ve nuclei.

. , 1,. 1 4-. .1- :1 - m ,—-~ ,."1 ' .- ° -- .. 1 -L- J. J 1
could we CE“ nss°et6u 1n -. ale w1tn ell suestra-es uses

phos1hatc and that cells

1.:
C‘

Q
(fiJ

1 . _ 0 o 1 L-_..-‘ V " _ o o .
suo1ected “ o 1171111111101“ sues L.- .nces rfc J:1e:1tl3 ' e...-1b au-

as did most control slides. These servet10ns

U)
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The lack of nuclear stainin in the ago-dye procedure

was pointed out by Go ori (1951) and Eovihoff (1952 . EKJeri-

.4. 1
flee"

k

U)
(Z)

1.2.
i
d-
f 1‘
1.1.
m

‘u
’J
J
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H
(a
d-
(D
r;
e
O
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I

n nta.l ev:Lcc :Jco er

' \

. 1

nent Wltfl their observations in this respect. Artifactual

nuclear staining with the Ca—cobalt procedure, however, does

not rule out the usefulness of this technique. Critical cor-

oarisons of nuclear staining in cells incuoate d in the

y.-. .3

1

[.4
"J

as d to the pre—

~ 1-1 jA'T‘ L- .‘J‘ . '0 ‘ . n J‘ n n
sarel (1 onsUrLtion Oi a non—Lrtifactual nL1cleJr reaction

etc. The ATP rea tions were darker than t ese obtaineJ in

these sues tra es, and ATP controls n ver exhi Jitea nuclear

.L‘;
b3

ty under

v Q
\ "9

‘1

fl
k

UClCL

‘.
.L

10

ClL

A
k-

-
f

*fied.

‘1

(N

. '1
2C

CO

(1U? —~. '.'. 0‘7
J.

‘ 1
L2 -- .31; L4.

1. Specimens of 2. fl leii us ed for studies of alkaline

A, J-" 1 a V .1. _ ° :3 ... J. -
use ”81 e OD 3413100. lI‘OJ 133C onlZl—lI‘Ce cultures ran

080 hours. In most ixp*fi iments

C)
Pl)
H
O
V
l J
5
,C}
J
3)
d.
O
H
9

culture ag» varied from 72 hours to 360 hours. The tecnniques
of Gomori (195 ) were used throughout this study to demon—
strat e a_lka ine p‘; ospha'as .

2. Seven plosyhononoesters WOT; used in these exec ri-
rents: sodium glyceropEOSphote, muscle ercrrli acid, yeast
adenylic acid, acenos ir e triphosoaoric acid, glucose-l-pnos—
enate (potass iun salt), creatine 3h033het e (calciuu salt),
and socii ium al3..—:apithyl acid 9 -033hete.

3. Three t3pes of reaction granules vese ootaincd by

C
U)
0
O
*"3
ct
. f
C)
S
I
O
O
(‘3
l...)
l-
O
c %
O
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g.)
v
r.
".3
O
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J
4..
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:3
H
d
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C)
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1-1-
d
[—30
c—f.
' 1
Cl"
0
U)

‘ . ‘ ‘ '9 '. fl . ”" ‘0 'F 7‘. : ‘ ’. ‘ I‘ f O) N "’ l‘w'.
(2) an lflteTflCLLQEG type oi piocloitate; and (3) laroe granu-
lar precipitates containing srall spherical vesicles. Tnese

could be produced experiuentally.

4. It was possible to demonstrate a number of phOS3ho—
nonoesterascs active ii the alkaline an" : :lycerOpnosona—
tase, AlPase, G—l—Pase, Cr—Pase, and A—j-Pase. variable

1 0

results "ere o:3 , incd witi A—3—Pase. These enzyme 3».
forentiated Tron one azotlcr by their difierent

res 3onses to activators and in

in he relative intensities of their reactions. Localisation

could not be relied upon to indicate intracellu ar differ-
ences in enz"ue .

5. Eagn:siun chloride did not 330 r es sentie_l for full

a -L: 3.1-- .' - 2-, .9 r"! F. ,‘3: 7* '
n.os3ilt( e act v_ y in soec33ens oi i. ifilhll. “o'ever,

f“ ‘T -’ n A? -- Ar“ 3 v ’. ¢ "._"~) 91A
uCn 0.0lh) cauSee lflOngSCfl Ht vlty of Ai-aa“.

