Enzymatic Protein Carboxyl Methylation in Rat Posterior Pituitary: Neurophysins in Rapid-Turnover Pool Determine Methyl Accepting Capacity

In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70-80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionine in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (less than 7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.     

Liver glutathione S-transferase polymorphism in Japanese and its pharmacogenetic importance

Summary A total of 168 autopsy liver extracts from Japanese individuals were examined for the glutathione S-transferase (GST) isozymes by means of starch gel electrophoresis. The gene frequencies of GST1^*1, GST1^*2, and GST1^*0 in Japanese were 0.252, 0.057, and 0.691, respectively. GST1^*3 was detected as a rare variant allele. The incidence of GST1 0 in 41 liver biopsy samples from patients suffering from various liver diseases was investigated using polyacrylamide gel isoelectric focusing. The GST1 0 phenotype was found more frequently in livers with hepatitis and carcinoma than in control livers. The isozymes coded by different GST loci were partially purified and characterized to study their biochemical properties. The apparent Km values with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate for the isozymes at the GST1, GST2, GST3, and GST4 loci were 604, 1345, 776 and 591 M, respectively.     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

Structure of cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from a fern (Asplenium cataractarum rosenstock,aspleniaceae), and its comparison with those of various seed plants

We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution rate per year per site for RuBisCO SSu was calculated to be 0.81×10⁻⁹ assuming that the separation between pteridophytes and seed plants arose 380 million years ago.     

Local distribution of the lycaenid butterfly,Jalmenus evagoras,in response to host ants and plants

Summary The caterpillars of Jalmenus evagoras are tended by ants as they feed upon Acacia trees. In the area of Brisbane, Australia, J. evagoras require ants of the Iridomyrmex anceps species group; predation and parasitism are so intense that larvae and pupae deprived of attendant ants cannot survive (Pierce 1983). We investigated the efficiency with which J. evagoras locate and exploit the host ant resource by sampling 737 quadrats in 30 sampling grids and six study sites containing appropriate host plants; ants were collected at baits located in the center of each quadrat. J. evagoras was found in all habitats where I. anceps cooccurred with host Acacia. Nine of the ten sampling grids which had three or more I. anceps/Acacia host quadrats also had colonies of J. evagoras present (or immediately adjacent), including sites as far as 35 km apart. Of 19 sampling grids on which host quadrats were rare (i.e., less than three quadrats), none had J. evagoras (P<0.001). Within sample grids, I. anceps was distributed indepedently from Acacia trees, suggesting that they are not dependent for their survival on either Acacia or on J. evagoras. Within montane pasture habitats, I. anceps and at least one other ground-dwelling Iridomyrmex species were distributed in mutually exclusive ant mosaic territories which were stable during a one month period. I. anceps did not colonize or tend pupae of J. evagoras experimentally placed in adjacent territories of a different, nontending species of Iridomyrmex, demonstrating the integrity of territory boundaries. Sampling of ants in Acacia trees revealed that, in the absence of J. evagoras, Iridomyrmex workers are not common above ground level, and that their numbers decline in larger trees (P=0.02). In I. anceps territories, eight of nine J. evagoras pupae placed in trees over 3.0 m tall were not found after 24 h whereas all ten controls placed in low trees were found and tended (P=0.00012). This may explain why J. evagoras tends to oviposit in trees less than 2.0 m tall. An alternative hypothesis, that smaller trees have higher content of total nitrogen, and are threfore more nutritious, was not supported. We conclude that the local distribution and host tree selection by J. evagoras is dependent upon the distribution, patchiness, and foraging behavior of the host ant, I. anceps, and its spatial overlap with a number of species of host Acacia.     

The purification and properties of a penicillinase whose synthesis is mediated by an R-factor in Escherichia coli

1. The penicillinase (beta-lactamase) from Escherichia coli strain TEM has been purified and its activity against a range of penicillin and cephalosporin derivatives measured. 2. The enzyme shows little resemblance to penicillinases from Bacillus cereus, Bacillus licheniformis and Staphylococcus aureus. 3. The molecular weight of the enzyme is 16700+/-5%, which is about half the value obtained for other penicillinases. 4. The enzyme is most similar in properties to a crude preparation of a penicillinase from Klebsiella (Aerobacter) aerogenes, but clearly different from crude enzyme preparations from other strains of E. coli. 5. Since penicillinase synthesis in E. coli strain TEM is mediated by an R-factor known to infect many other species of Enterobacteriaceae, the appearance of similar enzymes in other Gramnegative species is not surprising.     

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.     

Purification of the microsomal Ca2+-ATPase from rat liver

Summary The Ca²⁺-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose treatment, column chromatography with agarose-hexane adenosine 5-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca²⁺-ATPase was stable for at least two weeks when stored at –70°C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca²⁺-ATPase. Further characterization of the ER Ca²⁺-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca²⁺-ATPase cross-reacted with the purified Ca²⁺-ATPase from rat liver ER membranes.