Proton nuclear magnetic resonance spectroscopy of horseradish peroxidase isoenzymes: correlation of distinctive spectra with isoenzyme specific activities

High-resolution proton NMR spectra are reported for the paramagnetic ferric native and cyano complexes of the five major horseradish root peroxidase (HRP) isoenzymes (A1, A2, A3, B, and C). Axial imidazole resonances are observed in the native and cyano-complex spectra of all the isoenzymes, thus indicating the presence of a common axial histidine ligand. Proton NMR spectra outside the usual diamagnetic region are identical for sets of A1 and A2 isoenzymes and for the B and C isoenzyme set. Variation in heme residue chemical shift positions may be controlled in part by porphyrin vinyl side chain-protein interactions. Diverse upfield spectra among the isoenzymes reflect amino acid substitutions and/or conformational differences near the prosthetic group, as signals in this region must result from amino acid residues in proximity to the heme center. Acid-base dependence studies reveal an "alkaline" transition that converts the native high-spin iron (III) porphyrin to the low-spin state. The transition occurs at pH 9.3, 9.4, 9.8, and 10.9 for respective HRP A1, A2, A3, and C isoenzymes, respectively. Significantly, this ordering also reflects specific activities for the isoenzymes in the order A1 = A2 greater than A3 greater than B = C. Identical proton NMR spectra for A1/A2 and B/C isoenzyme sets parallel equivalent specific activities for members of a particular set. Proton NMR spectra thus appear to be highly sensitive to protein modifications that affect catalytic activity.     

The placenta as a site of cytomegalovirus infection in guinea pigs.

The development of cytomegalovirus (CMV) infection in the placenta was studied in Hartley guinea pigs inoculated at midgestation, and its role in determining the outcome of fetal CMV infection was assessed. A hematogenous spread of CMV from the mother to the placenta occurred early during the course of the infection. However, the virus remained present in placental tissues long after CMV had been cleared from maternal blood (i.e., 3 and 4 weeks postinoculation). At that time, the virus was able to replicate in placental tissues in the presence of specific maternal antibodies. Viral nucleocapsids were seen within nuclei of trophoblastic cells, and virions were present surrounding infected cells. In addition, typical CMV-induced histopathological lesions bearing CMV antigens were consistently localized at the transitional zone between the capillarized labyrinth and the noncapillarized interlobium. Whenever CMV infection of the fetus occurred, virus was isolated from the associated placenta. Among placental-fetal units with CMV-infected placentas, only 27% of the fetuses were found to be infected. In addition, there was a delay in the establishment of the infection in the fetus in relation to the placenta, although frequencies of virus isolation in placental and fetal tissues peaked at 3 weeks after CMV inoculation. These results suggest that during primary CMV infection of pregnant guinea pigs, the placenta not only serves as a reservoir for CMV but also acts to limit transmission of the virus to the fetus.     

pH-dependent processes in proteins

Recent improvements in the understanding of electrostatic interactions in proteins serve as a focus for the general topic of pH-dependent processes in proteins. The general importance of pH-dependent processes is first set out in terms of hydrogen ion equilibria, stability, ligand interactions, assembly, dynamics, and events in related molecular systems. The development of various theoretical treatments includes various formalisms in addition to the solvent interface model developed by Shire et al. as an extension of the Tanford-Kirkwood treatment. A number of detailed applications of the model are presented and future potentialities are sketched.     

Producing the cortical phenomenon of "kindling" during acute experiments on cats

Acute experiments on cats enabled spontaneous epileptogenic foci to be produced by means of electrical stimulation of the cerebral cortex. The central area of the suprasylvian gyrus was stimulated by 5-sec stimuli at 3-sec intervals. The strength of neuronal response gradually increased until spontaneous electrical activity set in as stimulation was set at a fixed intensity which had originally evoked local residual discharge at stimulation foci only, i.e., the phenomenon of "kindling" was observed. When "kindling" was produced in the cortical area under study, bursts of spindle activity were recorded on all ECoG with increasing frequency. Recordings of spindle activity then changed to a "spike-wave" pattern of activity. The results of these investigations, which were performed on an isolated strip of cortex, point to the involvement of subcortical structures in mediating "kindling" of the cortical focus.Institute of Clinical and Experimental Neurology, Ministry of Public Health of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 601–606, September–October, 1985.     

Single Doses of Acrylamide Reduce Retrograde Transport Velocity

Abstract: Single doses of acrylamide (0–1.3 mmol/kg) produced a dose-dependent decrease in the transport of ¹²⁵I-tetanus toxin to the perikarya of sensory neurons in dorsal root ganglia and motor neurons in ventral spinal cord. Acrylamide was a more potent inhibitor of retrograde transport in sensory axons than in motor axons. Substantially greater doses of N,N '-methylene-bis-acryl-amide, a reportedly non-neurotoxic analog of acrylamide, were required to alter the axonal transport of ¹²⁵I-tetanus toxin. Velocity of retrograde transport was assessed by determining the position of the leading edge of transported¹²⁵I-tetanus toxin at times following single doses of acrylamide. Acrylamide reduced the velocity of ¹²⁵I-tetanus toxin transport in a dose-dependent manner by up to 75%. No change in neuronal uptake of ¹²⁵I-tet-anus toxin was detected. It is concluded that single doses of acrylamide produce profound alterations in retrograde transport which precede the appearance of structural changes in affected nerve fibers.     

The stability of the yeast plasmid pJDB248 depends on growth rate of the culture

Summary The stability of the plasmid pJDB 248 has been measured in theS. cerevisiae strain S150-2B growing in a chemostat under conditions of glucose limitation. It was found that reducing the growth rate of the culture led to a more rapid loss of the plasmid from the cells.     

The pH dependence of hydrogen exchange in proteins

The static accessibility modified discrete charge model for electrostatic interactions in proteins is extended to the prediction of the pH dependence of hydrogen exchange reactions. The exchange rate profiles of buried amide protons are shown to follow the calculated pH dependence of the electrostatic component of protein stability. Rate profiles are calculated for individual buried amide protons in ribonuclease S and bovine pancreatic trypsin inhibitor. The electrostatic free energy of stabilization of the protein and the energy required to bring the catalytic ion to an exchange site are expressed as an apparent, pH-dependent contribution to the activation energy. Changes in the electrostatic stabilization of the proteins affect the calculated exchange rate for buried amide protons by more than 1000, while local field effects raise or lower the predicted exchange rates by less than 100. The pH dependence of exchangeable protons at the protein surface, such as the C-2 imidazole protons, is shown to follow the estimated energy required to introduce the catalytic ion at the exchange site. These calculations are discussed in terms of current models for proton exchange which incorporate the dynamic nature of the structure to explain exchange data from the interior of a protein.