# CELL DIVISION AND SURFACE GROWTH IN A MODEL SYSTEM

> **NIH NIH R01** · TEMPLE UNIV OF THE COMMONWEALTH · 1989 · $149,709

## Abstract

Stimulation of human neutrophils induces a "respiratory burst"
causing production of 02- and H202.  This response is crucial to
normal bacterial killing but an inappropriate response contributes
to neutrophil-mediated tissue damage in many diseases.  Stimulation
of the respiratory burst involves activation of an electron
transport complex, the NADPH oxidase.  Mechanisms of oxidase
activation are largely undefined.  Even the components of the
oxidase are controversial, although data strongly support that it
at least involves an NADPH-binding flavoprotein and a cytochrome.
The cytochrome is being intensively studied elsewhere, but the
flavoprotein has not been purified in quantity and its mechanisms
of activation and electron transfer are unknown.

This project will complete the development of a novel NADPH-
affinity method for purification of the NADPH-binding oxidase
flavoprotein.  It will also employ new methods for assessing
purification:  1) The ability to form a borohydride-reducible
adduct between the protein and 2,3-dialdehyde-NADPH.  2) The
ability to reconstitute NADPH-dependent 02- production in
liposomes.  For this model, flavoprotein purification fractions
will be combined with solubilized membrane (the membrane being
specifically depleted of functional NADPH-binding flavoprotein but
providing all other components of the oxidase complex), for co-
reconstitution of all oxidase components in liposomes by removal
of detergent.

Redox couples (FAD, FMN, thiols, iron-sulfur, transition metal)
within the flavoprotein we be determined.  Spectral, fluorescent
and electron paramagnetic resonance methods will be used for
titration of the flavoprotein with NADPH versus dithionite to
determine stoichiometry of NADPH to flavin electron transfer and
formation of intermediate redox states.  Parallel studies of
flavoprotein from resting and stimulated cells will test the
hypothesis that oxidase activation involves increased substrate
affinity (anaerobic NADPH binding) or catalysis (electron
transfer).

Tryptic peptides derived from the protein will be sequenced.  The
sequences will allow synthesis of peptides for raising anti-
flavoprotein antibodies and for synthesizing cDNA probes, each of
which can be used for molecular cloning of the flavoprotein cDNA.

## Key facts

- **NIH application ID:** 3124872
- **Project number:** 5R01AI010971-18
- **Recipient organization:** TEMPLE UNIV OF THE COMMONWEALTH
- **SAM.gov UEI:** QD4MGHFDJKU1
- **Principal Investigator:** MICHAEL L HIGGINS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1989
- **Award amount:** $149,709
- **Award type:** 5
- **Project period:** 1978-05-01 → 1993-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3124872

## Citation

> US National Institutes of Health, RePORTER application 3124872, CELL DIVISION AND SURFACE GROWTH IN A MODEL SYSTEM (5R01AI010971-18). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3124872. Licensed CC0.

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