Dependence on blood acetate concentration of the metabolic effects of ethanol in perfused rat liver

Unprotected oligonucleotides and oligodeoxynucleotides terminated with an unhindered 5'-phosphate group react with nucleoside 5'- phosphorimidazolides in aqueous solution to give 'capped' pyrophosphates in at least 70% yield. If adenosine 5'- phosphorimidazolide is used as a substrate in the reaction, ligase intermediates are obtained as products.     

The identification of serum tuftsin by reverse-phase high-performance liquid chromatography and mass spectrometry

We describe for the first time a method for unambiguously identifying the phagocytic stimulating tetrapeptide tuftsin from trypsinized human serum. The method consists of separating serum tuftsin by reverse-phase (RP)-HPLC, collecting HPLC fractions corresponding to the synthetic tuftsin retention time, and then subjecting those fractions to mass spectrometry/mass spectrometry (MS/MS) analysis, which provides optimal molecular specificity to the measurement. Although quantification was not the goal, it was estimated that the amount of tuftsin found by RP-HPLC and MS/MS was in the hundreds of nanograms per milliliter.     

Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons

External invertase is the product of the SUC2 gene of Saccharomyces cerevisiae. The deduced sequence of this enzyme (Taussig, R., and Carlson, M. (1983) Nucleic Acid Res. 11, 1943-1954) reveals it to contain 14 potential N-linked glycosylation sites, or sequons, although only 9-10 appear to be glycosylated (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine the location of the glycosylated sequons, external invertase was deglycosylated with endo-beta-acetylglucosaminidase H and its component peptides analyzed by both fast atom bombardment mass spectrometry (FABMS) and classical peptide isolation procedures. By use of the former technique most of the glucosamine-containing sequons could be located and by the latter sufficient amounts of small glucosamine-containing peptides were isolated to enable their quantitation. From the combined FABMS and glucosamine analyses, it was established that eight of the sequons in a subunit of invertase are either completely or almost completely glycosylated, while five others are glycosylated to the extent of about 50% or less. In the case of two overlapping sequons (4 and 5), which include Asn92-Asn93-Thr-Ser, only the first Asn was glycosylated. Thus, all but one of the sequons of external invertase are glycosylated to some extent, giving an appearance of only 9-10 N-linked oligosaccharides/subunit. The sequence identity of both external and internal invertase was verified by FABMS and by peptide sequence analysis. In only one site was an amino acid found to differ from that deduced from the DNA sequence of the SUC2 gene. This occurred at position 390 where a proline was found in place of alanine, which could result from a single base change in the triplet specifying the latter amino acid.     

Structure of yeast external invertase Man8-14GlcNAc processing intermediates by 500-megahertz 1H NMR spectroscopy

A series of high mannose oligosaccharides with the size range Man8-14GlcNAc was purified from Saccharomyces cerevisiae invertase, and the composition of each was determined by chemical analysis. Purity and composition were verified by 1H NMR spectroscopy at 500 MHz, and structures were assigned on the basis of chemical shifts in C1-H and C2-H protons of similarly substituted compounds of known structure. Such analyses showed that these invertase oligosaccharides were a homologous series of homogeneous compounds, each related to the next member by addition of 1 mol of mannose in a specific alpha-linked configuration. Man8GlcNAc purified from the total glycoprotein fraction of disrupted yeast was the smallest species found and had the same homogeneous structure as that previously reported for the Man8GlcNAc from invertase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Digestion of Man8-13GlcNAc species from invertase with Aspergillus satoi alpha 1,2-mannosidase provided products that were consistent with the structures assigned by 1H NMR as did fast atom bombardment-mass spectroscopy fragmentation analysis of the Man9,10GlcNAc oligosaccharides. These results lead to the proposal that Man8GlcNAc is the only trimming intermediate in Saccharomyces sp., and the remaining Man9-14GlcNAc oligosaccharides are biosynthetic intermediates which define the principal pathway of single-step mannose addition in the formation of the inner core of yeast mannan.     

Amplified expression of streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product

The endo-beta-N-acetylglucosaminidase H (Endo H) gene from Streptomyces plicatus has been cloned into the Escherichia coli plasmid pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature 272, 128-132), thus placing expression of this gene under control of the strong lambda promoter pL. The construction, pKCE3, which includes a properly positioned E. coli ribosome binding site from the lac operon (Robbins, P.W., Trimble, R. B., Wirth, D.F., Hering, C., Maley, F. Maley, G. F., Das, R., Gibson, B.W., and Biemann, K. (1984) J. Biol. Chem. 259, 7577-7583), was used to transform an E. coli strain lysogenic for a lambda prophage containing a temperature-sensitive repressor. By shifting cultures of pKCE3 lysogens to 42 degrees C, the production of Endo H commenced and was linear for about 1 h. Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S. plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure. Although most of the cloned Endo H was secreted into the periplasmic space by E. coli, its 4 kDa leader sequence peptide (Robbins et al. (1984] was only partially removed during processing. As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S. plicatus. Despite the residual approximately 2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E. coli or from S. plicatus. Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants.     

