A 43-kDa secreted glycoprotein from the intracellular parasitic nematode Trichinella spiralis has been considered as a factor involved in the formation of the Nurse cell in infected muscle. The closely related intracellular parasitic nematode Trichinella pseudospiralis that also infects muscle cells, does not form Nurse cells and was thought not to secrete the 43-kDa glycoprotein. This implied a unique role for the 43-kDa glycoprotein in T. spiralis infection and supported the hypothesis of involvement of the 43-kDa glycoprotein in Nurse cell formation. Following cloning of a full length cDNA encoding the 43-kDa protein, antibodies were raised against several domains of the 43-kDa glycoprotein. Here we show that a protein related to the 43-kDa glycoprotein exists in T. pseudospiralis. Immunohistochemical studies reveal important similarities in the distribution of the 43-kDa glycoprotein and the related protein from T. pseudospiralis in muscle infections with either of the two parasites. The 43-kDa glycoprotein may therefore play a common role in the life cycles of these two parasites and probably is not involved in Nurse cell formation.

Trichinella spiralis is an intracellular parasitic nematode that infects skeletal muscle cells. Infection results in loss of tissue specific characteristics and conversion of the muscle cell to a Nurse cell. The characteristic changes leading to the formation of the Nurse cell appear complete by day 12 after intramuscular infection. Proteins synthesized in the stichocytes (secretory cells) of T. spiralis and secreted in the host cell are believed to be involved in the process of Nurse cell formation. One secreted glycoprotein of 43 kDa has been considered as a candidate factor involved in Nurse cell formation. We determined the timing of synthesis and secretion of the 43-kDa glycoprotein and its temporal correlation to the changes of the infected host cell, to gain an understanding of the role of the 43-kDa glycoprotein in T. spiralis infection. We show that the 43-kDa glycoprotein is first expressed on day 11 following intramuscular infection, several days after the changes in the infected muscle cell have been initiated. Protein(s) immunologically related to the 43-kDa glycoprotein but not the 43-kDa glycoprotein itself are detected in the nuclei of mature Nurse cells. During the intramuscular stage the 43-kDa glycoprotein appears to be stored in the alpha-stichocytes of T. spiralis and appears to be secreted immediately following invasion of the intestinal columnar epithelial cells by the L1 larva. The role of the 43-kDa glycoprotein remains unknown, however, these findings argue against involvement of the 43-kDa glycoprotein in Nurse cell formation.

The L1 larvae of the parasitic nematode Trichinella spiralis invade skeletal muscle and initiate a process that has been interpreted to represent skeletal muscle dedifferentiation. In this process, the infected region of the muscle cell is converted into a unique structure, called the Nurse cell. The nematode T. spiralis can survive for tens of years within the cytoplasm of the Nurse cell and secretes proteins into the cytoplasm that are believed to play a role in mediating the Nurse cell formation or maintenance. We have cloned a cDNA encoding the T. spiralis-derived, 43-kDa secreted protein. Structural analysis of the predicted 344-amino acid sequence revealed an N terminally located signal peptide and a potential helix-loop-helix motif in the main body of the protein. Antibodies raised against the 43-kDa recombinant protein were used in immunocytolocalizations of T. spiralis-infected skeletal muscle sections. These antibodies strongly stained the Nurse cell nuclei and the nematode itself. Specific, though slightly weaker staining also occurred in the Nurse cell cytoplasm. In Western blots, the antibodies react with the 43-kDa protein but also detected at least two other T. spiralis-secreted proteins. DNA hybridizations reveal at least one additional 43-kDa-related sequence encoded in the T. spiralis genome. We conclude that either the 43-kDa protein and/or a closely related 43-kDa family member is secreted into the muscle and translocates to the muscle-derived nuclei. This model may provide insights into the mechanisms involved in Nurse cell formation.

Antibodies were elicited against a purified antigen with an apparent molecular weight of 43K. This antibody preparation also detected a second antigen consisting of a group of closely related components of 45–50K. These antigens are stage specific for the infective first stage larva of Trichinella spiralis and are among the repertoire of secreted antigens originating from the stichosome. Antibody raised against the 43K antigen reacted with the stichosome and cuticle of the mature larva and the cytoplasm and nucleoplasm, but not nucleolus, of all nuclei of infected host cells (Nurse cells) in sections of infected tissues. Studies on sections of synchronously infected muscle tissue revealed that antigen was present only within the worm on Day 7 of the infection. On Day 9 after infection, the stichosome and cuticular surface of the larva and the cytoplasm and nucleoplasm of each nucleus of the Nurse cell reacted with antibody. Nurse cell cytoplasmic and nuclear reactivity increased in intensity until Day 18 after infection. These results suggest that stichocyte-specific antigens are synthesized during the early phase of infection in the muscle, and that as the Nurse-parasite complex develops, some of the antigen is secreted into the milieu of the Nurse cell. The presence of antigen in the cytoplasm and nucleoplasm of the infected host cell is discussed in relation to Nurse cell formation and maintenance.

