Subject: FlyBase query
Dear Dr. Pinter,
I am curating your paper for FlyBase:
Pinter et al., 1998, Insect Biochem. Molec. Biol. 28(2): 91--98
and I have some questions about the relationships between the various
Drosophila calpain genes.
TER94 corresponds to the Ca2+-activated neutral protease (CANP) activity
purified and studied in Pinter et al, 1992, Biochemistry 31: 8201--8206.
I would like to know what the relationship is between this CANP activity
and the Ca2+-dependent proteolytic activities studied in Pinter and
Friedrich, 1988, Biochem. J. 253: 467--473.
In Pinter and Friedrich, 1988, Biochem. J. 253: 467--473, 2
Ca2+-dependent-proteinase activities are purified. These are a calpain
II-like activity (rightmost peak in Figure 2.) and a calpain I-like
activity (Figure 3).
Is the calpain-II like activity in this paper the same as the CANP activity
in Pinter et al, 1992, Biochemistry 31: 8201--8206 (i.e is it TER94) ?
Do you know whether the calpain-I like activity in Pinter and Friedrich,
1988, Biochem. J. 253: 467--473 corresponds to any of the other known
Drosophila calpain genes, which I list below, or is it a different calpain
gene ?
a) CalpA gene, at 56C-56D (this was cloned in J. Biol. Chem.
269: 25137--25142, where it is referred to as Dm-calpain).
b) Calp14B, at 14C. This comes from a PhD thesis:
Rutherford, 1995, Ph.D. Thesis, University of California at San Diego, CA
'The genetic and biochemical characterization of the major cyclophilin
isoform in Drosophila melanogaster.'
where a region of homology to a vertebrate calpain was found proximal to
the Cyp-1 gene.
c) sol - small optic lobes
this gene also has homology to vertebrate calpains, and maps to 19F.
I look forward to hearing from you,
Gillian Millburn
\--------------------------------------------------------------
Gillian Millburn.
FlyBase (Cambridge),
\--------------------------------------------------------------
>
Dear Gillian,
	I attempt to answer your questions. It might well be You won't
find my answers satisfying, - You won't get simple yes or no. Though I try
my best. And You are always wellcome to continue asking.
Best regards,
Marianna
> Dear Dr. Pinter,
>
> I am curating your paper for FlyBase:
>
> Pinter et al., 1998, Insect Biochem. Molec. Biol. 28(2): 91--98
>
> and I have some questions about the relationships between the various
> Drosophila calpain genes.
>
> TER94 corresponds to the Ca2+-activated neutral protease (CANP)
activity
UNFORTUNATELY I CAN NOT SAY SO. I HAVE CLONED TER94 BY THE AIDS OF AN
ANTI-CANP ANTIBODY. TER94 ITSELF SHOWS NO SIMILARITY TO CALPAINS OR ANY
OTHER PROTEASES. IN SUCH A CASE ONE SHOULD PROVE THAT TER94 IS REALLY A
PROTEASE. I HAVE NOT SUCCEEDED: THE EXPRESSED GST-TER94 DID NOT SHOW
CA2+-ACTIVATED PROTEASE ACTIVITY.
> TER94 corresponds to the Ca2+-activated neutral protease (CANP) activity
> purified and studied in Pinter et al, 1992, Biochemistry 31:  8201--8206.
> I would like to know what the relationship is between this CANP activity
> and the Ca2+-dependent proteolytic activities studied in Pinter and
> Friedrich, 1988, Biochem. J. 253: 467--473.
CANP (BIOCHEMISTRY) HAS BEEN PURIFIED. THE TWO CA2+-ACTIVATED PROTEASE
(BIOCHEM J.) HAS BEEN DETECTED RATHER, OR IF YOU LIKE: PARTIALLY PURIFIED.
THE RESINS USED FOR LIQUID CHROMATOGRAPHY ARE DIFFERENT: IT DOES NOT HELP
TO IDENTIFY CANP (BIOCHEMISTRY) AS ANY OF THE PREVIOUSLY DETECTED
CA2+-ACTIVATED PROTEASE (BIOCHEM.J.). IN BOTH CASES (BIOCHEM. &
BIOCHEM.J.) I HAVE USED CYTOSOL FRACTION TO START WITH AND THE
CA2+-DEPENDENT PROTEASE (BIOCHEM.J.) WITH HIGHER CA2+ CONCENTRATION
REQUIREMENT AND CANP (BIOCHEM.J.) SHOWS SIMILAR CA2+-SENSITIVITY. THESE
TWO MAY BE THE SAME.