Effects of haematocrit value and glucagon on the metabolism of perfused rat liver

Liver from adult male rats were perfused in situ for 30 min with either undiluted, defibrinated rat blood (haematocrit value 38%) or the same blood diluted with buffer to give a haematocrit of 20%. Perfusion with diluted blood lowered the PO2 of the effluent perfusate but this was insufficient to prevent the fall in O2 consumption due to the reduction in haematocrit. Glucagon (5 X 10(-9) M) increased hepatic O2 consumption with whole blood but not with diluted blood. perfusate K+ was increased by perfusion with diluted blood and glucagon. Bile flow was depressed and biliary K+ increased by glucagon but only in experiments with whole blood. Perfusate glucose was raised by lowering of hepatic O2 consumption but the hormonal stimulation of glucose output was the same at both haematocrits. Net ketogenesis was increased with perfusion with diluted blood and by glucagon. In the absence of glucagon there was a net secretion of triacylglycerols which was depressed by lowering of the haematocrit. Glucagon inhibited triacylglycerol secretion and the effect was greater with whole blood so that there was net uptake. While effects of glucagon were obtained during perfusion at a lower haematocrit, it would appear that whole blood was the medium that allowed their fullest expression.     

Bacteriology of manganese nodules: III. Reduction of MnO(2) by two strains of nodule bacteria

MnO₂ reduction by aerobic growing cultures of Bacillus 29 and coccus 32, isolated from ferromanganese nodules, was assessed for 7 days. A 1-day lag was observed before the onset of MnO₂ reduction by either culture. Addition of HgCl₂ to a final concentration of about 10^-3 M caused a rapid cessation of MnO₂ reduction by the growing cultures. Neither culture reduced MnO₂ when grown under continued anaerobiosis from the start of an experiment. However, if conditions were made anaerobic after MnO₂ reduction was initiated, reduction continued at a rate only slightly lower than that under aerobic conditions. Resting-cell cultures reduced MnO₂ equally well aerobically and anaerobically, provided that ferricyanide was present to serve as electron carrier. These findings showed that oxygen is needed for culture adaptation to MnO₂ reduction, and that oxygen does not interfere with microbial MnO₂ reduction itself. Both cultures caused sharp drops in the pH of the medium during MnO₂ reduction: with coccus 32, during the entire incubation time; with Bacillus 29, for the first 3 days. The E_h of the medium fluctuated with either culture and never fell below 469 mv with Bacillus 29 and below 394 mv with coccus 32. The rates of glucose consumption and Mn²⁺ release by Bacillus 29 and coccus 32 were fairly constant, but the rates of lactate and pyruvate production were not. Although acid production undoubtedly helped in the reduction of pyrolusite (MnO₂) by the bacteria, it did not appear to be important in the reduction of manganese oxide in ferromanganese nodules, as shown by the results with a nodule enrichment.     

Bacteriology of Manganese Nodules: III. Reduction of MnO2 by Two Strains of Nodule Bacteria1

MnO₂ reduction by aerobic growing cultures of Bacillus 29 and coccus 32, isolated from ferromanganese nodules, was assessed for 7 days. A 1-day lag was observed before the onset of MnO₂ reduction by either culture. Addition of HgCl₂ to a final concentration of about 10^-3 M caused a rapid cessation of MnO₂ reduction by the growing cultures. Neither culture reduced MnO₂ when grown under continued anaerobiosis from the start of an experiment. However, if conditions were made anaerobic after MnO₂ reduction was initiated, reduction continued at a rate only slightly lower than that under aerobic conditions. Resting-cell cultures reduced MnO₂ equally well aerobically and anaerobically, provided that ferricyanide was present to serve as electron carrier. These findings showed that oxygen is needed for culture adaptation to MnO₂ reduction, and that oxygen does not interfere with microbial MnO₂ reduction itself. Both cultures caused sharp drops in the pH of the medium during MnO₂ reduction: with coccus 32, during the entire incubation time; with Bacillus 29, for the first 3 days. The E_h of the medium fluctuated with either culture and never fell below 469 mv with Bacillus 29 and below 394 mv with coccus 32. The rates of glucose consumption and Mn²⁺ release by Bacillus 29 and coccus 32 were fairly constant, but the rates of lactate and pyruvate production were not. Although acid production undoubtedly helped in the reduction of pyrolusite (MnO₂) by the bacteria, it did not appear to be important in the reduction of manganese oxide in ferromanganese nodules, as shown by the results with a nodule enrichment.