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Aspergillus niger van Tieghem microsomes contain an enzyme that catalyzes mannose transfer from GDP-mannose to polyprenylphosphate. The studies of the specificity of this enzyme for both the sugar donor (nucleoside diphosphate sugar) and the acceptor (polyprenylphosphates that were made available to the enzyme by means of the fusion of acceptor-loaded liposomes with the microsomal membranes) gave the following results. i) All the polyprenylphosphates from C15 to C120 were acceptors except retinylphosphate. ii) The specificity of the enzyme for both the sugar and the base is very strict.

The non-nuclear membranes and the nuclei prepared from L1210 cells catalyze the in vitro transfer of N-acetyl(14C)glucosamine from UDP-N-acetyl(14C)glucosamine to endogenous glycoprotein acceptors. Adequate analysis of these acceptors have demonstrated that the nucleus has its own N-acetylglucosaminyltransferase system that leads to the formation of N-N'-diacetylchitobiosylated proteins.

This work demonstrates that (N-acetyl[14C]glucosamine)2 is transferred from dolichyl pyrophosphate-(N-acetyl[14C]glucosamine)2 to endogenous nuclear glycoproteins. The (N-acetyl[14C]glucosamine)2 moiety is N-linked, since it can be released from the tryptic glycopeptides by N-glycosidase F and by hydrazinolysis, but not by beta-elimination. The biological significance of this direct transfer of N,N'-diacetylchitobiose to nuclear proteins remains to be elucidated.

1. Nuclei were prepared from rat hepatocytes. A biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. 2. The transfer of [14C]N-acetylglucosamine to endogenous acceptors were studied comparatively in the nuclei and in the other subcellular fractions of rat hepatocytes. 3. In this report we describe the presence of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. We found 21% of this transfer in the nucleus fraction with an enrichment of 26 in comparison to homogenate.

Aspergillus niger postmitochondrial fraction, which contains high GTPase activity and high GTP binding capacity, has been subjected to subcellular fractionation on a sucrose gradient. A cytosolic and four membranous populations have been separated according to their relative density. The main difficulty has been the characterization of the plasma membrane of the fungus. This fraction, which does not contain any typical enzyme, has been identified after iodination of the outer proteins of protoplasts from A. niger. The immunological detection has shown the occurrence of cytosolic G proteins and membranous small G proteins located not only in the plasma membrane but also in the membranes of the endoplasmic reticulum.

Aspergillus niger van Tieghem microsomes contain an enzyme that catalyzes mannose transfer from GDP-mannose to polyprenylphosphate. The studies of the specificity of this enzyme for both the sugar donor (nucleoside diphosphate sugar) and the acceptor (polyprenylphosphates that were made available to the enzyme by means of the fusion of acceptor-loaded liposomes with the microsomal membranes) gave the following results. i) All the polyprenylphosphates from C15 to C120 were acceptors except retinylphosphate. ii) The specificity of the enzyme for both the sugar and the base is very strict.

The enzyme properties of a soluble uridine 5'-diphosphate (UDP) glucose: mycosporin-2 glucosyltransferase from spores of Ascochyta fabae Speg. (Fungi imperfecti) were studied. The optimal conditions for the glucose transfer from UDP-glucose to the mycosporin-2 (the amide form being the best acceptor) were determined; for maximal activity the glucosyltransferase requires a pH of about 8.5 and the presence of divalent cations (Mn(2+) being more efficient than Ca(2+) or Mg(2+)). The reaction was not reversible in presence of large amounts of UDP.

Rat liver microsomes catalyze the transfer of mannose from GDPmannose to both retinyl phosphate and dolichyl phosphate to form mannosylphosphorylretinol, mannosylphosphoryldolichol and GDP. The two reactions differ in term of reversibility. In fact, a 200-fold isotopic dilution of GDP[14C]mannose by unlabeled GDPmannose causes mannosylphosphoryldolichol labeling to disappear almost completely, while mannosylphosphorylretinol labeling remains at the same level. The same observation can be made if the mannose donor is removed by centrifugation and replaced by excess GDP; again mannosylphosphorylretinol is stable, but mannosylphosphoryldolichol drops down to one-third of its initial level, as expected for, respectively, a non-reversible and a reversible reaction. Placed in an aqueous medium, mannosylphosphorylretinol releases mannose 1-phosphate (beta configuration) whereas it is quite stable when kept in a membranous environment. These results strongly suggest that mannosylphosphorylretinol as soon as it is formed is segregated in such a way that it is no longer available to the back-reaction; the functional consequence of this segregation would be the possibility for mannosylphosphorylretinol to mannosylate some non-polar regions of certain protein chains.

Membrane fusion is a fundamental and wide-spread phenomenon in the functioning of cells. Many studies were carried out concerning fusion of plasma membranes as for example cell-cell fusions or uptake by cells of lipid-enveloped viruses. The present study deals with the interaction of intracellular membranes of Aspergillus niger with artificial membranes (liposomes). Association is monitored by the uptake of radioactive liposomes by fungal microsomal membranes. The discrimination between aggregation and pure fusion is done by layering the liposomes-microsomes mixture on a continuous sucrose gradient. The accurate quantitation of the fusion phenomenon is monitored with a fluorescent assay based on resonance energy transfer (Struck, D.K. et al. (1981) Biochemistry 20, 4093-4099). Both methods show that, at physiological pH, there is a spontaneous fusion of microsomes with cholesterol-free liposomes. This phenomenon is protein dependent as trypsinized microsomal membranes are no longer able to fuse with liposomes. Biological significance of the fusion process has been demonstrated using microsomal intrinsic protein mannosylation assay; the enhancement of the lipid to protein ratio due to the fusion of liposomes with microsomes of A. niger results in an increase in the rate of endogenous proteins mannosylation. Moreover, cytosolic proteins of A. niger promote the fusion of any kind of liposomes with microsomes.

The influence of various local anaesthetics has been studied on the mannose transfer enzymic system which is localized in Aspergillus niger van Tieghem microsomal membranes. The n-alkanes and tertiary amines do not seem to react on the first step of the reaction: i.e. the polyprenylphosphate-mannose biosynthesis. However two amines, the dibucaine and to a lesser extent the tetracaine, inhibit the mannose transfer from the polyprenylphosphate-mannose to the endogenous proteins.

A purified eel liver microsomal fraction catalyses the transfer of mannose, from GDP-mannose, to endogenous lipid and proteins. The mannolipid is identified as a polyprenol-phosphate-mannose and its role is discussed.