Glycosylation of ribonuclease A catalysed by rabbit liver extracts.

Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.     

Regulation of in vitro translation by double-stranded RNA in mammalian cell mRNA preparations.

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.     

Characterization of reactions catalysed by yeast phosphatidylinositol synthase

The nature of reactions catalysed by yeast phosphatidylinositol synthase expressed in E. coli has been investigated. The single enzyme is shown to carry both CDP-diacylglycerol-dependent incorporation of inositol into phosphatidylinositol (K_m for inositol of 0.090 mM) and a CDP-diacylglycerol-independent exchange reaction between phosphatidylinositol and inositol (K_m for inositol of 0.066 mM). The exchange reaction and reversal of phosphatidylinositol synthase were both stimulated by CMP, but had different optimum pH and requirements for substrates. These results suggest that CMP-stimulated exchange and CMP-dependent reverse reactions are distinct processes catalysed by the same enzyme. phosphatidylinositol synthase.     

Glycosylation of endogenous lipids and proteins by preparations of chicken embryo fibroblasts.

Glycosylation of endogenous phosphoisoprenyl lipids and membrane-associated proteins was shown to occur in preparations of chicken embryo fibroblasts incubated with GDP[14C]mannose and UDP-N-acetylglucosamine. The two preparations used were cells released from the culture dishes by buffered saline containing EDTA and crude membranes from those cells. Both beta-mannosyl-phosphoryldolichol and oligosaccharide-phosphoryl lipids with five to eight sugar residues were labelled under the conditons employed. The oligosaccharide isolated from the octasaccharide-lipid fraction was shown to be heterogeneous after an analysis of the products formed by treatment of the oligosaccharide with glycosidases. Some of the oligosaccharides appeared to contain N-acetylglucosamine at positions external to that of [14C]mannose. Lipids with oligosaccharide moieties of different structures were made by the two preparations. The results of pulse-chase experiments were consistent with the glycosylated lipids being intermediates in glycoprotein biosynthesis.     

Glycosylation abolishes an in vitro insulin-like action of prolactin

Ovine prolactin stimulated 14C-CO2 production from labeled glucose in adipose tissue of hypophysectomized rats in vitro, an insulin-like activity. Glycosylation of the hormone by attachment of a carbohydrate unit at asparagine31 abolished this in vitro insulin-like action. However, neither nonglycosylated nor glycosylated prolactin exhibited in vivo insulin-like action, as they did not lower serum glucose or non-esterified fatty acids in fasted hypophysectomized rats. Hindrance of receptor binding by the carbohydrate unit may account for the absence of in vitro insulin-like activity in glycosylated prolactin, but the dichotomy between in vivo and in vitro insulin-like actions for prolactin remains obscure.     

Glycosylation of pea cotyledon membranes

Pea cotyledons were injected with d-[(14)C]mannose or d-[(14)C]-glucosamine and incubated for 1 to 1.5 hours. Cotyledons were homogenized and subcellular fractions were isolated by differential centrifugation followed by linear sucrose density gradient centrifugation.Radioactivity that was precipitated by trichloroacetic acid was associated most extensively with rough endoplasmic reticulum, Golgi membranes, a membrane with a density of 1.14 grams per cubic centimeter (possibly plasma membrane) and an unidentified subcellular component with a density of 1.22 grams per cubic centimeter. Lower levels of incorporation were observed in protein bodies and mitochondria.Isolated membrane fractions were lipid-extracted to determine which components of the membrane contained the label. Rough endoplasmic reticulum contained the most extensively labeled lipids which had similar properties to the lipid intermediates thought to be involved in glycoprotein assembly. The lipid free residues of the various membrane fractions contained radioactivity that was released by protease treatment. Acid hydrolysis of the residues indicated that most of the radioactivity was associated with mannose or glucosamine. It appears that various subcellular components of the pea cotyledon possess glycoproteins that contain mannose and glucosamine.     

Glycosylation engineering in yeast: the advent of fully humanized yeast

Yeasts have been extensively used as model organisms to elucidate cellular processes and their mechanism in lower eukaryotes. Consequently, a large number of powerful genetic tools have been developed to engineer yeast and improve its utility. These tools and the development of efficient large-scale fermentation processes have made recombinant protein expression in yeast an attractive choice. However, for the production of glycoproteins for human use, native high-mannose yeast glycosylation is not suitable and therefore represents a major limitation for yeast based protein expression systems. Over the last two decades several groups have attempted to overcome this problem, yet with limited success. Recently however, major advances in the glycoengineering of the yeast Pichia pastoris, have culminated in the production of fully humanized sialylated glycoproteins.     

DNA polymerization catalysed by a group II intron RNA in vitro.

The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.     

The stereospecificity of the reduction of nitrate by reduced nicotinamide-adenine dinucleotides catalysed by Candida utilis preparations.