‘ rn‘ t ' ‘ . H Li“ L .4 '2 _0 W “ - Q I
e. lee type and age of tne culture Leela €.)lOJ:Q in-
c’ P ’ - q_‘ A ‘1 - 4‘ 4 . o .0 -L o _ A o .. _ .
fluenCed .ll.lioe pnos3xatase actiVity in speci OHS of
m . ‘ o I A, r o m 4- v7_ .3. _ trf‘fin v,‘ 7 .0 ‘ s ‘ro a _o ‘
;. releii. sells “sired in t333tone-n-el -e.ia ELLiDutGd

 

'V A

high glyceroolosdhatase activity tm‘r u":out culture liie.
On the other hand, n—fi-Pase activity was erratic. Revise—-

M .1. o 1-, -‘. - _._ 1....03 . ,- . .1. ° .3. -... v- . ,-, .- w
-ent Oi tee p.os3iate mailer present is tr33tone media vitn

7' 'J-- ~~ r" L: -. a 4—1, 1 . .1. :;:°4.° .
a 0.0ln Cisrate ei_£er (3H 3.3) ail tee sueseqaent adeitioe

1 . ‘ 1 1 _ -‘ -- _ 'V J. o ’0 J. 3'; - V n o .0 cc"
of LSClq reduced 3hos l;tise QCelVlb . 4-5-Rase actiVity was

’3
F.)
O
«:1-
d
:3
(_
A
‘1
H
.3

arsent or ne rly so. Glycer03HOS3latas trfotone,

tryptone—IgClq, vitemin- enriched tryptone, and tryptone-cit—

rate redia with QCLOQ ggolg was niseer than that of A—E—Pase.
However, the oo3osite effe t was observed in vitamin —enriched
tryetone solution with sed_eo cl I;Clq.
.- LL
7. In the absence 0: glycer 3h S3ha ese activitv, no

..,- 4.. V. 4.: . . 3 " 1.2...'.,.- :1 -. . .,
en"*“tlc reactions CO‘le De oat lASu l r “—5—?ase 01d

‘. r' — J... , +'-,—:.u .33 4- r .2 :»
n-3—Pase. .1; coco) o3ese active? (_:,.1_ 3. ‘iot 333(31r to -

0 fi- . J, r 1 a“ r\ . . J- . - fl - n a [q . J-
o. -r ' i ;. w-leii COlt“lQCQ 3re- r 3. 3 os313 es

4-3 .2- , - .1 J- ,1 , °.z_‘, 2-3.. *3 ~ ,- .1. .1 3 .0 m - 3
wife ' €36 .O;T--’3..l“r.=-DlC-3 “'le L3 L€ DC—CeLLle {CC ”If"? Oi J 337.2.

/‘\"" 3‘2,1\]‘Lf"-: ‘1 rip-sap «(‘54 rj 3‘V14‘r‘ O .DT”V “071'.“(3': 3. fit“ 1‘ r‘ 4'f‘r‘ T't 7"“6‘
-4 *‘4 -LJ\\ .LL“J.L .. \‘4‘ ‘ L 0 ma K) A. .A» -- \ uL ' \. 4‘ 5..) . u:- k...L.)

..--. J. 3 "m4- J-fi‘ -. a- .. - *.
Slebgesteo.+‘-r. sgis finale e,33.3mu3 d.e .AD'ULG 110:

a J- e~~ 7v -\ - -. -: 1 1 v- "1" '2 J." ‘* 3'. '7‘. r1" . n A
ohate acceotors n.or ell; si33lied e3 tne vle3,ins, 331g 3,_
' ., 1 ' 4“ 4‘ - ~ 1’ - ‘ ‘3 -'- . -' 1.7.2. .23,- '1 ‘7’
.3 5-1-1. bcr‘. 111 £3.18 S {301.2193 0.“- I), .OSUhf. C: 3.. L3-...i". brie CCl.9-. -LOY"C?V?I',
0 1 .1- ' . .0 n. . ,0 1.. ‘1 - ..,_ . -3 .1. ' .,.- ., 1 '
linoer:.ion oi phospnate iron cellular COQQOflGfluS l LCLlCtOly

-- .n- -k

- ° - - n .... 4-3. .. -, ,— .3 . - 3 - p :- .-. 3
3:?ior to .i etion was not ruled out as a source or preiorned

.
1'“ fl ' 75H 1
j 43s3e.t€.

./._._

v... ---.v -' ' — --r-\ D TU .u ' .—-— -.
9. Eng: eti c ac iJity in S3eci;ens oi -. ,Clell Jpgeared

' . 1‘ ~ . —‘ -.~ r‘ ’3- \—-1 : A 1 ~~ -~
'tc3 be loo“ted in tie UOGJuTlOI of tee cell Teolillubs o. tee

Q 1». -- n ‘1 . -. " LAN ' ‘4'”: . '. . "1‘ J“ r'- ‘. W '4 f
SLinetrate Lsed. nZO-DJG tests utilizino sue orbaaic por ion

5 ‘- 2" .QNWQ ('3 -. ."xr‘. r) 3 J‘T‘." . I’.‘ 4 4' ‘21 A ‘ ' ‘, fi 1
<3- tee “0’1 <13 - nap Unjl aCid ;3h033lew e Molecule tended

bl.-- I. -

.3— - .9: -0. -‘!~ -- -'— °
cC) co“ ir Luis location.

