When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with I'5NIH.4, prior to supplying the inhibitor. Analyses of the 'IN abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [I5NIH4', are '4N enriched and are not apparently derived from '5N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in rivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.It is now well established that GS2 occupies a central position in plant N metabolism (12, 13). The GS-GOGAT cycle is thought to be responsible for the assimilation of most, if not all, of the ammonia derived from nitrate reduction and photorespiration (2-4, 10, 18, 19, 21, 23, 25). Studies with the potent inhibitor of GS, MSO, appear to rule out any major contribution of GDH to ammonia assimilation (1,3,8,19,21,23). However, recent investigations with isolated plant mitochondria suggest that a small fraction of the ammonia generated from glycine decarboxylation can be directly reassimilated into glutamic acid via a mitochondrial GDH (26). The quantitative significance of this latter pathway in vivo still remains obscure. '

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Cotyledons of Phaseolus aureus contain protein-bound glu. cosamine which is metabolized during germination. The glucosamine is present in storage glycoproteins, and these are concurrently metabolized along with the glucosamine. These glycoproteins are associated with protein bodies. Characterization of the glucosamine-containing storage proteins showed them to be identical with vicilin and legumin, the major storage proteins of the Leguminosae. Extraction of Proteins. The cotyledons were homogenized either in 0.4 M NaCl or in 20 mm sodium borate, pH 9.0, using 2 ml of medium per g of tissue. The homogenate was centrifuged at 30,000g for 20 min, and the supernatant was collected. The pellet was extracted three times with the same medium, and the extracts were combined with the initial supernatant.Quantitative Determination of Protein. Proteins were usually assayed according to the procedure of Lowry et al. (13) using bovine serum albumin as a standard. The protein content of peak III glycoprotein was also determined with ninhydrin (27) after acid hydrolysis of the protein.Quantitative determination of hexosamine. The samples to be assayed for protein-bound hexosamine were precipitated with 7.5% trichloroacetic acid, washed with 5% trichloroacetic acid, alcohol, and ether. The dried samples were resuspended in 1 N HCI. Samples to be assayed for total hexosamine were homogenized in 1 N HCl. Hydrolysis was carried out at 90 C for 24 hr. Under these conditions there was no detectable loss of hexosamine. Hexosamines were separated from interfering chromogens by passage through a small Dowex 50 cation exchange column according to the method of Boas (4). This step is absolutely necessary to obtain reproducible results. Eluates from the columns were dried by evaporation under vacuum at 85 C, and the residue was dissolved in water. The hexosamine content was determined by the method of Elson-Morgan as modified by Johnson (11

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The enzymic processes involved in glycoprotein synthesis have been studied using crude extracts obtained from developing cotyledons of Phaseolus vulgaris harvested at the time of active deposition of vidin. Radioactivity from GDP-['4CJmannose can be incorporated by crude extracts into a single chloroform-methanol-soluble product as well as into insoluble product(s). Mannose is the sole '4C-labeled constituent of the lipid. The kinetics of incorporation of 14C, as determined by pulse and pulse-chase experiments using GDP-['4Cjmannose, as weU as direct incorporation from added ['4CImannolipid, shows In those systems where glycoprotein synthesis has been studied in more detail, the mechanism of lipid transfer of sugar to protein seems to depend upon the type of carbohydrate-peptide linkage that the finished glycoprotein will have. In the case of the formation of O-glycosidic linkages between carbohydrate and serine or threonine, such as those studied in yeast (3), the first sugar is added via a polyprenol monophosphate (in this case dolichol monophosphate) and all subsequent sugars seem to be added directly to the final acceptor from nucleoside diphosphate sugars. However, in glycoproteins which have the carbohydrate attached by an N-glycosidic linkage between N-acetyl-D-glucosamine and asparagine, the mechanism of attachment is more complex. The N-acetyl-D-glucosamine residue which will be directly attached to the protein is transferred from UDP-N-acetylo-glucosamine to form a polyprenol pyrophosphate derivative.The rest of the sugars that go to make up the carbohydrate moiety are transferred from nucleoside diphosphate sugars to polyprenol monophosphate lipids and from these lipids to the polyprenol pyrophosphate-N-acetyl-u-glucosamine. In this way, a lipid-bound oligosaccharide is made that is transferred, in its entirety, to the protein acceptor. In the animal systems where this mechanism has been demonstrated, the polyprenol monophosphate is dolichol monophosphate and the polyprenol pyrophosphate is dolichol pyrophosphate (12).Vicilin, the major storage protein of Phaseolus vulgaris seeds, is a glycoprotein containing mannose and glucosamine (29). Because this glycoprotein is made in large quantity at a specific time, developing seeds of this plant provide an excellent system in which to study glycoprotein synthesis. Here, we show that enzyme preparations from the developing seeds of P. vulgaris (kidney bean)