# SITE-SPECIFIC MUTAGENESIS OF THE CA2+-ATPASE

> **NIH NIH R01** · UNIVERSITY OF TORONTO · 1992 · $104,060

## Abstract

The long term objective is to understand the mechanism whereby the Ca2+ -
ATPase of sarcoplasmic reticulum transports Ca2+ to luminal spaces.  The
expression and mutagenesis of full length cDNA encoding the Ca2+ -ATPase,
followed by assay of the function of the product, is being used to address
questions of structure/function relationships in the protein.  Further
research is proposed under four headings:
I.  Development of an expression system for large scale production of
normal and mutant forms of the Ca2+ -pump:  In order to complement the
present expression/mutagenesis system in which Ca-ATPase cDNA is expressed
in COS-1 cells, development of a Baculovirus expression system that will
allow production of large amounts of protein is proposed.  Purification of
the expressed mutant Ca2+-ATPase protein in one step using a monoclonal
antibody affinity column with the IgM antibody A20 is also proposed.
II.  Determination of the topology of the transmembrane domain of the Ca2+
-ATPase.  Luminal and cytoplasmic epitopes in the Ca2+-ATPase have been
distinguished with monoclonal antibodies against defined sequences.  This
analysis will be extended to other sequences predicted to lie on lumenal or
cytoplasmic sides.  Alternatively, a specific epitope from the fast-twitch
Ca2+-ATPase will be inserted at various sites in the sequence of the slow-
twitch Ca2+-ATPase in order to determine topology.  The boundaries of
membrane spanning sequences will be determined by insertion of basic
clusters at each end.
III.  Site-specific mutagenesis to define amino acids critical to Ca2+
transport.  Mutation is proposed of:  (a) residues in transmembrane
sequences 4,5,6 and 8 to determine how they relate to Ca2+ binding and Ca2+
transport; (b) residues throughout the proposed nucleotide binding domain
to identify those involved in ATP binding; (c) residues potentially
involved in conformational changes in the molecule; (d) residues that will
permit measurement of spatial arrangements in the Ca2+-ATPase; (e) Gly,
Pro, Cys and Trp residues throughout the molecule.
IV.  Kinetic analysis of mutant function.  Since many mutants retain
partial reactions of Ca2+ transport, equilibrium and kinetic analyses of
these partial reactions will be carried out.  Ca2+ and ATP binding
constants, rates of formation and breakdown of phosphorylated intermediates
from ATP and Pi, and rates for overall Ca2+ transport will be measured
where possible.

## Key facts

- **NIH application ID:** 3159285
- **Project number:** 5R01AR039280-05
- **Recipient organization:** UNIVERSITY OF TORONTO
- **SAM.gov UEI:** NBMSSFM3HP56
- **Principal Investigator:** DAVID H MACLENNAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1992
- **Award amount:** $104,060
- **Award type:** 5
- **Project period:** 1988-05-01 → 1994-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3159285

## Citation

> US National Institutes of Health, RePORTER application 3159285, SITE-SPECIFIC MUTAGENESIS OF THE CA2+-ATPASE (5R01AR039280-05). Retrieved via MORVS 2026-10-24 from https://api.morvs.ai/grant/nih/3159285. Licensed CC0.

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