Two protein antigens were isolated from excretory-secretory products of Trichinella spiralis by biochemical methods and characterized with respect to their chemical and immunological properties. One antigen, of apparent Mr 43 000, is an abundant secreted protein of infective L1 larvae, while the other, of 45–50 kDa, is present in smaller amounts. Yields, extinction coefficients, isoelectric points, amino acid compositions, and partial N-terminal amino acid sequences for each are reported. Partial amino acid sequences of peptides derived from the 43-kDa protein by cyanogen bromide cleavage have been determined. Treating a reduced-pyridylethylated derivative of the 43-kDa protein with glycopeptidase F (N-glycanase) resulted in formation of a transient product, of 37 kDa followed by a stable polypeptide of 32 kDa (by SDS-PAGE), suggesting the presence of two N-linked carbohydrate groups. A similar result was obtained with the 45–50-kDa protein, which gave a transient doublet of 38 and 40 kDa and a final, stable product of 33 kDa, with a minor component of 35 kDa. Two glycosylation sites of the 43-kDa protein and one site of the 45–50-kDa protein can be identified in the amino acid sequences. Polyclonal antibodies prepared against the two proteins cross-reacted extensively, but failed to react with the doubly deglycosylated polypeptides in Western blots. The dominant epitopes present in the reduced-pyridylethylated polypeptides are, therefore, N-linked carbohydrate, although the presence of peptide epitopes in the native proteins cannot be excluded.

Glycogen branching enzyme was isolated from rabbit liver. The highly purified enzyme shows a monomer molecular weight of 71 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and apparent molecular weights of 93 000 by sucrose density gradient sedimentation and 52 000 by gel-exclusion chromatography on Sephacryl S-300. No glucosamine, mannosamine, galactosamine, or sialic acid was detected in the protein. An amino acid analysis is reported. The spectrum of branching enzyme is that of a simple polypeptide, with A1%280nm = 24.6. Highly purified branching enzyme consists of several closely related active enzyme forms that can be resolved by isoelectric focusing in polyacrylamide gel. The major species of pI 5.7 is flanked by less abundant forms of pI 5.6 and 5.8. Seemingly identical enzyme forms are observed in crude extracts of rabbit liver, skeletal muscle, brain, and heart, although the absolute and relative concentrations vary among the tissues. Branching enzyme apparently does not exhibit tissue-specific isoenzymes.

Glycogen branching enzymes (EC 2.4.1.18) in extracts of cerebral cortex from a patient with Lafora myoclonus epilepsy and several normal subjects were compared in terms of activity and physical properties of the proteins. Branching-enzyme activity in Lafora cortex tissue was significantly higher than in controls. Gel-exclusion chromatography revealed a single branching-enzyme species of identical elution volume in normal and pathological tissues. A molecular weight of 95,000 was estimated for the enzyme from normal cerebral cortex by means of sucrose density gradient sedimentation. A specific staining method for detection of branching-enzyme activity in polyacrylamide gel slabs was developed for the analysis of electropherograms of tissue extracts. Normal cortex extracts showed the presence of two or more closely spaced bands of branching activity; Lafora cortex extracts showed four strongly stained bands which migrated more rapidly toward the anode than those from the normal tissue. We conclude that Lafora myoclonus epilepsy does not involve a deficiency of total branching-enzyme activity but the protein appears to be of a slightly more acidic nature than that of normal tissue. This may reflect alterations in another system involved in protein processing.

FEBS LettersVolume 132, Issue 2 p. 179-182 Full-length articleFree Access Occurrence of high and low M r forms of glycogen phosphorylase in extracts of human brain Carola Ponzetto Zimmerman, Carola Ponzetto Zimmerman Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USASearch for more papers by this authorAllen M. Gold, Allen M. Gold Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USASearch for more papers by this author Carola Ponzetto Zimmerman, Carola Ponzetto Zimmerman Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USASearch for more papers by this authorAllen M. Gold, Allen M. Gold Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USASearch for more papers by this author First published: September 28, 1981 https://doi.org/10.1016/0014-5793(81)81155-6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 D. Proux, J.-C. Dreyfus, Clin. Chim. Acta, 48, (1973), 167– 172. 2 D. Proux, M. Vibert, M.C. Meienhofer, J.-C. Dreyfus, Clin. Chim. Acta, 57, (1974), 211– 216. 3 R.G. Martin, B.N. Ames, J. Biol. Chem., 236, (1961), 1372– 1379. 4 J.L. Hedrick, E.H. Fischer, Biochemistry, 4, (1965), 1337– 1343. 5 H.-Y. Hu, A.M. Gold, Biochemistry, 14, (1975), 2224– 2230. 6 K. Titani, A. Koide, J. Hermann, L.H. Ericsson, S. Kumar, R.D. Wade, K.A. Walsh, H. Neurath, E.H. Fischer, Proc. Natl. Acad. Sci. USA, 74, (1977), 4762– 4766. 7 V.L. Seery, E.H. Fischer, D.C. Teller, Biochemistry, 6, (1967), 3315– 3327. 8 J.A. Johnson, J.D. Nash, R.M. Fusaro, Anal. Biochem., 5, (1963), 379– 387. 9 K. Satoh, F. Imai, K. Sato, FEBS Lett., 95, (1978), 239– 242. 10 K. Feldmann, H. Zeisel, E. Helmreich, Proc. Natl. Acad. Sci. USA, 69, (1972), 2278– 2282. Volume132, Issue2September 28, 1981Pages 179-182 ReferencesRelatedInformation

