From: Jacob Lebowitz <lebowitz@helix.nih.gov>
  To  : rasmb@alpha.bbri.org
  Date: Thu, 27 Apr 2000 18:15:16 -0400

glycoproteins

There has been a fair amount of work on glycosylated proteins using AU.
One recent paper covers the characterization in a quite detailed way
(Fairman et al. 1999 Anal. Biochem. 270, 286-295). The latter makes use
of Shire's sed.equil. approach to obtaining the fraction carbohydrate
composition and this allows for Mw determinations.)

We (Pete Schuck and myself) have been studying the oligomerization states
of the HIV-1 gp120 and gp 140. For our case a combination of the
molecular weight from MS (peak of the distribution) allowed us to obtain
a good estimate of the partial specific volume using an approach of Lewis
and Junghans which will be published in Methods of Enzymology. 
Sedimentation equilibrium data for different fractions from SEC of
non-interacting oligomeric forms could be globally fit to give the
molecular weights of the oligomeric species.  By combining several
approaches (see the above Fairman ref.) a  characterization of oligomeric
states of cell surface proteins and viral proteins can be accomplished.
Our paper on gp 120 will be out in the J.Virol. in May. We have been
given an honary Virology license. In summary,  molecular weight analysis
of glycoproteins is possible and important. Obviously studying the
interactions of glycoprotein populations with different carbohydrate
compositions is far more challenging. 



Jack Lebowitz

<bold>National Institutes of Health

</bold>  <bold>Molecular Interaction Resource Bldg. 13 Rm. 3N17

  13 South Drive - MSC 5766

  Betheda MD 20892

  (301) 435-1955

  Fax: (301) 496-6608

  Office 3E49

  email: lebowitz@helix.nih.gov

</bold>











Priority: Urgent

From: Jim Bloom <<Jim.Bloom.B@bayer.com>

To: "        -         *rasmb@alpha.bbri.org" <<rasmb@alpha.bbri.org>

Subject: Incompetence..

Date: Thu, 27 Apr 2000 16:29:51 -0400


 Many thanks to Borries, Tom and Yujia Xu for their lucid explanations

of incompetence and competence.  I have had an interesting thought for
the

biochemically inclined.  I do much more ESI-MS analysis than AUC and
therefore

see lots of subtle differences in proteins.  We also work with
glycosylated

proteins.  Examples are an interleukin that contained four forms:  a
full

length non-glycosylated, des1-2 non-glycosylated (ie. biochemists

jargon....lacking the first two amino acids on the N-terminal), and two

glycosylated full length versions.  We also have another protein with a
full

length non-glycosylated population and  glycosylated at one N-linked site
and

glycosylated at two sites.   So, with this background comes a topic of
interest

to me: are these forms of the same molecule structurally "the same".   In
the

experience of the RASMBer's, has anyone seen a difference...for example
maybe

one might see aggregation with the non-glycosylated forms but not the

glycosylated?

 One other comment...in a discussion with John Philo at one time he

mentioned to me that in a sense using AUC with glycosylated proteins is
a

"waste of time" ie. too much heterogeneity.  Any comments on this 
issue?

 Jim