Substrate Specificity of Barley Cysteine Endoproteases EP-A and EP-B

The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P₂-P₁-P₁′-P₂′-tyrosine(NO₂)-aspartic acid, in which cleavage occurs between P₁ and P₁′, showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P₂. Arginine was preferred to glutamine at P₁, whereas proline at P₂, P₁, or P₁′ greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower K_m values.     

Conversion of N-acetylglucosamine to CMP-N-acetylneuraminic acid in a cell-free system of rat liver

A soluble fraction of rat liver converts glucosamine and N-acetylglucosamine in the presence of ATP and UTP to N-acetylneuraminic acid. This system, when supplemented with CTP, forms CMP-N-acetylneuraminic acid in high yield. Nicotinamide was found to enhance the synthesis of UDP-N-acetylglucosamine and N-acetylneuraminic acid. Kinetic analysis reveals N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, N-acetylmannosamine, N-acetylmannosamine 6-phosphate and N-acetylneuraminic acid 9-phosphate as intermediates. Under certain experimental conditions, however, an epimerisation of N-acetylglucosamine to N-acetylmannosamine was seen.     

Effect of nitrogen nutrition on endosperm protein synthesis in wild and cultivated barley grown in spike culture

In normal growth conditions, total protein percent (salt soluble plus hordein fractions) in the endosperm at maturity in barley cultivar Hordeum vulgare L. cv `Ruth' was about 14%, whereas in an accession of wild barley, Hordeum spontaneum Koch line 297, it was about 28%. Spike culture experiments were conducted to ascertain whether there were basic differences between the two genotypes under conditions of widely different nitrogen supply. Spikes of each genotype were grown from 8 to 25 days after flowering in in vitro culture in a growth medium containing 0 to 4 grams per liter nitrogen supplied as NH<sub>4</sub>NO<sub>3</sub>. Spikes were pulse-labeled at intervals from 12 to 24 days after flowering with 3.7 megabecquerel of [<sup>3</sup>H]leucine to determine relative rates of synthesis of hordein-1 and hordein-2 polypeptides. At low nitrogen levels `Ruth' had a lower protein content than 297, but at increasing nitrogen levels its protein content increased rapidly and reached a maximum (35%) higher than 297 (30%). The relative contribution of the hordein fraction to total protein increased mainly with time, and hordein-1 to total hordein increased mainly with nitrogen level, in both genotypes. There appeared to be no fundamental limitations in the capacity of `Ruth' to accumulate protein; 297 appears to have a greater basal level of nitrogen availability under normal conditions.     

Structural studies on heparins with unusually high N-acetylgucosamine contents

Studies bearing on the variations of N-acetylglucosamine contents of heparins have been made. Such variations may be considerable in heparin fractions isolated from the same tissue source. An N-acetylglucosamine concentration of 27% was found in a heparin fraction from hog mucosa, indicating that such a high content of this residue is not unique for whale heparin. Beef lung heparin, previously found only to contain low contents of N-acetylglucosamine, was isolated with 19% of this constituent. The data suggests that high N-acetylglucosamine contents in heparins is more widespread than thought previously.Chemical studies show that the distribution of N-acetylglucosamine residues in whale heparin is similar to that in beef lung or hog mucosa preparations, regardless of the concentration of N-acetyl groups in the latter.     

Primary structural requirements for N- and O-glycosylation of yeast mannoproteins

Primary structural requirements both for N- and O-glycosylation have been studied using a series of synthetic peptides and a membrane fraction from Saccharomyces cerevisiae. N-Glycosylation: the tripeptide sequence Asn-Xaa-Thr/Ser was found to be necessary for the transfer of saccharide units from oligosaccharide-lipid to asparagine. Substitution of asparagine by aspartic acid or glutamine, or replacement of threonine by valine in the hexapeptide Tyr-$\text{Asn}$-Leu-$\text{Thr}$-Ser-Val prevents its glycosylation. Also, a proline residue in the position of Xaa makes the peptide unable to function as an acceptor. Transfer onto asparagine occurs only efficiently if both the α-amino group of asparagine and the α-carboxyl moiety of the hydroxy amino acid are blocked. Yield of glycosylation improves with increasing peptide chain length. With regard to the glycosyl donor dolichyl diphosphate-bound GlcNAc₂Man₉Glc₃ is the preferred substrate. Non-glucosylated glycolipid Dol-PP-GlcNAc₂Man₉ is a poor donor, whereas smaller precursors Dol-PP-GlcNAc₂ and Dol-PP-GlcNAc₂Man₁ allow reasonable transfer. O-Glycosylation: no marker sequence can be derived for the formation of an O-glycosidic linkage via Dol-P-Man. Introduction of a proline residue in vicinity to the hydroxy amino acid leads to a significant improvement of glycosyl transfer. It is postulated that accessibility of potential O-glycosylation sites rather than a specific sequence may be a prerequisite for O-glycosylation.     