The kinetic mechanism of rabbit muscle glycogen synthase I was investigated by determining isotope-exchange rates at chemical equilibrium between uridine diphosphoglucose (UDPG) and glycogen and between UDPG and uridine 5'-diphosphate (UDP). The rates were followed simultaneously by use of UDPG labeled with 14C in the glucose moiety and with 3H in the uracil group. They were found to be independent of the concentrations of glycogen and the UDPG-UDP pair, averaging 6 X 10(-9) mol min-1 mg-1, with a ratio of UDPG-glycogen exchange to UDPG-UDP exchange of 0.85-0.95. The conclusion is that glycogen synthase has a rapid equilibrium random bi bi mechanism. The previously reported slow activation of glycogen-free synthase in the presence of glycogen was examined kinetically. The activation rate appears to be independent of glycogen concentration over a wide range, while the maximum activation is related to the third or fourth root of the glycogen concentration. This suggest that the slow bimolecular reaction mechanism proposed for human polymorphonuclear leucocyte glycogen synthase I [Sølling, H., & Esmann, V. (1977) Eur. J. Biochem. 81, 129] does not apply to rabbit muscle synthase I. The rate of exchange of glycogen molecules in the complex between glycogen and rabbit muscle synthase I under conditions where the enzyme is catalytically active was estimated by a novel method. The enzyme-glycogen complex was treated with [glucose-14C]UDPG and glycogen of different molecular weight. The distribution of isotope between the two forms of glycogen was determined after their separation by agarose gel chromatography. A rate constant of 0.3 min-1 was estimated for the exchange. It can be calculated, on the basis of the specific activity of the enzyme (20 mumol min-1 mg-1) and its action pattern, that hundreds of individual chains in the glycogen molecule must be available to the enzyme during the average lifetime of the complex. A mechanism is proposed for this process.

The requirement of muscle phosphorylase for branched polysaccharide substrates was investigated by kinetic studies on semisynthetic branched saccharides. One series of saccharides was prepared from maltoheptose by oxidizing the reducing group to a carboxyl group and coupling this with an amino group of ethylenediamine. The resulting aminooligosaccharide was coupled with p-nitrophenyl esters of mono-, di-, tetra-, and polycarboxylic aicds to produce saccharides containing one, two, four, and approximately 52 maltodextrin chains per molecule. A similar series of saccharides was prepared from a heterogeneous maltodextrin of average chain length 11.7. Kinetic constants were determined for the reaction with phoshorylase a in the direction of chain elongation. Michaelis constants are equilibrium constants for dissociation of saccharide from the enzyme-AMP-glucose-1P-saccharide complex. The Michaelis constants, expressed in terms of the concentration of nonreducing end groups, are independent of maltodextrin chain length but decrease considerably as the number of chains per molecule increases. Maximum velocities do not differ greatly from that for glycogen. Among the synthetic saccharides, only the polymer behaves similarly to glycogen in exhiiting a decreasing reaction rate as the chains are elongated. The kinetic constants are quantitatively consistent with a model in which two chain termini from the same saccharide molecule bind to the phosphorylase molecule simultaniously, Differences in binding between saccharides having different numbers of equally accessible chains are caused solely by statistical factors in the equilibrium. Highly branched substrates bind better because of their greater multiplicity of two end-group pairs.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMultiple forms of colicin E3 from Escherichia coli CA-38 (col E3, col I)Jane M. Glick, Sylvia J. Kerr, Allen M. Gold, and David SheminCite this: Biochemistry 1972, 11, 7, 1183–1188Publication Date (Print):March 1, 1972Publication History Published online1 May 2002Published inissue 1 March 1972https://pubs.acs.org/doi/10.1021/bi00757a011https://doi.org/10.1021/bi00757a011research-articleACS PublicationsRequest reuse permissionsArticle Views32Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

5-Gluconolactone is a potent inhibitor of rabbit muscle phosphorylase (I in the presence of saturating AMP.

