# PROTEASES THAT FUNCTION IN NK-LYMPHOCYTE KILLING

> **NIH NIH R01** · UNIVERSITY OF NEVADA RENO · 1993 · $30,857

## Abstract

DESCRIPTION:  (Adapted from applicant's abstract):  The long term objective
of Dr. Hudig's research is to determine how serine proteases control
lymphocyte-mediated killing.  She hypothesizes that the proteases cleave
their natural substrates during calcium initiation of granule-mediated
killing and that these natural substrates regulate perforin damage.  The
specific aims of the project are:  1) To isolate, to characterize
biochemically, and to inhibit each serine protease of rat RNK-16 granules,
focusing particularly on the chymases required for lysis.  Percoll
gradients will be used to obtain RNK-16 leukemia cell granules.
Isoelectric focusing will remove proteoglycan while cation exchange and
heparin, benzamidine and other affinity columns will be used to separate
proteins.  The Mr, pI, N terminal amino acid sequence, substrate subsite
map and inactivation rates with irreversible inhibitors will be determined
for each protease.  2) To complete nucleotide sequencing cDNA clones of the
serine proteases of rat RNK-16 cells, new clones will be sequenced and
additional genes will be isolated in addition to a new, NK associated
serine protease already sequenced by the applicant.  Derived amino acid
sequences will be used for protease identification, prediction of substrate
binding sites, and comparison with reported T cell proteases.  3) To block
lysis of proteases with cytolytic function with antibodies to the protease
S' substrate binding sites.  Granule proteases vary uniquely in a 13-15
residue region that binds the part of a protein substrate which becomes the
new amino terminus after cleavage.  Antibodies to S' peptides should
prevent protease access to protein substrates and slow granule lysis if the
S' protease participates in killing.  4) To isolate RNK-16 granule proteins
that may regulate or control lysis mediated by unfractionated granules.
Dr. Hudig will purify a protein that may limit granule-mediated lysis, is
inactivated by granules in the presence of Ca++, is protected from
inactivation by protease inhibitors and may be a natural substrate.  5) To
determine the natural substrates of the RNK-16 proteases.  It will be
determined if RNK-16 perforin is reduced in size during lysis by
unfractionated granule proteins or after incubation with purified RNK-16
proteases.  It will also be determined whether lysis-restricting proteins
are cleaved during granule-mediated lysis.  (animals:  rats and rabbits)

## Key facts

- **NIH application ID:** 3177450
- **Project number:** 3R01CA038942-08S1
- **Recipient organization:** UNIVERSITY OF NEVADA RENO
- **SAM.gov UEI:** WLDGTNCFFJZ3
- **Principal Investigator:** Dorothy Hudig
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1993
- **Award amount:** $30,857
- **Award type:** 3
- **Project period:** 1984-05-11 → 1994-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3177450

## Citation

> US National Institutes of Health, RePORTER application 3177450, PROTEASES THAT FUNCTION IN NK-LYMPHOCYTE KILLING (3R01CA038942-08S1). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3177450. Licensed CC0.

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