-‘n—

-3 --._ .3- -: -, -, r. 3 ' 3-3,. ' .J-wn ' -. .7? ' n - ,, 7' -7 , -
lO. Desert/.3. .ons . ee 1-x {31.18 tut-.37 i.e.e.ic,...tee eat C..—
‘ ‘- 4"“ "Ln " 1 ' "L' _-. -. “ 1 I“ J" P- yf‘ p "'\ 'L ’. 1
<3c>t3alt . s_3;at_sc and flOb ago-dye glospna use has lUHCbiOfl-
o _ "n‘ o -- 1 .L. r o . 1- ‘ J- .0 _| '0 _o v- __fi 1.:
£11. in OTUqfllSFS cultured in tr;p cone solut oas ior Sii eontns

’. -' -' -' ~- f'\ NnQ f‘ ‘1J._. - ‘ .‘ o. J. _- . n . .-
(31* longer. no ei r, r unis 1s cultured l; Vitau-1—enricnec

4— —— - J- . .- .23...- .n .-.1. .3 - . .° '7 .0 J-‘-. --‘_.‘1'.:_
b33377) 1.30.10 .‘...;L,..L:.l..1 4.01“ €..e -l;:_C( QCJ’lOCS Oi- H-153 -;-—.-7._L-.31L.CL; El

. -—\ .. . f. . 3- -r. a. 3-, :. 3 ' 4-3,. 7. - (V x .I- 1 ..
'tJ-Cuis for en "gnathse Detainee Vita tne va-COwalt tec1nieue

4.3. ~ “(3.0: , 1.1-.- .0 1‘ -
0 die a--.-n-1..3 o3. tee nucl ‘S

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d-

‘rrx 320. L ~- '17 . 3

-«~1?e are; ac s, *fObdle cu
‘ .. -r..- .,.., ._ ‘Jn h—.—’? .n -\ ..\-u \fi

1‘ Ce 34o33late. lot-W r, demoasuratlon oi a nuclear reac—

ieved to repre—

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LITERATURE CITED

Bailey K. l9h2 Myosin and adenosinetriphOSphate. Biochem.

Bevelander, G., and P. L. Johnson 1949 Alkaline phosphatase
in amelogenesis. Anat. Rec., lQH3125-l35.

Bourne, G. l9h# The distribution of alkaline phOSphatase in
various tissues. Quart. J. Exp. Phys., 3g;l—l9.

Bouwman, F. L. 1952 Histochemical evidence for the presence
of phosphatase activity in Tetrahygena geleii. Mich. Acad.
of Science, Arts, and Letters, 32:129-132.

Bradfield, J. R. C. 1950 The localization of enzymes in
cells. Biol. Rev.,‘35:ll3-l57.

Chevremont and Firket 1953 Alkaline phosphatase of the
nucleus. Internat. Rev. of Cytol., g;26l-288.

Clark, L. 0., Jr., E. I. Beck, and N. W. Shock 1950 Serum
alkaline phosphatase in middle and old age. J. Gerontol.,

éa7-12.

Danielli, J. F. l9h6 A critical study of techniques for
determining the cytological position of alkaline phOSpha-
tase. J. Exper. Biol., gg;llO-ll7.

Danielli, J. F. 1950 Cytolo ical demonstration of alkaline
phOSphatase. Nature, 165:7 2—763.

Danielli, J. F. 1951 Alkaline phosphatase and contractile
proteins. Nature, 168:H6h~%65.

Danielli, J. F. 1953 Cytochemistry. John Wiley and Sons,
NeW'York.

Danielli, J. F., and D. G. Catcheside 1945 PhOSphatase and
chromosomes. Nature, 156:294.

Dounce, A. L. l9h3 Enzyme studies on isolated cell nuclei
of rat liver. J. Biol. Chem., l42:685—698.

Doyle, W. L., J. H. Omoto, and M. E. Doyle 1951 Estimation
of phosphatase in histological preparations. Exp. Cell.
Research, 1;:20-38.

63

DuBois, K. P., H. G. Albaum, and V. R. Potter l9h3a Adeno-
sine triphosphate in magnesium anesthesia. J. Biol. Chem.,

;EZ:699-70h.

DuBois, K. P., and V. R. Potter 19H3b The assay of animal
tissues for respiratory enzymes. III. Adenosine triphos-
phatase. J. Biol. Chem., 159:185-195.

