Optically detected magnetic resonance studies of porcine pancreatic phospholipase A2 binding to a negatively charged substrate analogue

The direct binding of porcine pancreatic phospholipase A2 and its zymogen to 1,2-bis(heptanylcarbamoyl)-rac-glycerol 3-sulfate was studied by optical detection of triplet-state magnetic resonance spectroscopy in zero applied magnetic field. The zero-field splittings of the single Trp3 residue undergo significant changes upon binding of phospholipase A2 to lipid. Shifts in zero-field splittings, characterized mainly by a reduction of the E parameter from 1.215 to 1.144 GHz, point to large changes in the Trp3 local environment which accompany the complexing of phospholipase A2 with lipid. This may be attributed to Stark effects caused by the binding of a charged group near Trp3 in the enzyme-lipid complex. The cofactor, Ca2+, which is strongly bound to the enzyme active site, has an influence on the bonding, as reflected by smaller zero-field splitting shifts. A relatively small change in the Trp environment was observed for the interaction of the zymogen with lipid.     

Changes in Targeting Efficiencies of Proteins to Plant Microbodies Caused by Amino Acid Substitutions in the Carboxy-terminal Tripeptide

It has been demonstrated that the carboxyl terminus of microbodyenzymes functions as a targeting signal to microbodies in higherplants. We have examined an ability of 24 carboxy-terminal aminoacid sequences to facilitate the transport of a cytosolic passengerprotein, ß-glucuroni-dase, into microbodies in greencotyledonary cells of trans-genic Arabidopsis. Immunoelectronmicroscopic analysis revealed that carboxy-terminal tripeptidesequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as amicrobody-targeting signal, although tripeptides with prolineat the first amino acid position and isoleucine at the carboxylterminus show weak targeting efficiencies. All known micro-bodyenzymes that are synthesized in a form similar in size to themature molecule, except catalase, contain one of these tripeptidesequences at their carboxyl terminus. (Received April 14, 1997; Accepted April 8, 1997)     

Separate Intramolecular Targets for Protein Kinase A Control N-Methyl-d-aspartate Receptor Gating and Ca2+ Permeability

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca²⁺ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca²⁺ permeability (P_Ca/P_Na) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca²⁺ conductance, because neither Na⁺ conductance nor Ca²⁺-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca²⁺ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca²⁺ permeability.     

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.     

Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase alpha activity

One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.     

DNA binding of the mouse class II major histocompatibility CCAAT factor depends on two components.

A CCAAT-binding factor that recognizes a CCAAT sequence (Y box) located upstream of the major histocompatibility class II gene I-A beta has been partially purified. This CCAAT-binding factor was found to consist of two components, designated factors A and B, both of which were required for efficient binding to the DNA. Factor A had an apparent molecular size of 34 kilodaltons, and factor B had an apparent molecular size of 42 to 46 kilodaltons.     

Effects of cyclopiazonic acid and aflatoxin singly and in combination on selected clinical,pathological and immunological responses of guinea pigs

Cyclopiazonic acid (CPA) and aflatoxin are known sometimes to coexist in nature but little is known of possible biological interaction in mammals that consume mixtures of these two mycotoxins. Guinea pigs were dosed orally with CPA (2.2 mg/kg) or aflatoxin (0.045 mg B₁/kg) singly or in combination. Effects of toxin consumption were determined on clinical health, body weight gain, pathological change, and several immunologically related parameters including delayed cutaneous hypersensitivity, antibody response, complement hemolytic titer, intracutaneous mitogen (PHA) and in vitro lymphocyte blastogenesis. In contrast to an earlier study by others, significant synergy between these two toxins was demonstrated in reduced rate of body weight gain, lethality and histologic changes (vacuolization) in hepatocytes. Reductions in complement titer, intradermal PHA, delayed cutaneous hypersensitivity response and in vitro lymphocyte blastogenesis were related to aflatoxin activity. No effects on antibody formation to Brucella abortus were observed with either toxin or the combination of toxins. Cyclopiazonic acid appeared to restore the suppressive effects of aflatoxin in delayed cutaneous hypersensitivity response and in vitro lymphocyte blastogenesis.