Michael Blumenstein

Perfusion of cell spheroids for study by NMR spectroscopy

Abstract Many anchorage-dependent types of cells can be cultured in suspension as small clumps of defined size known as spheroids. These spheroids are particularly suited for study with NMR spectroscopy because, unlike suspensions of single cells, they can be perfused without being carried along with the perfusate, thus eliminating the need for filters or gels. We have successfully maintained human breast cancer cells (MCF-7) and Chinese hamster cells (V 79) in a physiological steady state as spheroids for more than 80 hours in a 10 mm NMR tube using a simple and inexpensive perfusion setup which requires no modification of NMR instrumentation. Although we were primarily interested in obtaining 31P NMR spectra, cell mass was sufficiently high that natural abundance 13C NMR spectra were also readily obtained.

The tyrosine ring of oxytocin undergoes hindered rotation when the hormone is bound to neurophysin.

Abstract Tyrosine specifically enriched with 13C in the meta positions has been chemically synthesized and incorporated into oxytocin via solid phase peptide synthesis. The 13C nmr spectrum of a 1:1 mixture of the enriched hormone complexed to neurophysin was obtained. The spectrum consisted of three peaks. The two outer peaks, representing 85% of the total intensity, were of equal area, had shifts of −0.9 and +2.4 parts per million relative to the free peak, and each had a linewidth of 100 hz at 20°C, with increasing linewidths at higher temperatures. These two peaks arise from a binding mode in which tyrosine ring rotation is hindered by interaction with neurophysin. The rotation rate at 20°C is 130s−1, and at 42°C is 900s−1. The central peak occurred at the position of the resonance due to free hormone, had a temperature independent linewidth of 30–40 hz, and represented about 15% of the total intensity. We believe this peak is due to a binding mode in which tyrosine ring rotation is rapid, 104–108s−1.

31P NMR studies on the interaction of deoxyuridylate with thymidylate synthase

The 31P nuclear magnetic resonance signal of deoxyuridylate was studied in the presence and absence of thymidlate synthase. In the absence of enzyme the chemical shift of deoxyuridylate is pH dependent with a pKa of 6.25. In the presence of enzyme, a peak corresponding to the dianioinc form of deoxyuridylate is observed which is independent of pH between pH 5.7 and pH 7.4. The pKa of the phosphate in the deoxyuridylate-thymidylate synthase complex is therefore less than 5. The release of inorganic phosphate from deoxyuridylate catalyzed by contaminating phosphatase was also observed.

19F NMR studies of the binding of 5-fluoro-2′-deoxyuridylate to thymidylate synthase

SummaryIn the presence of thymidylate synthase, the 19F signal of 5-fluoro-2′-deoxyuridylate is shifted upfield 0.6 ppm or 4.5 ppm depending on the enzyme preparation used. The bands at these positions represent different species of binary complex. When either binary complex is reacted with methylenetetrahydrofolate a ternary complex is formed with a 19F signal shifted 12.5 ppm upfield and broadened to 120 Hz.Substitution of the hydrogen atoms of the methylene group of methylenetetrahydrofolate with deuterium atoms results in line-narrowing of the spectrum of the ternary complex from 120 to 80 Hz indicating the close proximity of the methylene group to the fluorine atom in the ternary complex. A model compound, 5-fluoro-6-hydroxy-5-methyl-5, 6-dihydrouracil, gives a chemical shift in the same direction and of similar magnitude to that seen with the ternary complex.

IgA1 protease cleaves heavy chains independently in dimeric human IgA1.

Bacterial IgA1 proteases have substrate specificity for human IgA1 immunoglobulin, and cleave both the heavy (alpha) chains where they are paired by disulfide bonds in the hinge region. To determine if the close apposition of the alpha chains allows a single enzyme-substrate-binding event to cleave both hinge region peptides we quantitated the relative levels of intermediate products during the course of complete hydrolysis of an IgA1 paraprotein. The substrate had four Fab regions, analogous to a secretory IgA dimer. The experimental data were then compared to computer-generated models in which various levels of cooperativity among Fab regions were tested. The results most closely conformed to a model in which each individual alpha chain is proteolyzed independently, without regard to the total number of hinge region peptides available in the substrate IgA1. These results will be used to guide the design of IgA1 hinge region peptide analogues as IgA1 protease inhibitors.

NMR Studies of the Conformation of Tuftsin and a Pentapeptide Tuftsin Inhibitor in Solutiona

A complete understanding of the mode of action of tuftsin requires a knowledge of its biologically active conformation. As with most peptides, a direct measurement of the active conformation of tuftsin has not been possible, so indirect methods have been employed. Numerous tuftsin analogues have been synthesized, and measurements of activity and of inhibitory properties have led to some conclusions concerning obligatory structural features (reviewed in Siemion & Konopinska ). A variety of physical techniques have been used to study the structure of tuftsin in solution. Investigations have been performed on tuftsin as well as tuftsin analogues, in aqueous and nonaqueous solutions. Among the spectroscopic techniques employed have been nuclear magnetic resonance (NMR),24 circular dichroism ( ~ d ) ~ . ~ and infrared (IR). Theoretical calculations have also been carried out.. In light of the rather short length of tuftsin, it is somewhat surprising that there has been no general agreement on its structure in solution. Among the proposed structures are those involving a B t ~ r n , ~ , ~ a quasi-cyclic molecule, and a hairpin with split ends. We have employed H and C NMR to study the structure of tuftsin and pentapeptide inhibitor, Thr-Lys-ProPro-Arg. Our results indicate that in aqueous solution tuftsin exists in a random conformation, while in dimethyl sulfoxide (MeSO), a somewhat more ordered, but as yet undetermined, structure exists. In this paper, I will review our results, as well as the results from other laboratories. In particular, I will show that a wealth of available evidence precludes the possibility that tuftsin exists in a P-turn structure in aqueous solution.

Chapter 7 – Nuclear Magnetic Resonance Studies of Peptide–Macromolecule Interactions

Publisher Summary This chapter presents an overview of the types of experiments that can be performed, the systems that can be studied, and the results that can be obtained in applying nuclear magnetic resonance (NMR) to the study of peptide-macromolecule interaction. The NMR of peptide–macromolecule interactions usually involves experiments of a different type than those used to study free peptides. The investigation of peptide–macromolecule interaction has the potential of giving information that is more useful for understanding peptide action than that obtained from studies of free peptides. Improvements in NMR instrumentation and in isolation and purification of important receptor molecules should greatly increase the scope and significance of NMR studies of peptide–macromolecule interactions undertaken in the coming years. This will be one of the most important areas of NMR research and will also lead to an enhanced understanding of peptide action.