Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

Outer membrane proteins as major antigens of Fusobacterium nucleatum

The immunochemical reactions of rabbit polyclonal antibodies directed to different preparations of Fusobacterium nucleatum i.e, whole cells, peptidoglycan associated proteins, a peptidoglycan-protein complex and a purified 40 kiloDalton (kDa) protein, were investigated on outer membrane preparations of Fusobacterium species and a restricted number of Leptotrichia buccalis after their separation on sodium dodecyl sulphate polyacrylamide gels and electrotransfer to nitrocellulose. All F. nucleatum strains had identical reaction patterns with the immune sera tested. Surface exposed parts of a restricted number of proteins with apparent molecular weights at 70 kDa (a doublet band), 60 kDa, 55 kDa and 40 kDa seemed to be major immunogens. Antigenic related proteins either of identical or slightly deviating electrophoretic mobilities to the 40-kDa protein were observed with the other members of Bacteroidaceae tested. The characteristic 70-kDa protein doublet seemed to be restricted to F. nucleatum although single protein bands of near identical molecular weights belonging to the other species tested also reacted. The data also indicate that the 60-kDa and 55-kDa polypeptides might be present in other species of Fusobacterium.     

Outer membrane proteins as major antigens of Fusobacterium nucleatum

Abstract The immunochemical reactions of rabbit polyclonal antibodies directed to different preparations of Fusobacterium nucleatum i.e, whole cells, peptidoglycan associated proteins, a peptidoglycan-protein complex and a purified 40 kiloDalton (kDa) protein, were investigated on outer membrane preparations of Fusobacterium species and a restricted number of Leptotrichia buccalis after their separation on sodium dodecyl sulphate polyacrylamide gels and electrotransfer to nitrocellulose. All F. nucleatum strains had identical reaction patterns with the immune sera tested. Surface exposed parts of a restricted number of proteins with apparent molecular weights at 70 kDa (a doublet band), 60 kDa, 55 kDa and 40 kDa seemed to be major immunogens. Antigenic related proteins either of identical or slightly deviating electrophoretic mobilities to the 40-kDa protein were observed with the other members of Bacteroidaceae tested. The characteristic 70-kDa protein doublet seemed to be restricted to F. nucleatum although single protein bands of near identical molecular weights belonging to the other species tested also reacted. The data also indicate that the 60-kDa and 55-kDa polypeptides might be present in other species of Fusobacterium .     

Characterization of the protein kinase activity in beet root plasma membranes

Two protein kinase activities were found in plasma membrane-enriched preparations from red beet ( Beta vulgarix L.). The kinases in these preparations produced the phosphorylation of several membrane polypeptides. These kinases also phosphorylated histone III-S and casein. The activities of two different kinases could be distinguished: one was half-maximally stimulated by 1 μ M free Ca²⁺ phosphorylated histone III-S better than casein, showed half-maximal activity at an ATP concentration of 0.071 m M . had an optimum pH of 7, and was poorly inhibited by GTP, CTP or UTP. Another, much lower, kinase activity that phosphorylated casein was also observed; it was Ca²⁺ independent, showed half-maximal activity at ATP concentrations of 0.017 and 0.287 m M , exhibited a broad pH optimum about pH 7 and was inhibited by GTP, CTP, UTP or GDP to a greater extent than the calcium-stimulated activity. When plasma membrane proteins were solubilized with lysophosphatidyicholine and treated with [γ-³²P]ATP at several dilutions, a 125-kDa polypeptide was autophosphorylated in the absence of Ca²⁺, while 77-, 71- and 65-kDa polypeptides were autophosphorylated in its presence. Autophosphorylation in gels after electrophoresis showed a Ca²⁺-stimulated phosphoprotein band at 64 kDa.     

Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR

To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.     

The secretory nature of the lesion of carrot cell variant ts11, rescuable by endochitinase

The carrot cell variant ts11 is unable to form somatic embryos at the non-permissive temperature of 32 °C, but the block can be overcome by the addition of a 32-kDa acidic endochitinase to the medium. In this work we conducted a cyto-histological analysis of the blocked embryo forms. The morphology of the endomembrane system is altered; in particular, the ER is dilated and may show electron-dense precipitates and continuity with the plasma membrane. These morphological alterations do not occur in the presence of externally-added endochitinase. We also noticed modifications of the culture medium that are probably related to the morphological observations: the total amount of secreted proteins is reduced and pulse-chase experiments revealed that, compared with wild-type cells, the secretion of major polypeptides is reduced while new minor polypeptides are secreted. Western blot analysis revealed the presence of the binding protein BiP, a resident of the ER and of glutamine synthase, a cytosolic protein, in the medium of ts11 but not wild-type cells. These results indicate that ts11 is altered in the secretory pathway but do not clarify the role of endochitinase. Received: 13 January 1997 / Accepted: 21 March 1997     

Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken

Abstract: The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell-cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P-40-solubilized retina membranes had two components as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 45.5-kDa doublet and a 69-kDa polypeptide. Immunoreactive bands of 46-50 kDa were recognized following SDS-PAGE of detergent-solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N-glycosidase F (EC 3.2.2.18) converted the 45.5–50-kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N-Glycosidase Ftreatment also converted the 69-kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32-kDa species. Treatment with endo-β-N-acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo-β-N-acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5-kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O-glycosidase (endo-α-N-acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for α2, 6-linked sialic acid) was confined to the higher molecular mass species of the 45.5-kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue-specific posttranslational modifications, particularly differences in sialylation of antigen-bearing polypeptides.     

