Incorporation of carbohydrates into endogenous acceptors of liver microsomal fractions.
TL;DR: Radioactive sugar-nucleotides reacted with the microsomal fractions of rat and rabbit liver to form glycoproteins with endogenous acceptors to reveal two products; one was very labile to acid hydrolysis and could be extracted with lipid solvents, while the other was protein-bound sugar.
About: This article is published in Archives of Biochemistry and Biophysics.The article was published on 1970-05-01. It has received 75 citations till now.
Citations
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TL;DR: Tunicamycin inhibited the production of the [3H]G1cNAc-lipid but did not affect the synthesis of the 14C-mannolipid, which accounts for the effect of the antibiotic upon glycoprotein formation in vivo.
821 citations
346 citations
TL;DR: This hypothesis is rejected in favour of the idea that the attached carbohydrate determines the extracellular fate of the protein molecule.
Abstract: It has been suggested that glycosylation is a means of labelling proteins for intracellular recognition and export. This hypothesis is rejected in favour of the idea that the attached carbohydrate determines the extracellular fate of the protein molecule.
202 citations
TL;DR: The formation of an enzyme-acceptor complex as the effective intermediate in platelet: collagen adhesion provides a simple model for studying the primary step in haemostasis and may provide a rational basis for the design of anti-thrombogenic drugs.
Abstract: The formation of an enzyme-acceptor complex as the effective intermediate in platelet: collagen adhesion provides a simple model for studying the primary step in haemostasis and may provide a rational basis for the design of anti-thrombogenic drugs
164 citations
TL;DR: The results are consistent with the assumption that dolichol monophosphate is involved in theMannosylation of a specific position in yeast glycoproteins, i.e. in the formation of mannosyl linkages to serine and/or threonine.
Abstract: 1 In a crude particulate fraction from yeast mannose is transferred from GDP-mannose to an endogenous “lipid” fraction, the monophosphates of dolichol-14 to dolichol-18 The same membrane fraction also catalyzes the mannosylation of glycoproteins More than 80% of the total radioactivity in the glycoprotein fraction obtained from GDP-[14C]mannose is released by β-elimination The small-sized radioactive products of β-elimination are mannose, mannobiose and mannotriose
2 Ageing of the particulate fraction leads to a drastic loss of mannosyl transfer activity from GDP-[14C]mannose to dolichol monophosphate To the same extent the amount of [14C]mannose obtained after β-elimination of the glycoproteins decreases The amount of radioactive mannobiose and mannotriose is, however, much less affected
3 With decreasing GDP-mannose concentrations the amount of [14C]mannose obtained after β-elimination increases as compared to the amount of radioactive mannobiose and mannotriose The Km-value for the incorporation of mannosyl groups directly linked to serine and/or threonine, therefore, is lower than the Km-value for the transfer of subsequent mannosyl residues
4 The mannosylation of dolichol monophosphate from GDP-[14C]mannose proceeds in the presence of Mg2+ and Mn2+ ions as well When solely Mg2+ is present in the incubation mixture, β-elimination of the glycoprotein produced yields almost exclusively mannose, and it is only in the presence of Mn2+ that the normal β-elimination pattern is observed
5 Incubation of the particulate fraction with dolichol-monophosphate-[14C]mannose results in the formation of radioactive glycoprotein; again most of the radioactivity is released by β-elimination but only [14C]mannose is obtained as product When dolichol-monophosphate [14C]mannose is incubated together with non-radioactive GDP-mannose, β-elimination yields radioactive mannose, mannobiose and mannotriose Mannobiose is found to be labelled in the reducing end only
6 The results are consistent with the assumption that dolichol monophosphate is involved in the mannosylation of a specific position in yeast glycoproteins, ie in the formation of mannosyl linkages to serine and/or threonine The subsequent mannosylation proceeds directly from GDP-mannose in a reaction obligatorily requiring Mn2+
7 After β-elimination of the methanol-insoluble material mannosylated in the presence of dolichol-monophosphate [14C] mannose the residual insoluble radioactivity is to a large extent transformed into dialyzable material by pronase Mannose, therefore, is transferred from dolichol-monophosphate mannose also to glycoprotein positions not involving OH-groups of serine or threonine
144 citations
References
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TL;DR: In this paper, the authors described a simplified version of the method and reported the results of a study of its application to different tissues, including the efficiency of the washing procedure in terms of the removal from tissue lipides of some non-lipide substances of special biochemical interest.
59,550 citations
TL;DR: Modifications are introduced, based on a test given by Feigl for reducing sugars, which eliminate the heating step, and in which the reagents are applied in organic solvents, thus removing the danger of migration of the sugar spots.
Abstract: THE ammoniacal silver nitrate spray1 used for the detection of sugars has several disadvantages; to those mentioned by Partridge2 should be added the necessity for very careful control of the heating step, particularly important in laboratories lacking special apparatus. We have introduced modifications, based on a test given by Feigl3 for reducing sugars, which eliminate the heating step, and in which the reagents are applied in organic solvents, thus removing the danger of migration of the sugar spots. The method has been in use for more than a year, and has proved easy to handle and extremely reliable.
3,526 citations
TL;DR: Radioautography after injection of galactose-H3, as after glucose- H3, indicates that synthesis of complex carbohydrates takes place in the Golgi region of many secretory cells.
