th  - 1988 - 51st ENC Conference

V FERFUSION PROBE FOR A BRUKER AM-400 WIDE-BORE SPECTROMETER.
134 J Mark E. Anderson e# , Michael #Chob a ni an A, Ed S. Mooberry ~. John L.
Markley # and Carlos Ar~s ~. National Magnetic Resonance Facility
at Madison and Department of Biochemistry, College Of AAgriculture and Life Sciences,
University of Wisconsin-Madison, Madison, Wl 53706. University of Wisconsin,
School of Medicine, Madison, WI 53792, ~b Department of Biochemistry, Autonomous
University of Barcelona, Barcelona, Spain.
We constructed a probe for the Bruker AM-400 wide-bore spectrometer that
permits the perfusion of kidney proximal tubules. The design was inspired by the
work of Y. Boulanger, et al., but we chose a solenoidal coil geometry and doubly-
tuned the probe to P-31 and H-2 [1]. The deuterium channel facilitates the shimming
of the uncommon geometry. Since the proximal tubules of the kidney are very oxygen-
dependent, flow rates on the order of 200 ml/min are necessary in a perfused system.
The probe's design permits flow rates up to 1,000 ml/min. The construction of the
perfusion chamber permits easy sample access and minimizes the chances for leaks and
disruptions of coil geanetry. The temperature is monitored by a thermocouple
located in the effluent side of the perfusion chamber. We have tested the system
and have found that it is possible to maintain cell viability for over 12 hours.
The proximal tubules are isolated and injected into hollow dialysis fibers that are
then inserted into the perfusion chamber. The ATP levels of the cells rose to a
steady state value after two hours of perfusion and remained at those levels for the
duration of the experiment. With this system and modifications to the electronics,
a wide range of metabolic experiments of sensitive cells are possible.
[1] Y. Boulanger, P. Vinay, M.T. Phan Viet, R. Guardo and M. Desroches, Magn. Reson.
Ned. 2, 495-500 (1985).
[Supported by: U.S.-Spain Joint Grant CCA-8510/098, NIH grants RR02301 and RR027gl,
NSF Grant PCM-84504g, NKF 133M007, the U.S. Department of Agriculture, and the
University of Wisconsin.]
BO,~IONUCLEAR TWO DIMENSIONAL saC DOUBLE GUANTUM CORRELATION
V SPECTROSCOPY (2D xJC[x~C]DOC) AND IH-{13C]HETCOR AS PRIMARY TOOLS
135 J FOR SPIN SYSTEM AND HE~ ASSIGNmeNTS IN CYTOCHROME Csss: Michael
D. Reilye, Eldon L. Ulrich, William M. Westler and John L. Markley, Department of
Biochemistry, College of Agricultural and Life Sciences, 420 Henry Mall, University of
Wisconsin-Madison, Madison, WI 53706.
The first step in sequence-speciflc resonance assignments, identification of
individual spin systems, has traditionally involved time-consuming acquisition and
analysis of several 2D experiments that correlate scalar-coupled proton networks. We
present an alternative method for spin system identification that relies mainly on
scalar a3C-IsC and I~C-IH coupling. The IJC[ISC}DOC experiment is first used to
assign carbon spin systems. Next, the IH[13C]HETCOR experiment is used to extend
these assignments to carbon-bound proton resonances. Amide and amine NH resonances
are then identified and sequential assignments made by XH(I:C)MR-HECTOR(HMBC) or a
combination of NOESY and COSY. This approach has several advantages over proton-
proton methods. First, there are eighteen unique amino acid xsC-IsC coupling patterns
and only eight unique IH-IH spin systems. Second, primary assignments are based on
conformation-independent one-bond 13C-I~C and x:C-ZH connectivities. Proton methods
rely on dihedral-angle-dependent three bond coupling, and so expected cross peaks may
be weak or nonexistent. Third, proteins have fewer carbons than protons, and these
have a larger chemical shift range~ this simplifies analysis for larger proteins or
for proteins that have a significant amount of random coil structure. Fourth, xJC-IsC
connectivities currently provide the only general means of making unambiguous aromatic
side chain assignments. Finally, the new method reduces the time needed for resonance
assignments. Isotope-enriched proteins can be obtained inexpensively by the use of
modern blotechnology methods. We demonstrate the technique for cytochrome csss from
Anabaena 7120 uniformly labeled to 26% in I~C. Computer programs are being developed
to automate first and second order assignments based on these data. [Supported by
USDA 85-CRCR-1-1589, NSF PCM-g4504g and NIH RR02301, RR02781.]
166