Glycosylation in vitro of an asparagine sequon catalysed by preparations of yeast cell membranes.

Preparations of yeast cell membranes can catalyse in vitro the N-acetyl-beta-D-glucosaminylation of the asparagine sequon at residues 34--36 of bovine pancreatic ribonuclease A. The relevant glycopeptides were isolated from tryptic hydrolysates of the glycosylated ribonuclease and analysed. The donor used was UDP-N-acetyl-D-glucosamine, although the mechanism of the transfer is unknown. Mn2+ ions at concentrations of 25 mM double the activity of the enzymic transfer.     

Homologous recombination intermediates between two duplex DNA catalysed by human cell extracts.

Using as substrates, 1: the replicative form (RF) of phage M13 mp8 in which the reading frame of the lac Z' gene was disrupted by insertion of an octonucleotide, and 2: a restriction fragment one kb long, containing the functional lac Z' gene (isolated from wild type M13 mp8), we show that nuclear extracts from human cells (3 lines tested) promote the targeted replacement of the altered sequence by the functional one. Following incubation with the extracts, the DNA's were introduced in JM 109 bacteria (rec A- and lac Z'-) which were grown in presence of a colorimetric indicator of beta-galactosidase activity. Homologous recombination gives rise to the genotypical modification: lac Z'+ instead of lac Z'- in the bacteriophage DNA. This is revealed by phenotypical expression of the lac Z' gene product in replicating bacteriophage, i.e. the formation of blue instead of white plaques. The frequency of recombination (blue/total plaques) is increased by a factor of 50-80 as a function of protein concentration and of incubation time. The maximal frequency observed is 5 X 10(-5). There is no increase over the background when extracts are boiled. Electrophoresis and electron microscopy of DNA's incubated with the extracts show the formation of recombination intermediates with single strand exchange. Restriction analysis of recombined DNA confirms that the process corresponds to targeted sequence exchange. These data allow to propose three steps for homologous recombination between two duplex DNA's: i) unpairing of the two duplexes; ii) single-strand exchange and synaptic pairing; iii) resolution of the cross-junctions. The three steps correspond to those predicted by the gene conversion model of Holliday.     

A study of the reaction catalysed by the liver branching enzyme

The mechanism of action of liver branching enzyme has been studied by using as substrate two polysaccharides in which the non-reducing ends had been labelled by incubation with phosphorylase and a trace amount of [¹⁴C]glucose 1-phosphate. After these polysaccharides had been treated with branching enzyme, their structure was analysed by periodate oxidation, by degradation with phosphorylase and amylo-(1→6)-glucosidase and by degradation with pullulanase. All the results indicate that the branching enzyme catalyses the transfer from (1→4)- to (1→6)-linkage of a chain of glucose units, the minimum length of which is six glucose units. A maltodextrin containing 16 glucose units was not acted on by the branching enzyme.     

Some characteristic reactions catalysed by the ribonuclease activity in sections from fixed tissues

Summary 1. The ribonuclease activity in sections from freeze-dried Carnoy-fixed rat pancreas has been characterized through studies on the products of interaction with synthetic nucleoside derivatives, nucleotide esters, and of the polynucleotide synthetic properties.2. Homogenates of the sections in McIlvaine's buffer atph 7.0 degrade pyrimidine 2:3-phosphates to the corresponding 3-dihydrogen phosphate, and the purine derivatives at a much slower rate.3. Synthetic cytidylyl-cytidine, and benzyl- and ethyl-3-esters of pyrimidine nucleotides are degraded to nucleoside 3-phosphates. All the purine, and the 2-esters of pyrimidine nucleotides are not affected during 24 hours of incubation.4. Under influence of the homogenates, ribopolynucleotides will readily be synthesized from cytidine-2:3-phosphate and cytidine, the 3-P-5dinucleotide links being identified in cytidylyl-cytidine and cyclic dicytidylic acid.5. The conclusion is reached that the activity of the sections coincides with the DEase and CPase activity of crystalline pancreatic RNase. Some aspects of the possibilities for the histochemical localization of the activity are briefly discussed.With 6 Figures in the TextAided by grants from the Royal Physiographic Society of Lund, the Nora Sörensson Foundation, and the J. V. and Charlotta Lundgren Foundation.     

Diadenosine triphosphate splitting by rat liver extracts.

A splitting activity on diadenosine triphosphate has been found in rat liver. One of the products of the cleavage is ADP. A Km of 10 μM has been found. This activity on diadenosine triphosphate seems to be specific as diadenosine tetraphosphate, a nucleotide previously described by others to occur in rat liver at very low concentration, is not a substrate of the reaction. The occurrence of diadenosine triphosphate in rat liver has not been so far reported, but a dinucleoside triphosphate structure has been described at the 5′ end of certain mRNAs. The possibility that this enzymatic activity may be involved in the hydrolysis of diadenosine triphosphate or in the processing of mRNAs is suggested.     

A ribonuclease activity is activated by heparin or by digestion with proteinase K in mitochondrial extracts of Leishmania tarentolae.

A ribonuclease activity in a 100,000 x g supernatant of a Triton lysate of a mitochondrial-kinetoplast fraction from Leishmania tarentolae is activated by incubation with heparin or by predigestion of the lysate with proteinase k or pronase. In vitro-transcribed pre-edited cytochrome b mRNA is cleaved at several sites. With time, complete degradation of the RNA occurs. All cleavages occurred within putative single-stranded regions of the RNA. No cleavage was observed with 9 S rRNA. The presence of a nonspecific nucleotide or nucleoside slows the rate of cleavage. The cleavage activity is inhibited by sodium dodecyl sulfate or phenol/chloroform extraction, is retained by a 10-kDa cutoff filter, and passes through a 30-kDa filter. Micrococcal nuclease inhibits the proteinase-induced activity but not the heparin-induced activity.     