Polysaccharide phosphorylase isolated from Idaho potatoes was the subject of a steady state kinetic study. The initial velocity of the reaction between α-d-glucopyranose 1-phosphate (glucose-1-P) and amylopectin, in the absence of orthophosphate, was determined as a function of substrate concentration. A similar study was carried out for the reverse reaction. The result is characteristic of a sequential reaction mechanism in which both substrates must add to the enzyme before any product can be liberated. Isotope exchange rates at chemical equilibrium were determined for the glucose-1-P:Pi:amylopectin system in the presence of phosphorylase. Exchange of 32P from glucose-1-P into Pi and exchange of 14C from glucose-1-P into amylopectin were followed simultaneously by the use of glucose-1-P containing both isotopes. Concentrations of glucose-1-P and Pi were varied together in their equilibrium ratio at constant amylopectin concentration, and the concentration of amylopectin was varied at fixed concentrations of phosphates. Exchange rates for the two isotopes were equal under all conditions and gave linear reciprocal plots. These results support a rapid equilibrium mechanism. Isotope exchange rates were also determined under nonequilibrium conditions. Exchange of 32P from glucose-1-P into Pi was followed as a function of amylopectin concentration at a fixed concentration of glucose-1-P and several fixed concentrations of Pi. The exchange was also followed as a function of glucose-1-P concentration at a fixed amylopectin concentration and several fixed Pi concentrations. These experiments are equivalent to conventional product inhibition experiments. The results indicate that Pi is a competitive inhibitor with respect to glucose-1-P and a noncompetitive inhibitor with respect to amylopectin. Our conclusion is that potato phosphorylase has a rapid equilibrium Random Bi Bi mechanism (Cleland, W. W., Biochim. Biophys. Acta, 67, 104 (1963)) involving binary complexes of enzyme with amylopectin, glucose-1-P, and Pi, and ternary complexes of enzyme with amylopectin and glucose-1-P and with amylopectin and Pi.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPeptide sequences and relative reactivity of the reactive sulfhydryl groups of rabbit muscle phosphorylaseAllen M. Gold and David BlackmanCite this: Biochemistry 1970, 9, 23, 4480–4486Publication Date (Print):November 1, 1970Publication History Published online1 May 2002Published inissue 1 November 1970https://doi.org/10.1021/bi00825a003RIGHTS & PERMISSIONSArticle Views22Altmetric-Citations12LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (833 KB) Get e-Alerts Get e-Alerts

Isotope-exchange rates at chemical equilibrium were determined for the glycogen-α-d-glucopyranose 1-phosphate-Pi system in the presence of phosphorylase a. Exchange of 32P from α-d-glycopyranose 1-phosphate (glucose-1-P) into Pi and exchange of 14C from glucose-1-P into glycogen were followed simultaneously by the use of glucose-1-P containing both isotopes. Concentrations of glucose-1-P and Pi were varied together in their equilibrium ratio at constant glycogen concentration, and the concentration of glycogen was varied at fixed concentrations of phosphates. Exchange rates for the two isotopes were equal under all conditions (with the possible exception of measurements at the highest concentrations of the phosphates) and gave linear reciprocal plots. One exception was noted in which the reciprocal plot was concave upward at low concentrations of substrate, probably because of allosteric effects. The results support a rapid equilibrium mechanism. Initial velocity of the reaction in the absence of product was determined by the use of an isotopic assay. The results were characteristic of a sequential mechanism. Isotope-exchange rates were also determined under nonequilibrium conditions. Exchange of 32P from glucose-1-P into Pi was followed as a function of glycogen concentration at a fixed concentration of glucose-1-P and several fixed concentrations of Pi. The exchange was also followed as a function of glucose-1-P concentration at fixed glycogen concentration and several fixed Pi concentrations. Similar experiments were done while following the exchange from Pi into glucose-1-P. These experiments are equivalent to conventional product inhibition experiments. The results indicate that the two phosphates are noncompetitive inhibitors with respect to glycogen, but are competitive with respect to one another. Our conclusion is that phosphorylase a has a rapid equilibrium Random Bi-Bi mechanism involving binary complexes of enzyme with glycogen, glucose-1-P, and Pi, and ternary complexes of enzyme with glycogen and glucose-1-P and with glycogen and Pi.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSulfhydryl groups of rabbit muscle glycogen phosphorylase b. Reaction with dinitrophenylating agentsAllen M. GoldCite this: Biochemistry 1968, 7, 6, 2106–2115Publication Date (Print):June 1, 1968Publication History Published online1 May 2002Published inissue 1 June 1968https://pubs.acs.org/doi/10.1021/bi00846a013https://doi.org/10.1021/bi00846a013research-articleACS PublicationsRequest reuse permissionsArticle Views50Altmetric-Citations42LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts