SUBSTRATE SPECIFICITIES AND INHIBITION OF INTRACELLULAR PHOSPHATASES

127 Pages·04.693 MB·English

by  KALLMAN, FRANCES LOU (GREEN)

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Xerox University Microfilms
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Kallman, Prances Lou (Green) I923-
1950
Substrste specificities and inhibi­
.K2
tion of intracellular phosphatases.
:«sw York, 19'(.9 .
86 typewritten leaves, illus.,
taVlea,dja^rs. 2gcn.
, -j-s : fH.D.) - Keiv York Univer­
sity, Graduate School, 1950.
Bibliography: p .80-86."
C50696
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WIYRRSITT HEIQHT9
\
SUBSTRATE SPECIFICITIES AND INHIBITION OF INTRACELLULAR PHOSPHATASES
by
Frances Orson Kallman
DeeemWr-1949
A dissertation in the department of biology
submitted in partial fulfillment of the re­
quirements for the degree of Doctor of
Philosophy at New York University*
The author wishes to express sincere appreciation to
Professor M.J* Kopao for his inspired guidance and invaluable
help in the planning and exeoution of this problem* Sincere
thanks are also due Professor Harry A* Charipper for facili­
tating this study and to Mr* Leon Dsiorney for expert teohnioal
assistance*
Facilities were made available by grants No. C-367 and C-843
of the U.S. Public Health Service, Cancer Research Grants Branch*
2 0
}
TABLE OP CONTENTS
Page
INTRODUCTION ............................................................................................................... 1
I. Centrifugal Separation of Cellular F&rtioles . . . . . . 1
II. Phosphatase Studies • 5
A* Specificities ...................................................... 5
B. Inhibitions and Activations ............................. 6
111. Phosphatase Activity in Cellular Partioles • • • • • • • 8
IV. Histoohemical Determination of Phosphatase ........................ 9
V. Function of Allcaline Phosphatase . . • • • . • • . . • . 10
MATERIALS AND METHODS . . . . . 13
1. Preparation of Tissues and Particulate Fractions . . . . 13
A* Tissues • • • • • • • • ........................................ . . . 13
B. Particulate Fraotionation • • • * • • .................... 14
Homogenizing Solution 19
II* Determination of Enzyme A otivity.................................................20
A* Reaction Mixture ..................... . . . . . 20
Substrate Solutions . . . . . ...................... . . . 2 2
Buffer Solution .................................................24
Inhibitor Solutions ....................................................... 25
B. Phosphate Determination.................... 26
C. Nitrogen Determination . . . . . . . . . . . . . . 2 9
D. Calculations . . . . . . . . . . . . . . . . . . . 3 0
III. Histoohemical Procedures ............................. 31
RESULTS...............................................................................................................34
I. Result* of Histoohemical Analysis .........................................
Page
II. Activity of Various Tissue Fartioulatea.................... . . 3 5
A. Relative Activity of Particulate Fractions of
Kidney . . . . . . . ....................................................... .36
III. Aotivity of Particulate Fractions of Intestinal Muoosa 37
A. Sffeot of Time on A otivity...............................................38
B. Effect of Conoentration of Substrate on Aotivity 39
C. Relative Aotivity of Particulate Fractions . . . 39
IV. Effeot of Inhibitors on Phosphatase Aotivity of
Particulate Fractions .........................................43
DISCUSSION ............................................................................................46
1. Histoohemical Analysis. ......................................... . . . 45
A. Intestine . . . .............................. . . . . . . . . . 4 5
B. Kidney . . . . . . . . . . . . . . • 48
II. Reliability of Quantitative Results ........................... . 48
A. Preparation of Tissue Partioulates . . . . . . . 48
B. Enzyme Determinations . . . . . . . . . . . . . . 5 0
III. Relative Activities of Particulates from Various
Tissues . . 5 3
IV. Aotivity of Kidney and Intestinal Muoosa Fractions
with Beta-glyoer©phosphate...............................................54
V. Substrate Speoifioities of Alkaline Phosphatases in
Particulate Fractions of Kidney and Intestinal
Muoosa . . ................................. .61
A. Fructose Diphosphate .................................. . . . . . . 6 2
B. Alpha-glyoerophosphate and Alpha-bata glycero­
phosphate ..................................................................................63
C. 01uoose-l-phosphate and Gluoose-8-phosphate . . . 65
D. Adenosine-3-phosphorio Acid and Nuoleio Aoid . • .67
VI. Inhibitions of Alkaline Phosphatase in Partioulate
Fractions . . 70
Page
VII. General Considerations 73
SUMMARY............................................................................* ..................................76
BIBLIOGRAPHY........................................................................................................80
APPENDIX
1
IHTBODPCIIOH