Microsomal metabolism of N-nitrosodi-n-propylamine: formation of products resulting from α-and β-oxidation

We have identified propionaldehyde, n-propanol, isopropanol and N-nitroso-2-hydroxy-propylpropylamine following incubation of N-nitrosodi-n-propylamine with a microsomal fraction from rat liver. Based on the yields of the various products, we have shown that β-oxidation occurs at about 15% of the level of α-oxidation. β-as well as α-oxidation was shown to be carried out by the microsomal mixed function oxidase system. N-nitroso-2-hydroxy-propylpropylamine is further oxidized by the microsomal preparation to yield N-nitroso-2-oxopropylpropylamine.     

Evolutionary relationship of the members of the sulphur-rich hordein family revealed by common antigenic determinants

Summary Five monoclonal antibodies raised against an enriched C hordein fraction have been characterized in detail and were found to be specific for the members of the sulphur-rich hordein family. Two antibodies specific for B hordein polypeptides were identified, one of which reacted predominantly with CNBr cleavage class III polypeptides. 1 hordein was recognized by two antibodies, of which one also reacted with 2 hordein and several members of the CNBr cleavage class II B hordein polypeptides. One antibody recognized 3 hordein but cross-reacted at higher antibody concentration with almost all of the B and C hordein polypeptides. The specificity of the monoclonal antibodies was confirmed by Western blotting of one- or two-dimensionally separated hordein from the B hordein-deficient mutant hor2ca and its wild-type Carlsberg II and the 3 hordein-deficient genotype Nevsky. The identification of the hordein-specific monoclonal antibodies was further supported by immune precipitation of in-vitro transcribed and translated 2 hordein, and hor2ca and Carlsberg II mRNA translation products. The monoclonal antibodies were used to screen for mutants in hordein synthesis. Two mutants, one deficient in 1 hordein synthesis and a second in 2 or closely related B hordein polypeptides were identified. A model is proposed for the evolution of the sulphur-rich hordein loci Hor5 and Hor2.     

Purification and biochemical properties of an N-hydroxyarylamine O-acetyltransferase from Escherichia coli

The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS–PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.     

The amino acid sequence of cytochrome c from Enteromorpha intestinalis

Proposed amino acid sequence of cytochrome c from Enteromorpha intestinalis is presented. The cytochrome is a basic protein, homologous with higher plant and animal cytochromes c of mitochondrial origin. Peptides from chymotryptic and tryptic digests were analysed by the dansyl-phenylisothiocyanate method and aligned by comparison with other homologous cytochromes c. The proposed sequence consists of a single polypeptide chain of 111 residues which is acetylated at its N-terminus. The sequence contains one residue of the unusual amino acid ?-N-trimethyllysine in position 80.     

Deacetylation of N8-acetylspermidine by subcellular fractions of rat tissue

The in vitro deacetylation of N⁸-acetylspermidine by an enzyme activity in rat tissues is described. This deacetylase activity occurs as a soluble, cytoplasmic enzyme in rat liver and was detected in the 100,000g supernatant fraction of all tissues examined. The highest specific activity was found in liver. Spleen, kidney, and lung were found to contain 20–50% of the activity in liver, while heart, brain, and skeletal muscle exhibited from 2 to 10% of the activity in liver. Serum contained only barely detectable levels of activity, much lower than any of the tissues studied. The in vitro metabolism of N¹-acetylspermidine differed from that observed for N⁸-acetylspermidine and does not appear to involve a simple deacetylation reaction.     