The reduction of nitrate by reduced nicotinamide-adenine dinucleotides, catalysed by extract of Candida utilis, exhibits an apparent high degree of stereospecificity for the 'B' methylene hydrogen atom of NADPH and mixed stereospecificity for the methylene hydrogen atoms of NADH. Purified nitrate reductase, on the other hand, exhibits 'A' stereospecificity for NADH and NADPH. The apparent switch of stereospecificity from the 'B' to the 'A' side of NADPH, which occurs after purification of the enzyme, is partly explained by the fact that in crude extracts nitrate is reduced completely to ammonia. Nitrite does not accumulate but is reduced to ammonia by nitrite dehydrogenase, which is 'B'-specific, so that up to 75% of hydrogen removed from NADPH during the reduction of nitrate could occur from the 'B' side. A further increase in the removal of hydrogen from the 'B' side of NADPH could be the kinetic isotope effect that is observed when ['A'-3H]NADPH is the reductant, the H--C bond being cleaved 2.3 times faster than the 3H--C bond. The mixed stereospecificity observed with NADH has been traced to an uncharacterized enzyme that catalyses a 'B'-specific exchange between NAD+ and NADH. This reaction is discussed in relation to the possibility that it may explain other cases of apparent mixed stereospecificity that have been reported.     

Glycosylation of endogenous lipids and proteins by preparations of chicken embryo fibroblasts

Glycosylation of endogenous phosphoisoprenyl lipids and membrane-associated proteins was shown to occur in preparations of chicken embryo fibroblasts incubated with GDP[¹⁴C]mannose and UDP-N-acetylglucosamine. The two preparations used were cells released from the culture dishes by buffered saline containing EDTA and crude membranes from those cells. Both β-mannosyl-phosphoryldolichol and oligosaccharide-phosphoryl lipids with five to eight sugar residues were labelled under the conditions employed. The oligosaccharide isolated from the octasaccharide-lipid fraction was shown to be heterogeneous after an analysis of the products formed by treatment of the oligosaccharide with glycosidases. Some of the oligosaccharides appeared to contain N-acetylglucosamine at positions external to that of [¹⁴C]mannose. Lipids with oligosaccharide moieties of different structures were made by the two preparations. The results of pulse-chase experiments were consistent with the glycosylated lipids being intermediates in glycoprotein biosynthesis.     

Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.     

Disruption of yeast membranes by methylphenidate

Methylphenidate blocked sorbose uptake and loss by yeast spheroplasts and, at higher concentrations (30 mm), disrupted the spheroplasts. At still higher concentrations (70 mm), methylphenidate also ruptured the membranes of whole yeast cells; sorbose and materials absorbing at 280 nm were lost from the cells, and methylene blue stained them. Intracellular structures were extensively affected, as shown by electron micrographs, and were more sensitive to disruption by methylphenidate than the external membrane. N-ethylmaleimide and Ca(2+) enhanced the rupture of external membranes by methylphenidate.     

Glycosylation of hesperetin by plant cell cultures

The biotransformation of hesperetin by cultured cells of Ipomoea batatas and Eucalyptus perriniana was investigated. Three glycosides, hesperetin 3'-O-beta-D-glucopyranoside (33 microg/g fr. wt of cells), hesperetin 3',7-O-beta-D-diglucopyranoside (217 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(beta-D-glucopyranosyl)]-beta-d-glucopyranoside (beta-gentiobioside, 22 microg/g fr. wt of cells), together with three hitherto known glycosides, hesperetin 5-O-beta-d-glucopyranoside (23 microg/g fr. wt of cells), hesperetin 7-O-beta-D-glucopyranoside (57 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(alpha-L-rhamnopyranosyl)]-beta-D-glucopyranoside (beta-rutinoside, hesperidin, 13 microg/g fr. wt of cells), were isolated from cultured suspension cells of E. perriniana that had been treated with hesperetin. Oligosaccharide chains were regioselectively formed at the C-7 position of hesperetin to afford beta-gentiobioside and beta-rutinoside. On the other hand, cultured I. batatas cells converted hesperetin into hesperetin 3'-O-beta-D-glucopyranoside (60 microg/g fr. wt of cells), hesperetin 5-O-beta-D-glucopyranoside (23 microg/g fr. wt of cells), and hesperetin 7-O-beta-D-glucopyranoside (110 microg/g fr. wt of cells).     

Glycosylation of exogenous protein by endoplasmic-reticulum membranes from castor-bean (Ricinus communis) endosperm.

Endoplasmic-reticulum membranes isolated from the endosperm tissue of 3-day-old castor-bean (Ricinus communis) seedlings catalysed the enzymic transfer of the sugar moiety from an oligosaccharide--lipid to a chemically unfolded form of ribonuclease A.