1. Studies were carried out to determine the cellular and subcellular site of biosynthesis of components of fraction I, an alpha-globulin fraction containing acidic glycoproteins isolated from guinea-pig serum. l-[U-(14)C]Leucine or -valine and d-[1-(14)C]glucosamine were used as precursors. 2. A lag of about 10min. occurred before appreciable label appeared in fraction I of serum after injection of leucine or glucosamine. Label in fraction I after 60min. labelling with glucosamine was present almost entirely in hexosamine and sialic acid. 3. Site of synthesis was investigated by studies in vivo up to 17min. after injection of precursor. Particulate subcellular fractions isolated from liver, spleen and kidney or homogenates of the latter two tissues were extracted with Lubrol. Extracts were allowed to react by double diffusion with antisera to fraction I or to subfractions isolated from it, and gels were subsequently subjected to radioautography. With either amino acid or glucosamine as precursor, only extracts of the microsome fraction of liver formed precipitin lines that were appreciably radioactive. 4. The role of the microsome fraction of liver in the synthesis of these glycoproteins was confirmed by immunological studies after incubation of liver slices with leucine or glucosamine. Incorporation of leucine was also investigated in a cell-free microsome system. 5. Material was also precipitated from certain Lubrol extracts of liver microsomes by direct addition of antiserum and its radioactivity measured. Degradation of material thus precipitated and use of heterologous immune systems showed that labelling of precipitin lines represented biosynthesis. 6. A study of extraction procedures suggested that the substances present in the microsome fraction of liver that react with specific antisera are associated with membranous structures. 7. Most or all precipitin lines formed by Lubrol extracts of liver microsomes interacted with precipitin lines given by guinea-pig serum or fraction I, immunological identity being apparent with some lines. The microsome-bound substances thus represent serum glycoproteins or precursors of them. 8. The distribution of label in various tissues and in the protein of subcellular fractions of liver after administration of [(14)C]glucosamine to the guinea pig was also studied. Some variation in results obtained with liver was found depending on the fractionation medium used.
Hobson & Rees, 1955) and served to identify them by synthetic as opposed to degradative means. Throughout the species investigated the phosphokinases predicted by the previous results were all proved to be present and capable ofphosphorylating the expected base. As was reported for some of the echinoderms (Needham & Baldwin, 1937), several annelids, e.g. Glycera gigantea, Nerei8 diversicolor and Myxicola infundibulum, contain a mixture of phosphagens and show that the enzyme systems for the synthesis and utilization of phosphagens in living muscle are completely duplicated. The experimental results again emphasize that creatine phosphate, acting as a phosphagen, is not confined to the echinoderms, protochordates and vertebrates, but is also present, often in considerable quantities, in the annelids. At the present time the distribution of creatine phosphate appears quite arbitrary. This fact lessens the value of the identification of the phosphagens in evolutionary studies in the animal kingdom. SUMMARY 1. A method of extraction of guanidine phosphokinases from annelid muscle is described.
1. Fraction I, a fraction containing acidic glycoproteins, isolated from guinea-pig serum, was digested with Pronase after removal of sialic acid and a major and a minor glycopeptide fraction were isolated by chromatography with Sephadex G-25 and G-50. 2. The major fraction was examined by various methods and shown to contain several glycopeptides. Estimates of molecular weight of the glycopeptide fractions were obtained. Although some variation appeared to occur, the glycopeptides were not grossly heterogeneous with respect to size. An average prosthetic group was estimated to contain about 15 sugar residues. 3. Aspartic acid was the principal amino acid present in the fractions and in all subfractions of the major fraction investigated. Where examined, ammonia was liberated on acid hydrolysis in approximately equimolar amounts to the aspartic acid present. The carbohydrate composition of the fractions was also determined. 4. The glycopeptides showed relatively little degradation in alkaline solution. 5. These results suggest that an N-acylglycosylamine bond involving aspartic acid forms the major type of linkage between carbohydrate and polypeptide. The isolation of a compound with the composition and chromatographic properties of 2-acetamido-1-(l-beta-aspartamido)-1,2-dideoxy-beta-d-glucose supports this view, and indicates that N-acetylglucosamine is the sugar involved in at least many linkages. 6. Fraction I contains some glycoproteins that are susceptible to Pronase and one or more others that resist digestion before the removal of sialic acid. A brief examination revealed some similarities between prosthetic groups derived from both kinds of glycoprotein.
I957 the eliminated protein can be recovered in the liver but very little in the lungs. 2. Iodoproteins injected intravenously into specifically immunized rabbits are eliminated from the blood at the same rate as in normal animals, but immunized rabbits with good antisera to 2-dichloroethyl sulphone-treated proteins eliminate these proteins from their blood more rapidly than do normal animals. 3. With both antigens the deposition in the liver and lungs is greater in immune animals than in controls. 4. One hour after injection of iodoprotein at least one-third of the radioactivity in the blood is no longer protein-bound. 5. The deiodination of iodoproteins in the tissues renders them unreliable for studies in vivo, even for comparatively short-term experiments. We are most grateful to Professor A. Wormall for his constant advice and encouragement. We are indebted to the Medical Research Council and the Central Research Fund of the University of London for grants (to Professor A. Wormall) which have partly covered the costs of these investigations. We are also thankful to Mrs A. E. Almond for assistance with many ofthe experiments described here, and to Miss A. Routledge for technical help throughout these investigations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.