ONE CAN SAY THAT THERE ARE TWO OR THREE CYTOSOLIC CA2+-DEPENDENT
PROTEASES, WHICH HAVE BEEN IDENTIFIED IN DROSOPHILA: ONE OF THESE NEEDS
LOW CA2+ CONCENTRATION TO BE ACTIVATED AND THE OTHER TWO (WHICH MAY BE THE
SAME) NEED HIGH.
>
> In Pinter and Friedrich, 1988, Biochem. J. 253: 467--473, 2
> Ca2+-dependent-proteinase activities are purified.  These are a calpain
> II-like activity (rightmost peak in Figure 2.) and a calpain I-like
> activity (Figure 3).
>
> Is the calpain-II like activity in this paper the same as the CANP activity
> in Pinter et al, 1992, Biochemistry 31: 8201--8206 (i.e is it TER94) ?
SEE MY PREVIOUS NOTE.
>
> Do you know whether the calpain-I like activity in Pinter and Friedrich,
> 1988, Biochem.  J. 253: 467--473 corresponds to any of the other known
> Drosophila calpain genes, which I list below, or is it a different calpain
> gene ?
>
> a) CalpA gene, at 56C-56D (this was cloned in J. Biol. Chem.
> 269: 25137--25142, where it is referred to as Dm-calpain).
PROBABLY NOT, SINCE CALPA IS NOT DETECTED IN THE CYTOSOLE WITH THE
ANTIBODY WHICH I HAVE USED TO CLONE IT.
>
> b) Calp14B, at 14C.  This comes from a PhD thesis:
>
> Rutherford, 1995, Ph.D. Thesis, University of California at San Diego, CA
>
> 'The genetic and biochemical characterization of the major cyclophilin
> isoform in Drosophila melanogaster.'
>
> where a region of homology to a vertebrate calpain was found proximal to
> the Cyp-1 gene.
I HAVE NEVER HEARD ABOUT THIS THESIS, THOUGH I WOULD BE VERY INTERESTED TO
READ. TO TELL THE TRUTH, I HAVE BEEN LOOKING FOR NEW PUTATIVE CALPAIN
GENES IN THE DATABASE RECENTLY AND I COULD NOT FIND ANY.
>
> c) sol - small optic lobes
>
> this gene also has homology to vertebrate calpains, and maps to 19F.
>
I HAVE CHECKED SOL STRAIN FOR CA2+-ACTIVATED PROTEASE ACTIVITY AND IT IS
INDISTINGUISABLE FORM WILD TYPE STRAIN (CANTON S). THE EXPERIMENT HAS BEEN
PERFORMED AS DESCRIBED IN THE BIOCEM.J. ARTICLE: CYTOSOLE FRACTION, PH4.5
FRACTIONATION, THEN ACTIVITY MEASUREMENT.
THE SOL PROTEIN SHOULD BE A PROTEASE. THOUGH NEITHER SOL OR OTHER GENES
WHICH SHOW HIGH SIMILARITY TO THE PROTEASE DOMAIN OF CALPAINS NOT
NECESSARILY ENCODES CA2+-DEPENDENT PROTEASES. CA2+-DEPENDENT PROTEASE
ACTIVITY HAS NOT YET PROVED IN THE CASE OF ANY OF THESE INTERESTING
PROTEINS. PERSONALLY I DON'T SEE ANY REASON TO CALL THEM CALPAIN: CALPAINS
ARE BY DEFINITION CA2+-ACTIVATED CYSTEINE PROTEASES.
>
> I look forward to hearing from you,
>
> Gillian Millburn
>
> --------------------------------------------------------------
> Gillian Millburn.
>
> FlyBase (Cambridge),
> --------------------------------------------------------------
>
\---------------------
Marianna Pinter
>
Subject: Re: FlyBase query
Dear Marianna,
thankyou for your reply, it was very helpful.  I would like to curate the
information in your e-mail as a personal communication from you to
FlyBase.
Here is what I propose:
1. We currently have the Ca2+-activated neutral protease (CANP) activity
purified and studied in Pinter et al, 1992, Biochemistry 31:  8201--8206 as
Ca-TPase.
I will create 2 new genes for the Ca2+-dependent-proteinase activities
studied in Pinter and Friedrich, 1988, Biochem. J. 253: 467--473.  Is it OK
if I call them CalpI and CalpII (for Calpain-I and Calpain-II) ? or would
you rather I call them Ca-PaseI and Ca-PaseII (for Ca2+-dependent
proteinase I and II) ?
I will put a comment saying that the relationship between each of these
genes and Ca-TPase is unknown.