Abstract: The radioautographic distribution of the label of galactose-H3 was compared with that of glucose-H3 in a series of secretory cells of the rat. Whereas the glucose label appeared in all mucous cells, the galactose label was incorporated only into certain mucous cells. Whenever either label was incorporated, however, it was located first in the Golgi region and later in the secretion product, mucus. Several lines of evidence, including extraction of glucose label with peracetic acid—beta glucuronidase, indicated that the material synthesized in the Golgi region was glycoprotein in nature. In chondrocytes, both the galactose and the glucose label appeared first in the Golgi region and later in cartilage matrix; extraction of glucose label with hyaluronidase indicated that much of it consisted of mucopolysaccharide. In all secretory cells, the extraction of glycogen by amylase had no effect on Golgi radioactivity. Such extraction did not eliminate the scattered cytoplasmic label also seen after glucose-H3 injection, but completely eliminated that seen after galactose-H3. Consequently, the galactose-H3 label in the Golgi region stood out more clearly, and was detected in many cells: pancreas, liver, epididymis, and intestinal columnar cells. In the latter, label later appeared in the surface coat. Thus, radioautography after injection of galactose-H3, as after glucose-H3, indicates that synthesis of complex carbohydrates takes place in the Golgi region of many secretory cells.
415 citations
TL;DR: The present study demonstrates the existence of specific binding sites for ADP and ATP at the mitochondrial membrane which can be attributed to the adenine nucleotide carrier and the specific binding is selectively sensitive to methylene blue-catalyzed photo-oxidation.
Abstract: The present study demonstrates the existence of specific binding sites for ADP and ATP at the mitochondrial membrane which can be attributed to the adenine nucleotide carrier. Parameters of the binding and various other properties of the carrier sites are determined.
1
The differentiation of the carrier sites is based on the assumption that atractyloside removes adenine nucleotides competitively from these sites. For the accurate evaluation and discrimination of the carriers three parallel binding samples are compared: (a) total binding; (b) atractyloside addition before adenine nucleotide, which prevents binding to the carrier and the exchange with endogenous adenine nucleotides; (c) addition of atractyloside after adenine nucleotide, which displaces adenine nucleotide from the carrier but not from the exchanged pool.
Total uptake of adenine nucleotides is thus differentiated into three portions: (a) binding to the carrier sites (= specific binding); (b) exchange; (c) binding to unspecific, atractyloside-insensitive sites.
2
Procedures have been developed for depleting mitochondria of endogenous adenine nucleotides to increase the proportion of the atractyloside-removable binding in relation to the magnitude of the exchangeable endogenous adenine nucleotide pool.
3
Kinetics of the binding at atractyloside-removable sites are very rapid compared to “binding” by exchange with endogenous adenine nucleotides, even at 0°.
4
The specificity of the carrier binding, as measured by competitive replacement of [14C]ADP with unlabelled nucleotides, shows binding confined to ATP, ADP, dADP and, to a lesser extent, adenosine tetraphosphate. The high specificity is in agreement with that measured for the adenine nucleotide exchange.
5
From the concentration dependence of the specific adenine nucleotide binding, non-linear Scatchard plots are obtained consistent with two sites of unequal affinity (Kd′= 0.3 μM; Kd”= 7 μM). The total number of carrier sites is C0/cyt. a= 1.3 (mole/mole). The ratio of high/low affinity types is C′/C”= 1:5. The corresponding values for rat heart are: Kd′= 0.1 μM, Kd”= 4 μM; C0= 2.2; C′/C”= 1:4. With ATP two binding sites of unequal affinity are also obtained. Both Kd′ and Kd” for ATP are 1.5 to 2 times higher than for ADP. ATP binding is not affected by uncouplers.
6
In arsenate depleted rat liver mitochondria a single binding site of high affinity (Kd= 0.5 μM) is found with C0/cyt. a= 1.2 (mole/mole). This is only half the content (per cyt. a) found in rat heart.
7
Some differentiation between specific and unspecific binding is also possible on the basis of SH and histidine reagents. Only the unspecific binding is sensitive to p-chloromercuribenzoate. The specific binding is selectively sensitive to methylene blue-catalyzed photo-oxidation. This fact, and the strong decrease in atractyloside-removable binding between pH 7.0 and 7.5, suggests the involvement of a histidine group in the specific binding.
8
Sonic particles retain some ability for uptake of adenine nucleotides, but this is no longer atractyloside-sensitive. The concentration dependence of total adenine nucleotide binding gives non-linear Scatchard plots which can be fitted by two binding sites with unequal affinities. The possible identity of the atractyloside-“insensitive” sites in sonic particles with the specific sites in intact mitochondria is discussed. Treatment with detergents also largely removes atractyloside-sensitive and atractyloside-removable binding.
9
The occurrence of two sites with different affinities, it is suggested, reflects inner and outer localizations of carriers. High affinity sites may be those which are in contact with residual endogenous adenine nucleotides and therefore apparently saturated early. The models may be discussed in terms of a carrier distribution in a mobile system or in terms of a double-faced dimer carrier.
403 citations
TL;DR: It has been shown that up to 50 g of soft, preminced, mammalian tissue can be satisfactorily homogenized in less than 30 min by the use of a pressure homogenizer.
166 citations