# MITOCHONDRIAL PROTEINS AND LEYDIG CELL STEROIDOGENESIS

> **NIH NIH R01** · TEXAS TECH UNIVERSITY HEALTH SCIS CENTER · 1994 · $139,022

## Abstract

INTRODUCTION:  While the rate limiting enzymatic step in
steroidogenesis is the conversion of cholesterol to pregenenolone by
the CSCC complex located in the mitochondria, the true rate
determining step in this process is the delivery of substrate to the
inner mitochondrial membrane where cleavage occurs.  This process
requires the synthesis of new proteins which are thought to be
indispensable for cholesterol transfer.  The search for this
regulatory protein(s) has spanned greater than two decades and has
not yet yielded unequivocal information on this problem.  Using
radioisotope labelling techniques followed by 2-D PAGE, we have
described several mitochondrial proteins in hormone stimulated MA-10
mouse tumor Leydig cells which may be candidates for the regulatory
protein(s).  Earlier findings from our laboratory greatly
strengthened the probability that these mitochondrial proteins were
involved in acute steroidogenic regulation.  In this proposal we add
further evidence of the importance of these proteins and describe a
model whereby they may function in the transfer of cholesterol to the
inner mitochondrial membrane.  Therefore, we propose to continue to
pursue the purification and characterization of these proteins since
they would appear to be extremely strong candidates for playing a
role in steroidogenesis.  SPECIFIC AIMS:  Specifically, it is
proposed:  1) to further characterize the synthesis, degradation,
phosphorylation and protein processing profiles of mitochondrial
proteins in MA-10 and R2C cells using two-dimensional polyacrylamide
gel electrophoresis.  2) to use FPLC technology to purify the
mitochondrial 30 kDa proteins from stimulated MA-10 cells.  3) to
utilize the purified protein(s) in reconstruction experiments with
isolated mitochondria in order to determine if they play a role in
the acute regulation of CSCC activity.  4) to utilize the purified
protein(s) for microsequencing and for the generation of polyclonal
antibodies which will then be used in further characterization of the
protein(s) with regards to function, rates of synthesis and
localization.  5) to clone and sequence the gene for these proteins.
METHODS:  Mitochondrial proteins and phosphoproteins will be studied
by radiolabeling, 2-D PAGE and fluorography.  Quantitation of
proteins will be performed using the Visage 2000 image analysis
system.  Steroid production will be measured by RIA.  Protein
purification will be accomplished using FPLC.  Antibodies will be
generated using standard polyclonal antibody production methods.
Immunocytochemistry will be performed using the protein-A gold
technique.  Microsequencing of proteins will be performed by a
commercial source.  Cloning and sequencing of the gene will be
performed using standard methodology.

## Key facts

- **NIH application ID:** 2197463
- **Project number:** 5R01HD017481-09
- **Recipient organization:** TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
- **SAM.gov UEI:** E4Z2NUYUMHF9
- **Principal Investigator:** DOUGLAS M STOCCO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1994
- **Award amount:** $139,022
- **Award type:** 5
- **Project period:** 1984-07-01 → 1996-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/2197463

## Citation

> US National Institutes of Health, RePORTER application 2197463, MITOCHONDRIAL PROTEINS AND LEYDIG CELL STEROIDOGENESIS (5R01HD017481-09). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/2197463. Licensed CC0.

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