The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases.

The crystal structure of chicken egg white cystatin has been solved by X-ray diffraction methods using the multiple isomorphous replacement technique. Its structure has been refined to a crystallographic R value of 0.19 using X-ray data between 6 and 2.0A. The molecule consists mainly of a straight five-turn alpha-helix, a five-stranded antiparallel beta-pleated sheet which is twisted and wrapped around the alpha-helix and an appending segment of partially alpha-helical geometry. The 'highly conserved' region from Gln53I to Gly57I implicated with binding to cysteine proteinases folds into a tight beta-hairpin loop which on opposite sides is flanked by the amino-terminal segment and by a second hairpin loop made up of the similarly conserved segment Pro103I - Trp104I. These loops and the amino-terminal Gly9I - Ala10I form a wedge-shaped 'edge' which is quite complementary to the 'active site cleft' of papain. Docking experiments suggest a unique model for the interaction of cystatin and papain: according to it both hairpin loops of cystatin make major binding interactions with the highly conserved residues Gly23, Gln19, Trp177 and Ala136 of papain in the neighbourhood of the reactive site Cys25; the amino-terminal segment Gly9I - Ala10I of bound cystatin is directed towards the substrate subsite S2, but in an inappropriate conformation and too far away to be attacked by the reactive site Cys25. As a consequence, the mechanism of the interaction between cysteine proteinases and their cystatin-like inhibitors seems to be fundamentally different from the 'standard mechanism' defined for serine proteinases and most of their protein inhibitors.     

Different forms of human cystatin C

Two isoelectric forms of human cystatin C with pI 9.2 and 7.8 have been isolated from urine of patients with different nephrological disorders. Treatment of both forms with alkaline phosphatase revealed that the difference between them is not due to the phosphorylation of some amino-acid residue. Further purification of cystatin C with pI 9.2 by hydrophobic chromatography and N-terminal sequencing showed that it consists predominantly of the full-length form of cystatin C with the N-terminal sequence SSPG-. Cystatin C with pI 7.8 was separated into two peaks. The first represented a pure form truncated by an octapeptide and beginning with the N-terminal sequence LVGG-. The second was a mixture containing 33% of the first peak and 66% of a truncated form with the N-terminal sequence VGGP-. Inhibitory activity of the full-length cystatin C and the pure truncated form has been measured against cathepsins B, H and L and show no significant differences in Ki values. These results further support the proposed mechanism of interaction of cysteine proteinases with their inhibitors cystatins (Bode, W., Engh, R., Musil, D., Thiele, U., Huber, R., Karshikow, A., Brzin, J., Kos, J. & Turk, V. (1988) EMBO J. 7, 2593-2599).     

Properties of acetylcholinesterase and non-specific cholinesterase in rat superior cervical ganglion and plasma

Amphiphile dependency, solubility in aqueous solutions, and sensitivity to proteolysis of acetylcholinesterase (AChE) and nonspecific cholinesterase (nsChE) in the rat superior cervical ganglion were studied and compared to properties of soluble plasma cholinesterases. Ganglion AChE shows strong amphiphile dependency: an amphyphilic substance must be present in the homogenizing medium in order to obtain maximal apparent enzyme activity. Apparent activity of AChE solubilized in Ringer's solution was also increased after subsequent addition of a detergent. The 4 S molecular form, predominant in this extract (corresponding to the fastest electrophoretic band), is very sensitive to papain proteolysis but can be protected by a detergent. This molecular form therefore carries an important hydrophobic domain and is probably membrane bound in situ. The 10 S form of ganglionic AChE, extracted in Ringer's solution, is probably a soluble enzyme since, like soluble plasma enzymes, it is not amphiphile dependent and is rather resistant to proteolysis. Ganglion nsChE is more water soluble, less amphiphile dependent and more protease resistant than AChE.     

The magnetic diver differential microgasometer

The magnetic differential microgasometer described here is basically an equalarm torsion balance for measuring the reduced weight of submerged objects. The changes of buoyancy (ΔV_gx;ΔRW) are compensated for by the magnetic force acting on the same arm where the change has occurred (substitution method of weighing). Two ampullae, one with the sample and the other with the blank, are placed into the loops mounted at the ends of the arms. The gas volume and the geometry of the ampullae are practically equal. Therefore, the instrument is stable and fairly insensitive to changes of barometric pressure and temperature, and it can be used for gasometric measurements in an open flotation vessel. The applicability of the method for experiments where high blanks cannot be avoided was demonstrated by respiration measurements of multiplying cells and by measurements of cholinesterase activity of muscle end-plate samples.     