Elliott, A. M., and R. L. Hunter 1951 PhOSphatase activity
in Tetrahymena. Biol. Bull., 190:165-172.

Emmel, V. M. 1946 A cytochemical and quantitative study of
the effects of potassium cyanide on alkaline phosphatase
ictiXity in the kidney and intestine. Anat. Rec., 2g;

23" 370

Emmel, V. M. 1950 Effects of HCl on alkaline phOSphatase
in kidney and intestine: histochemical and quantitative
study. Proc. Soc. Exptl. Biol. and Med., ijllh—ll7.

Fennell, R. A. 1951 The relation between growth substances,
cytochemical properties of Tetrahvmena geleii, and lesion
induction in the chorioallantois. J. Elisha Mitchell Sci.
800., @23219-229.

Fennell, R. A., and F. O. Marzke 1954 The relation between
vitamins, inorganic salts, and the histochemical character-
istics of Tetrahymena geleii w. J. Morph., 23:587—616.

Flavia, L. R., and B. Pearson 195% Alkaline phOSphatase
activity during carcinogenesis of mammary tumors in mice
implanted with stilbestrol pellets. J. Nat. Cancer Inst.,
lfizllZ3-ll33.

Gold, N. 1., and B. Gould 1951 Collagen fiber formation
and alkaline phosphatase. Arch. Biochem. and BiOphys.,

33:155—165.

Gomori, G. 1939 Microtechnical demonstration of phospha-
tase in tissue sections. Proc. Soc. Exp. Biol. and Med.,
Eg:23-26.

Gomori, G. 19H9a Histochemical Specificity of phosphatases.
Proc. Soc. Exp. Biol. and Med., 2937—11.

Gomori, G. l9H9b Further studies on the histochemical
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65

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68
PLATE I

Alkaline phOSphatase activity in specimens of T. geleii w.
Figures 1, 3, 5, and 6 stained for glycerophos hatase using
the Gomori Ca-cobalt technique; figures 2 and stained for
azo-dye phosphatase using the Gomori azo-dye technique. Mag-
nification X570. Micrometer scale insert: 1 space : 0.01 mm.

Fig. 1. 144-hour cells cultured in tryptone—MgC12
solution;

Fig. 2. l44—hour cells cultured in tryptone-Mg012
solution;

Fig. 3. 140-hour cells cultured in tryptone-Mg012
solution;

Fig. 4. 140-hour cells cultured in tryptone-Mg012
solution;

Fig. 5. 140—hour cells cultured in tryptone-Mg012
solution; and

Fig. 6. 144-hour cells cultured in tryptone-MgCl2
solution.

 

 

 

7O
PLATE II

Alkaline phosphatase activity in Specimens of T, geleii w.
Figures 7 8, and 9 stained for glycerOphosphatase, 10 and
.11 for A—5-Pase, using the Gomori Ca-cobalt technique; fig-
ure l2 stained for azo-dye phOSphatase using the Gomori
azo-dye technique. Magnification X570. Micrometer scale
inSert: 1 Space : 0.01 mm.

Fig. 7. l44-hour cells cultured in tryptone-MgCl2
solution;

Fig. 8. 792-hour cells cultured in tryptone sol-
ution;

Fig. 9. 144—hour cells subcultured in tryptone
solution for approximately six months;

Fig. 10. 288-hour cells cultured in tryptone sol-
ution;

Fig. 11. 216-hour cells cultured in tryptone-MgC12
solution; and

Fig. 12. 264uhour cells cultured in vitamin-en-
riched tryptone solution.

 

PLATE III

Alkaline phosphatase activity in specimens of T. geleii w.
Figure 13 stained for azo-dye phOSphatase using the Gomori
azo—dye technique; figures 14 and 19 stained for ATPase;
l5, G—l-Pase; l6, Cr-Pase; 17 and 18, glycerOphOSphatase,
using the Gomori Ca—cobalt technique. Magnification X570.
Micrometer scale insert: 1 Space : 0.01 mm.

Fig. 13.
Fig. 14.
Fig. 15.
Fig. 16.
Fig. 17.

Fig. 18.

Fig. 19.

144—hour cells cultured in tryptone—
citrate solution with added MgCl2;

72-hour cells cultured in tryptone sol—
ution;

432—hour cells cultured in tryptone sol-
ution;

216-hour cells cultured in tryptone sol-
ution;

l44—hour cells cultured in tryptone-
citrate solution with added MgClZ;

288-hour cells cultured in tryptone sol-
ution. Organisms immersed in 0.2M
citrate buffer for 15 minutes prior
to incubation in glycerOphOSphate for
24 hours; and

72-hour cells cultured in tryptone—
citrate solution with added MgC12.

 

 

 

 

 

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