Formation of polyprenol-linked mono- and oligosaccharides in Phaseolus aureus

A crude membrane preparation from Phaseolus aureus hypocotyls catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into a acid labile glycolipid and a methanol insoluble fraction. Addition of dolichyl monophosphate to the incubation mixture stimulated the formation of both the mannolipid and the methanol insoluble endproduct. Thin-layer chromatography of endogenous lipid and of the stimulated lipid fraction revealed that both compounds run identical. Ficaprenyl monophosphate also stimulates the incorporation of mannose; however, the ficaprenyl monophosphate mannose formed is not identical to the endogenous mannolipid. This suggests that the endogenous acceptor has the properties of an α-saturated polyprenyl monophosphate rather than those of the ficaprenyl phosphate type. The same membrane preparation also incorporates N-acetylglucosamine into an acid labile glyolipid as well as into a polymer fraction. Evidence is presented that the N-acetylglucosamine containing lipid consists of a mixture of dolichyl pyrophosphate N-acetylglucosamine and dolichyl pyrophosphate di-N-acetylchitobiose. It seems likely that the two compounds have a precursor-product relationship. Incubation of dolichyl pyrophosphate di-N-acetylchitobiose together with GDP-mannose gives rise to lipid-bound mannosyl-di-N-acetylchitobiose. Radioactivity from either the [¹⁴C]mannolipid or the N-acetyl[¹⁴C]glucosamine containing lipid is incorporated into a methanol insoluble product to 3.4 and 6.3%, respectively; it seems, at least in part, to be a glycoprotein.     

Conformational basis of N-glycosylation of proteins: conformational analysis of Ac-Asn-Ala-Thr-NH2

The structural requirements of the Asn-X-Thr(Ser) sequence for the N-glycosylation of proteins has been traced to a local conformation acting as a signal for the enzymatic process. The conformational space of the smallest in vivoN-glycosylation substrate, Ac-Asn-Ala-Thr-NH₂, has been thoroughly explored using energy calculations. All the lowest energy conformers have been characterized as bended structures.     

N-acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 μmol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP-N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [³H]glucosamine-HCl or $\text{N-[}{}^3\text{H]acetylmannosamine}$ showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.     

Intracellular localization of GDP-l-fucose: Glycoprotein and CMP-sialic acid: Apolipoprotein glycosyltransferases in rat and pork livers

A GDP-l-fucose:glycoprotein fucosyltransferase which transfers l-fucose to terminal β-N-acetyl-d-glucosaminyl residues of sialidase-, β-galactosidase-treated α₁-acid glycoprotein and a CMP-sialic acid:glycoprotein sialyltransferase acting on sialidase-treated apolipoprotein-Ala₁ from human very low density lipoprotein have been shown to be concentrated in rat liver Golgi apparatus preparations at enrichments of 40- and 45-fold, respectively, and in pork liver Golgi-rich fractions at enrichments of 35- and 20-fold, respectively. A second fucosyltransferase acting on sialidase-treated α₁-acid glycopretein was absent from rat liver and was enriched only 13-fold in a pork liver Golgi-rich fraction. The smooth-surfaced microsome fraction was the only other rat liver subcellular fraction with appreciable levels of the GDP-l-fucose: β-N-acetyl-d-glucosaminide fucosyltransferase and the lipoprotein sialyltransferase (enrichments of 2.6- and 5.2-fold, respectivley). This enrichment could not be attributed to the plasma membrane content of the smooth microsome fraction since plasma membrane fractions from rat liver were shown to have relatively low concentrations of these two transferases (enrichments of 0.3 or less). Rat liver plasma membrane was also shown to have similarly low relative specific activities for three other glycosyltransferases (sialyl-, galactosyl-, and N-acetylglucosaminyl-). The accurate determination of the glycosyltransferase activities of the plasma membrane fraction required the use of relatively low concentrations of plasma membrane and relatively high concentrations of nucleotide-sugars in order to avoid interference by the high nucleotide-sugar pyrophosphatase and hydrolase activities of this fraction.     

Enhancing activity of N-glycosylation for constitutive proteins secretions in non-polarized cells

Several fusion proteins of mouse Interleukins (mILs) and the enhanced green fluorescent protein (EGFP) were expressed in fibroblast and epithelial cells. Among these proteins, the mIL-31 derivative was the most efficiently secreted into the medium in a N-glycosylation-dependent manner. From the analysis of deletion mutants, the minimal structure for constitutive secretions consisted of a signal peptide and N-glycosylation. Introduction of the signal sequence from mIL-31 to human p53 protein failed to secrete the products, but further addition of the N-glycosylation site resulted in constitutive secretion of biologically active p53 protein into the medium in the N-glycosylated form. In this report, we showed the importance of N-glycosylation for constitutive protein secretions, especially using non-polarized cells.     