Cyanide formation from glyoxylate and hydroxylamine catalysed by extracts of higher-plant leaves

Extracts of spinach, maize and barley contain an enzyme which catalyses the formation of hydrogen cyanide from glyoxylate and hydroxylamine. The enzyme is dependent upon ADP and a divalent cation such as manganese. Glyoxylicacid oxime is a poor substrate for the enzyme. Carbon dioxide is another product of the reaction and is probably produced in 1:1 stoichiometry with hydrogen cyanide. The possible relationship of this enzyme to the regulation of nitrate reduction is discussed.     

Kinetic transients in the reduction of aldehydes catalysed by liver alcohol dehydrogenase.

The transient-state kinetics of enzymic reduction of acetaldehyde and benzaldehyde by NADH, catalyzed by horse liver alcohol dehydrogenase, have been examined under single-turnover conditions, obtained by carrying out reactions either with limiting amounts of enzyme in the presence of 20 mM pyrazole or with limiting amounts of substrate. Analysis of the variation with substrate, coenzyme, and enzyme concentrations of amplitudes and time constants for the exponential transients observed at 328 nm and 300 nm shows that the kinetics of enzymic aldehyde reduction are qualitatively and quantitatively consistent with the relationships derived in the preceding paper for an ordered ternary-complex mechanism involving identical and independent catalytic sites. It is concluded that there is no evidence whatsoever for the kinetic significance of a half-of-the-sites reactivity or any other kind of subunit interaction in the liver alcohol dehydrogenase system. The biphasic transients observed at 328 nm for the reduction of aromatic aldehydes such as benzaldehyde are a normal kinetic characteristic of the ordered ternary-complex mechanism, being attributable to accumulation of the ternary enzyme-NAD-product complex when product dissociation from this complex is slow in comparison to its formation by ternary-complex interconversion.     

Conjugation of phenylalanine hydroxylase with polyubiquitin chains catalysed by rat liver enzymes.

Phenylalanine hydroxylase (PAH, EC 1.14.16.1) is a highly regulated liver enzyme which catalyses the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the catabolic pathway of this amino acid. Among the approx. 400 different mutations of human (h) PAH, frequently associated with the metabolic disease phenylketonuria, a low stability is a characteristic property when expressed in eucaryotic cells. In this study, the pathway of hPAH degradation is addressed with focus on its conjugation with polyubiquitin chains catalysed by the ubiquitin-conjugating enzyme system (E1, E2, E3) isolated from rat liver by covalent affinity chromatography on ubiquitin-Sepharose. In the reconstituted in vitro ubiquitination assay, the enzyme system catalysed both the formation of free polyubiquitin chains and the polyubiquitination of wild-type (wt) hPAH and its 'catalytic domain' (DeltaN102/DeltaC24-hPAH) as visualized by two-dimensional electrophoresis. The ubiquitination of wt-PAH may play a role in the degradation of this liver enzyme notably of its many unstable disease-associated mutant forms. The present approach may also have a more general application in the study of liver proteins as possible targets for ubiquitination.     

Glycosylation of human pancreatic ribonuclease: differences between normal and tumor states

Characterization of the N-glycans from human pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3) revealed completely different glycosylation patterns. RNase 1 from healthy cells contained neutral complex biantennary structures, with smaller amounts of tri- and tetraantennary compounds, and glycans with poly-N-acetyllactosamine extensions, all extensively fucosylated. In contrast, RNase 1 glycans from tumor cells (Capan-1) were fucosylated hybrid and complex biantennary glycans with GalNAc-GlcNAc antennae. RNase 1 glycans from Capan-1 and MDAPanc-3 cells also contained sialylated structures completely absent in the healthy pancreas. Some of these features provide distinct epitopes that were clearly detected using monoclonal antibodies against carbohydrate antigens. Thus monoclonal antibodies to Lewis(y) reacted only with normal pancreatic RNase 1, whereas, in contrast, monoclonal antibodies to sialyl-Lewis(x) and sialyl-Lewis(a) reacted only with RNase 1 secreted from the tumor cells. These glycosylation changes in a tumor-secreted protein, which reflect fundamental changes in the enzymes involved in the glycosylation pathway, open up the possibility of using serum RNase 1 as a tumor marker of pancreatic adenocarcinoma.     

The role of lysine-41 in ribonuclease A studied by proton-magnetic-resonance spectroscopy of guanidinated ribonuclease A.

Ribonuclease A has been guanidinated at the lysine residues and the nona-guanidinated and deca-guanidinated (fully substituted) products separated. In confirmation of an earlier report by Glick and Barnard (1970), it has been shown by chemical procedures that the former derivative is not reacted at lysine-41. Guanidination of lysine-41 to produce the fully substituted product causes loss of enzymic activity without any apparent change of conformation, as tested by conformational comparisons (using proton magnetic resonance spectroscopy) including (a) difference spectroscopy, evidence for the involvement of lysine-41 in a catalytic role in the enzyme. Dimethylation of lysine-41 of nona-guanidinated ribonuclease A produces sharp proton resonances which shifts as the dimethylamino group is titrated and allow the determination of an apparent pK of 8.8 for unsubstituted lysine-41.     

A protein inhibitor of rabbit liver phosphorylase phosphatase.

Extracts of rabbit liver contain a heat-stable, non-dialysable inhibitor of phosphorylase phosphatase. The inhibitory activity is destroyed by trypsin treatment or by ethanol precipitation. The kinetics of the inhibition are non-competitive with respect to phosphorylase a. The inhibitory activity is polydisperse on gel permeation chromatography. The mechanism of the inhibition is due to a direct interaction of the protein inhibitor with the enzyme.