2. I will put a comment saying that it is likely that Ca-TPase, and the 2
proteinases from Pinter and Friedrich, 1988, Biochem. J. 253: 467--473 are
not the same as CalpA, as they are purified from the cytosol and CalpA is
not detected in the cytosol with an anti-CalpA antibody.
3. I will also put a comment saying that sol- flies have
Ca2+-activated protease activity that is indistinguishable from
wild-type.
I would be grateful if you could tell me whether the proposals I have made
for naming the various Ca-dependent proteinases above are OK.  Thankyou
once again for helping to sort this out,
Gillian
>
Subject: Re: FlyBase query
Dear Gillian,
I hope my answers on your points will help You in the work.
Regards,
Marianna
On Tue, 30 Jun 1998, Gillian Millburn wrote:
> Dear Marianna,
>
> thankyou for your reply, it was very helpful.  I would like to curate the
> information in your e-mail as a personal communication from you to
> FlyBase.
>
> Here is what I propose:
>
> 1. We currently have the Ca2+-activated neutral protease (CANP) activity
> purified and studied in Pinter et al, 1992, Biochemistry 31:  8201--8206 as
> Ca-TPase.
I would suggest to use the name CANP. It stands fo Ca2+-activated neutral
protease. To introduce a new name usually makes confusion. Here I don't
see any point to do that. (In addition, Ca-TPase rhymes with ATPase and I
don't find it especially good choice.)
>
> I will create 2 new genes for the Ca2+-dependent-proteinase activities
> studied in Pinter and Friedrich, 1988, Biochem. J. 253: 467--473.  Is it OK
> if I call them CalpI and CalpII (for Calpain-I and Calpain-II) ? or would
> you rather I call them Ca-PaseI and Ca-PaseII (for Ca2+-dependent
> proteinase I and II) ?
I think CalpI and CalpII would be a safe and traditional solution.
>
> I will put a comment saying that the relationship between each of these
> genes and Ca-TPase is unknown.
You might say a little more. CalpI is not the same as CalpII or CANP, -
that's for sure. And the relation between CalpII and CANP is unknown.
>
> 2. I will put a comment saying that it is likely that Ca-TPase, and the 2
> proteinases from Pinter and Friedrich, 1988, Biochem. J. 253: 467--473 are
> not the same as CalpA, as they are purified from the cytosol and CalpA is
> not detected in the cytosol with an anti-CalpA antibody.
That's fine, - but use CANP please instead os Ca-TPase.
>
> 3. I will also put a comment saying that sol- flies have
> Ca2+-activated protease activity that is indistinguishable from
> wild-type.
That's OK.
\---------------------
Marianna Pinter
Department of Molecular &Cellular Biology
The University of Arizona
>
Subject: Re: FlyBase query
Dear Marianna,
> > 1. We currently have the Ca2+-activated neutral protease (CANP) activity
> > purified and studied in Pinter et al, 1992, Biochemistry 31:  8201--8206 as
> > Ca-TPase.
> >
> I would suggest to use the name CANP. It stands fo Ca2+-activated neutral
> protease. To introduce a new name usually makes confusion. Here I don't
> see any point to do that. (In addition, Ca-TPase rhymes with ATPase and I
> don't find it especially good choice.)
I will use the name CANP, for Ca2+-activated neutral protease.
> >
> > I will create 2 new genes for the Ca2+-dependent-proteinase activities
> > studied in Pinter and Friedrich, 1988, Biochem. J. 253: 467--473.  Is it OK
> > if I call them CalpI and CalpII (for Calpain-I and Calpain-II) ? or would
> > you rather I call them Ca-PaseI and Ca-PaseII (for Ca2+-dependent
> > proteinase I and II) ?
>
> I think CalpI and CalpII would be a safe and traditional solution.
>
OK
> > I will put a comment saying that the relationship between each of these
> > genes and Ca-TPase is unknown.
>
> You might say a little more. CalpI is not the same as CalpII or CANP, -
> that's for sure. And the relation between CalpII and CANP is unknown.
I will add the extra information you suggest.
>
> >
> > 2. I will put a comment saying that it is likely that Ca-TPase, and the 2
> > proteinases from Pinter and Friedrich, 1988, Biochem. J. 253: 467--473 are
> > not the same as CalpA, as they are purified from the cytosol and CalpA is
> > not detected in the cytosol with an anti-CalpA antibody.
>
> That's fine, - but use CANP please instead os Ca-TPase.
I will use CANP.
Is all that OK ?
Gillian
>
Subject: Re: FlyBase query
That's all fine, Gillian.
Regards,
Marianna
\---------------------
Marianna Pinter
Department of Molecular &Cellular Biology
The University of Arizona