Oligomerization and the sensitivity of phosphoenolpyruvate carboxylase to inactivation by proteinases

Phosphenolpyruvate (PEP) carboxylase from leaves of Crassula argentea displays varying levels of sensitivity to inactivation by various proteolytic enzymes. In general, the native enzyme is sensitive to proteinases known to attack at the carbonyl end of lysine or arginine (trypsin, papain, or bromelain). The ineffective proteolytic enzymes are those which have low specificity or which attack at the N-terminal end of hydrophobic amino acids, or which cannot attack lysine. The lack of an effect of endoproteinase arginine C, which is specific for arginine, probably indicates that lysine is the critical residue. When the native enzyme, which is comprised of an equilibrium of dimers with tetramers in approximately equal quantities, is treated by preincubation with 5 millimolar PEP, the enzyme becomes much more resistant to proteolytic inactivation. When the preincubation is with 5 millimolar malate rather than buffer alone, the effect is to slightly increase (ca. 15%) the sensitivity of the enzyme to inactivation by trypsin as measured by estimates of the pseudo-first order rate constant for inactivation. PEP carboxylase from corn leaves appears to be relatively susceptible to inactivation by trypsin, but is unaffected by preincubation with malate or PEP. The sensitivity of this C₄ enzyme to inhibition by malate is also unaffected by preincubation with these ligands.     

Light-regulated methylation of chloroplast proteins

Protein carboxyl methyltransferases, which catalyze transfer of methyl groups from S-adenosyl-L-methionine to the free carboxyl groups of acidic amino acids in proteins, can be divided into two classes based on several characteristics, such as the stoichiometry of substrate protein methylation, base stability of the incorporated methyl group, specificity for substrate, and participation in a regulatory system with which methylesterases are associated. The presence of such an enzyme in a photosynthetic system was demonstrated in the present work. The extent of methylation of chloroplast proteins was stimulated 30% by light and then decreased by the same amount in the presence of the electron transport inhibitor 3-(3',4'-dichlorophenyl)-1', 1'-dimethylurea or uncouplers of phosphorylation, indicating a dependence of the methyltransferase activity on photosynthetic electron transport and the trans-membrane delta pH. The light-independent, as well as the light-dependent, activity is probably of chloroplast origin since the extent of light stimulation in the purified thylakoid membranes and the stromal fraction was similar, and at low concentrations of S-adenosyl-L-methionine the small subunit of ribulose-1,5-bisphosphate carboxylase:oxygenase was found to be the predominant substrate. The labeling pattern of chloroplast proteins and labeling of an exogenous nonchloroplast protein indicated that the methyltransferase activity was not substrate-specific, although at low concentrations of the methyl donor, the small subunit of ribulose-1,5-bisphosphate carboxylase:oxygenase was labeled almost exclusively. Based on the low stoichiometry (less than 100 pmol/mg protein) of the methylation, its base lability, irreversibility, and the lack of substrate specificity except at very low concentrations of methyl donor, it was inferred that the chloroplast methyltransferase is best classified as a class II system that may function as part of a repair mechanism to replace racemized amino acids.     

Inhibition by prostaglandin E2 of neurotransmission in rabbit but not human bronchus--a calcium-related mechanism?

Prostaglandins have been implicated in the development of airway hyperresponsiveness, and this may be mediated via modulation of neurotransmission. We compared the effects of prostaglandin E2 on the contractile response to electrical field stimulation in rabbit and human bronchus. Prostaglandin E2 produced marked inhibition in rabbit bronchus (mean % inhibition 35 +/- 17, P less than 0.05) but was without effect in human bronchus. The inhibition in rabbit bronchus was not the result of a direct effect on muscle tone and the site of action is likely to be pre-synaptic since prostaglandin E2 had only minor effects on exogenous acetylcholine. Since prostaglandins are known to affect calcium mobilization, we compared the dependence of cholinergic stimulation on the calcium voltage dependent channel (VDC) in the two species. Cholinergic stimulation was dependent on the VDC in rabbit but not human bronchus and this may be an explanation for the different effects of prostaglandin E2 in the two species.     

U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.     

Induction of T4 DNA ligase in a recombinant strain of Escherichia coli

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.     

An epizootic of Mycoplasma ovipneumoniae infection in captive Dall's sheep (Ovis dalli dalli)

In late spring of 1986, 10 of 23 Dall's sheep (Ovis dalli dalli) at the Metropolitan Toronto Zoo were moved to a new exhibit, where all developed severe respiratory signs refractory to anthelmintic and antibiotic therapy. In July, two animals died with chronic active bronch-pneumonia, and a third was euthanized because of pneumonia several months later. Bacteria were not isolated from the lungs of the first, steptococci and Pasteurella hemolytica were isolated from the other two, respectively; Mycoplasma ovipneumoniae was isolated from both. Pulmonary lesions in all three sheep were consistent with Mycoplasma sp. infection. Nasal swabs of the remaining animals yielded no consistent bacterial isolates; however, four of eight sheep were positive for M. ovipneumoniae. Viral cultures yielded an as yet unidentified herpesvirus. Sheep in the original and new herds had no serologic titers to parainfluenza-3, equine viral rhinopneumonitis, or infectious bovine rhinotracheitis, and had variable titers against bovine respiratory syncytial virus. No titers against M. ovipneumoniae were present in 13 sheep still in the original exhibit, but titers varied from 1:32 to 1:256 in eight pneumonic sheep. Sera taken from three sheep before or early in the outbreak were all negative for antibody to M. ovipneumoniae. Two of the affected Dall's sheep had been in contact with domestic sheep in the winter of 1985-1986, and M. ovipneumoniae was subsequently cultured from the domestic flock. Exposure to a new pathogen, and environmental and social stress in a new exhibit may have resulted in this severe disease in Dall's sheep.     

Subcellular Distribution of UDP-Galactose:Ceramide Galactosyltransferase in Rat Brain Oligodendroglia

Oligodendrocytes isolated from 18-19-day-old rat brain were homogenized in 0.32 M sucrose. The homogenate was centrifuged at 100,000 g for 50 min in a gradient containing 0.8, 1.05, and 1.3 M sucrose. Three discrete bands were obtained at the interfaces 0.32-0.8 (F1), 0.8-1.05 (F2), and 1.05-1.3 M (F3). The distribution of UDP-galactose:ceramide galactosyltransferase (CgalT) activity in each fraction was measured using liposomes containing normal fatty acid-containing ceramides (NFA-CgalT activity) or 2-hydroxy fatty acid-containing ceramides (HFA-CgalT activity). Although detection of both CgalT activities was possible in all fractions, HFA-CgalT activity was enriched in F1 and F2 fractions, which also showed an enrichment of Golgi and endoplasmic reticulum markers, respectively. It is interesting that NFA-CgalT activity was significantly enriched in the F2 fraction. These results suggest that hydroxylated and nonhydroxylated galactocerebrosides may be synthesized at different intracellular locations.     

Predator driven changes in community structure

Summary The zooplankton community of a small pond changed markedly with temporal variation in predation pressure. Long term changes in zooplankton community structure occurred following the replacement of planktivorous fish by phantom midge (Chaoborus americanus) larvae as the predominant predator of zooplankton. The interannual changes following the establishment of Chaoborus included the apparent or near extinction, of species ill adapted to the new predation pressure and the successful colonization of well adapted species. Seasonal changes in the species composition and size distribution of the zooplankton community correlate with temporal variation in predation intensity associated with temperature-activity patterns of the predator or changes in the stage structure of the predator population.