Cell markers in the recognition of acute myeloblastic leukaemia subtypes

The diagnosis of acute myeloblastic leukaemia (AML) is based on cell morphology, cytogenetic and molecular changes, cell markers and clinical data. Our aim was to establish whether morphology and cell markers are comparable in the evaluation of AML. Bone marrow smears were analysed, and flow cytometry and monoclonal antibodies were used to determine cell type and maturity. Morphology and cell markers correlated differently in different AML subtypes.     

How a modification (8-aza-3-deaza-2'-deoxyguanosine) influences the quadruplex structure of Hotoda's 6-mer TGGGAG with 5'- and 3'-end modifications

We have synthesized a modified 6-mer using Hotoda's 6-mer TGGGAG with 5'- and 3'-end modifications as a template. We have replaced from one to all four 2'-deoxyguanosines by 8-aza-3-deaza-2'-deoxyguanosine (c3z8dG, 1) in order to investigate the anti-HIV structure activity relationship (SAR). ODN 4 (TGGG*AG) is the only one that exhibits a moderate anti-HIV-1 activity.     

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.     

Comparison of natural and recombinant clitocypins, the fungal cysteine protease inhibitors

A member of the cysteine protease inhibitor clitocypin gene family from basidiomycete Clitocybe nebularis was expressed in Escherichia coli. Following careful optimization of the expression procedure the active inhibitor was purified from inclusion bodies and its properties examined and compared to those of the natural clitocypin. The CD spectrum of recombinant clitocypin was similar to that of natural clitocypin, indicating that protein was properly refolded during purification. In spite of some differences in primary structure, structural, functional and immunological equivalence was established. Kinetic analyses of the natural and recombinant clitocypins were performed. Both clitocypins inhibited a range of cysteine proteases to a similar extent, and demonstrated an unusually broad inhibitory spectrum, including distantly related proteases, such as papain and legumain, belonging to different protease families. The homogenous, biologically active recombinant clitocypin is obtained at levels adequate for further structure-function studies.     

Restoration of enzymatic activity in a Ser-49 phospholipase A2 homologue decreases its Ca(2+)-independent membrane-damaging activity and increases its toxicity

Ammodytin L (AtnL) is a Ser-49 secretory phospholipase A2 (sPLA2) homologue with myotoxic activity. By analogy to the Lys-49 sPLA2 myotoxins, AtnL has been predicted to be enzymatically inactive due to the absence of the conserved Asp-49 that participates in coordination of the Ca2+ cofactor. By substituting Ser-49 and three other residues in the Ca2+-binding loop of AtnL, we obtained the first two enzymatically active mutants of Lys-49/Ser-49 sPLA2 homologues. The mutants LW and LV, which differed only by the presence of Trp and Val at position 31, respectively, efficiently hydrolyzed phospholipid vesicles, while recombinant AtnL displayed no activity. In contrast to AtnL but similarly to ammodytoxin A (AtxA), a homologous neurotoxic sPLA2, both mutants exhibited catalysis-dependent membrane-damaging ability, involving vesicle contents leakage and fusion. However, LW and LV also exhibited the potent, Ca2+-independent disruption of vesicle integrity characteristic of AtnL, but not of AtxA, in which leakage of the contents is not associated with membrane fusion. Although LV and, especially, LW have the advantage over AtnL of being able to act in both Ca2+-independent and Ca2+-dependent modes, and display higher cytotoxicity and higher lethal potency, they have a lower Ca2+-independent membrane-damaging potency and display reduced specificity in targeting muscle fibers in vitro. Our results indicate that, in evolution, Lys-49 and Ser-49 sPLA2 myotoxins have lost their Ca2+-binding ability and enzymatic activity through subtle changes in the Ca2+-binding network without affecting the rest of the catalytic machinery, thereby optimizing their Ca2+-independent membrane-damaging ability and myotoxic activity.