A method for the isolation of cytidylate series from ribonuclease T1-oligonucleotides

An extracellular ribonuclease from Physarum polycephalum cultures was found to hydrolyze Cp-N linkages in T₁-oligonucleotides at a much slower rate than Up- or Ap-esters. Hence, it allows the characterization of all (Cp)_nNp series (where N is a U, A or G residue). This specificity nicely complements existing methods and therefore is very valuable for nucleotide sequence analysis of T₁-oligonucleotides.     

Regulation of n-acetylglucosamine uptake in yeast

Various yeasts have been investigated for their ability to grow on N-acetylglucosamine as the sole carbon source and only those which are associated with the disease, candidiasis, gave positive results. The yeasts unable to grow on N-acetylglucosamine lacked the capacity to transport the aminosugar across the cell membrane. In pathogenic yeasts, two systems of different affinity for substrate were found to operate in the uptake of N-acetylglucosamine. In glucose-grown cells a constitutive, low affinity uptake system was present, but upon addition of inducer, a specific high affinity uptake system was synthesized. Experiments with the inhibitors of macromolecule synthesis suggested that the synthesis of RNA and protein is necessary for induction whereas the synthesis of DNA is not.In glucose-grown Candida albicans cells which are devoid of N-acetylglucosamine enters into the cells as phosphorylated form using a constitutive uptake system. Uranyl acetate (0.01 mM) which binds to cell membrane-associated polyphosphates, inhibited completely the inducible uptake of N-acetylglucosamine. Labelling experiments, designed to determine the temporal sequence of appearance of N-acetylglucosamine in intracellular free sugar and sugar-phosphate pools, indicated that N-acetylglucosamine first appeared in the cells as phosphorylated form. Similar results were obtained with Saccharomyces cerevisiae 3059 and some other yeasts which are devoid of N-acetylglucosamine kinase in both uninduced and induced conditions. These results are consistent with the model of van Steveninck that involves phosphorylation during transport. Furthermore, inhibitors of energy metabolism (arsenate, azide and cyanide), proton conductor (m-chlorocarbonylcyanide phenylhydrazine) and dibenzyl diammonium ion (membrane permeable cation) inhibited the inducible N-acetylglucosamine uptake in C. albicans.     

Subcellular localization of endo-β-N-acetylglucosaminidase and high-mannose type free N-glycans in plant cell

Subcellular distribution of plant endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase) and high-mannose type free N-glycans produced by the endoglycosidase has been analyzed using cotyledons of pumpkin seedlings as the model plant cells. Each organelle in the cotyledons was fractionated by ultracentrifugation with the sucrose density gradient system and the endo-β-GlcNAc-ase activity in each fraction was assayed with fluorescence labeled N-glycans as substrates. The endoglycosidase activity was exclusively recovered in the soluble fraction (cytosol fraction) but not in other specific organellar fractions, suggesting that the endoglycosidase would reside predominantly in the cytosol. The quantitative analysis of high-mannose type free N-glycans occurring in each fraction showed that more than 70% of the free N-glycans was recovered from the soluble fraction, suggesting the endoglycosidase would work in the cytosol and the resulting free N-glycans would accumulate in the same fraction. The pumpkin endo-β-GlcNAc-ase (endo-CM) partially purified from the cotyledons showed optimum activity around pH 6.5, supporting this enzyme would reside in the cytosol. Furthermore, the detailed analysis of substrate specificity of endo-CM using various high-mannose type N-glycans showed that the pumpkin enzyme, as well as other plant endo-β-N-acetylglucosaminidases, were highly active toward the high-mannose type glycans bearing the Manα1-2Manα1-3Manβ1-structural unit.     

Alkaline and acid phosphatases of the marine diatoms Chaetoceros affinis var. willei (Gran) Hustedt and Skeletonema costatum (Grev.) Cleve

Alkaline phosphatase activity in cultures of the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt was higher than in Skeletonema costatum (Grev.) Cleve. The enzyme activity was localized in coarse cell particles. Acid phosphatase activity was found in the cytoplasmic fraction. Induction of alkaline phosphatase depended on the $\text{N}\text{P}$ ratio in the culture medium. A $\text{N}\text{P}$ ratio > 40 in dilution/batch culture and > 30 in large scale batch culture, respectively, induced alkaline phosphatase.Cell phosphorus showed a critical value below which alkaline phosphatase was induced. Alkaline phosphatase in natural phytoplankton from the Trondheimsfjord is unlikely to occur except possibly in special situations.