| Summary: | Thesis (Ph.D.)--Boston University === The sequence of reactions concerned in the formation of adrenocortical
steroids from cholesterol includes the cleavage of the
cholesterol side chain between C-22, 23 to form 5 -pregnenolone. This
step is accelerated by adrenocorticotrophic hormone (ACTH) administration,
whereas the later hydroxylation of progesterone to form corticosterone
and cortisol are not. These hydroxylations are known to be dependent
upon triphosphopyridine nucleotide (TPNH), but the cofactor requirement
for cholesterol side chain cleavage has not been defined. In view of
the dominant role proposed fro TPNH by currently held theories of the
mechanism of action of ACTH, it is important to know if the only ACTH
responsive step in the corticosteroidogenetic sequence requires TPNH
as an essential cofactor.
The conversion of cholesterol to c-21 steroids was investigated
in subcellular fractions of bovine adrenal cortex tissue incubated with
cholesterol-4-c^14 and the extent of conversion estimated by separating
the radioactive products from unchanged cholesterol-4-c^14 on silicic
acid columns. The radioactive products were tentatively identified on
the basis of their chromatographic behavior in paper and partition column
systems. The subcellular fractions were prepared by conventional differential
centrifugation techniques with 0.44M sucrose, with and without
ionic additions, for homogenization and isolation media. Nuclei, mitochondrial,
microsomal and soluble fractions were prepared from the ionicsucrose
media and in the case of pure sucrose homogenates the "fluffy layer".
of the mitochondrial pellet was treated as an additional fraction.
The mitochondrial fraction from either homogenate required only
Mg++ and fumarate for cholesterol conversion activity. Succinate or
a-ketoglutarate could replace fumarate and aerobic conditions were
necessary. The requirement for citric acid cycle intermediates could be
met by the "extemal" TPNH generating system, glucose-6-phosphate (G6P),
its dehydrogenase (G6P+DH) and TPN. Acetone dried mitochondrial fractions
from pure sucrose homogenates were fully active with fumarate and Mg++,
but after restricted dialysis against phosphate buffer, TPN was required
in addition to fumarate. The activity could be obtained in a soluble,
non-particulate form by extraction of the acetone dried mitochondrial
fraction with M/15 phosphate buffer pH 6.8, followed by centrifugation
at 105,000g. The products of cholesterol-4-c^l4 conversion by the dialyzed
or soluble extract of acetone dried mitochondrial fractions were largely
accounted for by ~5 -pregnenolone and progesterone, with the relative
amounts of the two intermediates depending upon the endogenous level of
DPN in the preparation or the presence of this cofactor in the medium.
Distribution studies based on the activity of acetone dried
fractions were run in order to avoid problems connected with unequal
mixing of tracer and endogenous cholesterol. The results indicated that
approximately equal amounts of the total activity were present in the
nuclei, mitochondrial and "fluffy layer" fractions. The microsomal and
soluble fractions were inactive. The highest activity per mg. protein
was found in the "fluffy layer" with the mitochondrial fraction next,
a result possibly related to the greater ease of cholesterol-4-C^14 entry
into the disorganized structure of the "fluffy layer".
Soluble extracts from the acetone dried fractions were active on
addition of Mg++, fumarate and TPN, but only the mitochondrial fraction
gave acetone powders active in the absence of TPN. The "bound" cofactor
in fresh mitochondrial or "fluffy layer" fractions was not available for
TPNH generation by G6P and its dehydrogenase and only reacted to a
limited extent in acetone dtted preparations.
High concentrations of sucrose inhibited cholesterol conversion
by particulate preparations, but not the solubilized activity from
acetone powders. The effect was possibly due to a reduced entry of
cholesterol-4-c^l4, as the specific activities of the products of conversion
were reduced, while TPNH generation by either the "internal" or
"external" systems was inhibited to an equal extent.
The possible relationships of the findings co ACTH action are
discussed. [TRUNCATED]
|
|---|