The problems of correlating oytological structure with oellular
funotion are not easily approaohed by cell physiologists* The reasons
for this are at least tw o;first,cytologioal structures, as recognised
by classical staining procedures, may not accurately represent structures
doourring in the living oell and second, experimental study of these
structures inevitably involves ohanging their environment and aooordingly
their properties may be radioally ohanged from the native condition* The
present study, involving phosphatase determinations on isolated cyto­
plasmic granules together with histoohemical studies,is subjeot to the
same limitations* in the following report,the speoifioities and inhibi­
tions of alkaline phosphatases in various oellular partioulate fractions,
separated by differential centrifugation, were determined* These quanti­
tative determinations were coupled with histoohemioal studies* Intestinal
muoosa and kidney tissues taken from adult rabbits.Were used*
I.CENTRIFUGAL SEPARATION OF CELLULAR PARTICLES
The values of the use of centrifugation techniques in the separa­
tion of oytologioal structures are that analysis of separable constitu­
ents of the cell oan only be made after their separation, and sufficient
material oan be isolated by this prooess to allow for ohemioal investiga­
tion on a relatively maore scale* A wide variety of oellular constituents
hajp been separated by this method* Among the more prominent studies of
this nature are those of Dounoe on oell nuolei (32), Claude and Potter
on the isolation of chromatin threads from nuolei (26), and Graniok an
chloroplast isolation (46)•
The first auooaasful isolation of oytoplasmio granules by differ**
ential centrifugation was performed by Qensley and Hoerr (7)* In view
of tbe faot that oytoplasmio partioles of animal oells bad been recog­
nized for many years in advanoe of their isolation, one might well aslc
the reason for the lapse in time between their recognition and isolation*
This was largely due to the faot that many of the aiorosoopioally visible
partioles, suoh as aitoohondria, are of a highly labile nature, at least
in the oells of some animals* Mitoohondria rapidly disappear in dead
oells and in oells treated with organio solvents or fluids containing
aoetio aoid* An exoeption to this was found by Bensley and Oersh (6),who
demonstrated that the mitoohondria persisted in liver oells of Aablystoma
after freeze-drying the tissue, extraoting with organio solvents, treat­
ing with aoetio aoid or raising the temperature* Shortly after, the first
isolation of oytoplasmio partioles was performed by Bensley and Qoerr (7),
who separated mitoohondria from liver oells of guinea pig* Liver oells
were ground, put through bolting silk and suspended in physiological saline*
Subsequently the broken oells were subjected to a series of oentrifugations
and washings whioh ultimately produced a sediment of partioles whioh stain­
ed with aniline aoid fuohsin and methyl green and were identified as mito­
ohondria* During this prooedure the mitoohondria ohanged from rods to
round granules*
Miorosoopio granules have also been isolated by Claude (23)* The
oentrifugation preoedure used differs with the type of tissue* Using
rat or guinea pig livers, the miorosoopio or "large granules" may be
sedimented by oentrifuging several times for 30 minutes at 2,000 times
gravity or by oentrifuging at 18,000 times gravity for 3 to 6 minutes*
Both miorosoopio and aubmiorosoopio partioles have been separ­
ated from a wide variety of tissues by Claude and ooworkers* In his
early work (21) Claude discovered and separated submiorosoopio parti­
oles frost saline extraots of ohioken tumor oells and normal oells of
ohiok embryos* These same partioles were later (24) separated from a
large number of other tissues* Claude (21) originally considered that
suoh partioles might be mitoohondria or their fragments, but later he
abandoned this theory beoause the partioles were too small and did not
have the same staining properties as mitoohondria* Claude (23) proposed
that the term "miorosome" be applied to these granules and suggested (24)
that beoause they are rioh in ribonuoleio aoid, they may be self-dupli-
oating*
The importanoe of the proper ohoioe of medium into whioh the
partioles are liberated, is emphasised by Claude (22)* The granular
material in the solutions aggregates and, on standing, deteriorates
rapidly, whioh, according to Claude, may be due to the tendenoy of salt
solutions to beoome aoid on standing* For this reason, he used a salt
solution containing either 0*003 M phosphate buffer or 0*0002 H sodium
hydroxide*
In the mere reoamt work of Hogeboom, Sohneider and Pallade (52),
the effeots of various media were studied on the maintenance of mioro­
soopio or large granule fractions freed from rat livers* Maximum main­
tenance was found to ooour when the granules were liberated into 0*8 to
1*0 M suorose solution* Haintenanoe was judged by retention of sise
and shape of granules normally observed in the intaot oell and by re­
tention of the ability to stain with Janus green B»