Thrombin-reactive polypeptides of human blood. Some biochemical and immunological properties

Two polypeptides of 74 kDa and 55 kDa have been isolated from human platelets by immunoaffinity and lectin affinity chromatography and their effects on thrombin reactivity have been examined. These proteins in combination enhanced the aggregation of platelets by thrombin while aggregation induced by trypsin, collagen and adenosine diphosphate was not significantly affected. An enhancement in the action of thrombin on fibrinogen, N-benzoylarginine ethyl ester and H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride was also observed in the presence of the platelet proteins. Under similar conditions, the proteins did not influence the esterolytic activity of trypsin or plasmin. Studies at different thrombin and protein concentrations showed maximum enhancement of enzyme reactivity when the ratio between the peptides and thrombin was optimal. In the presence of these proteins, the affinity of thrombin for N-benzoylarginine ethyl ester was about twofold higher than in the control. Two polypeptides with properties similar to those described above have also been isolated from human plasma. Antibodies to the above proteins isolated from either platelets or plasma were raised in rabbits. Intact platelets solubilized in Triton X-100 or plasma showed two precipitin lines in immunoelectrophoresis against both of the above antisera and a similar pattern was observed with the isolated polypeptides. The polypeptides did not interact in immunoelectrophoresis with antisera to whole serum, antithrombin, C4 binding protein or protein S. These 74-kDa and 55-kDa polypeptides contained radioactivity when radioiodinated platelets were used suggesting that they are located on the cell surface. Fresh plasma was analyzed by gel electrophoresis under nondenaturing and denaturing conditions and the proteins were transferred to nitrocellulose sheets. Staining with antibody to these thrombin-reactive proteins and 125I-protein A showed several reactive plasma proteins under nondenaturing conditions with the major band migrating in the albumin area. In plasma treated with sodium dodecyl sulfate, the 74-kDa and 55-kDa components were observed. A prominent 74-kDa band and a fainter 55-kDa component were again observed when platelets solubilized in sodium dodecyl sulfate were analysed by the above procedure. It is proposed that human platelets and plasma contain polypeptides which may directly modulate thrombin reactivity.     

Low molecular weight GTP-binding proteins in cardiac muscle. Association with a 32-kDa component related to connexins.

Low molecular weight GTP-binding proteins and their cellular interactions were examined in cardiac muscle. Heart homogenate was separated into various subcellular fractions by differential and sucrose density gradient centrifugation. Various fractions were separated by sodium dodecyl sulfate-gel electrophoresis, blotted to nitrocellulose, and GTP-binding proteins detected by incubating with [alpha-32]GTP. Three polypeptides of M(r) 23,000, 26,000, and 29,000 were specifically labeled with [alpha-32P]GTP in all the fractions examined and enriched in sarcolemmal membranes. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 26- and 29-kDa polypeptides. A polypeptide of M(r) 40,000 was weakly labeled with [alpha-32P]GTP in the sarcolemmal membrane and tentatively identified as Gi alpha by immunostaining with anti-Gi alpha antibodies. Cytosolic GTP-binding proteins were labeled with [alpha-32P]GTP and their potential sites of interaction investigated using the blot overlay approach. A polypeptide of 32 kDa present in sarcolemmal membranes, intercalated discs, and enriched in heart gap junctions was identified as a major site of interaction. The low molecular weight GTP-binding proteins associated with the 32-kDa polypeptide through a complex involving cytosolic components of M(r) 56,000, 36,000, 26,000, 23,000, and 12,000. A monoclonal antibody against connexin 32 from liver strongly recognized the 32-kDa polypeptide in heart gap junctions, whereas polyclonal antibodies only weakly reacted with this polypeptide. The low molecular weight GTP-binding proteins associated with a 32-kDa polypeptide in liver membranes that was also immunologically related to connexin 32. These results indicate the presence of a subset of low molecular weight GTP-binding proteins in a membrane-associated and a cytoplasmic pool in cardiac muscle. Their association with a 32-kDa component that is related to the connexins suggests that these polypeptides may be uniquely situated to modulate communication at the cell membrane.     

A novel adenylylation process in liver plasma membrane-bound proteins

Rat liver plasma membrane contains five distinct polypeptides of apparent molecular mass of 130, 120, 110, 100, and 86 kDa which are labeled upon incubation with [alpha-32P]ATP as well as with [gamma-32P]ATP. Covalently bound adenosine 5'-monophosphate to some of the polypeptides was identified using nonhydrolyzable analogues of ATP. Chase experiments of alpha-32P-nucleotide-labeled polypeptides with different nonradiolabeled phosphocompounds and sensitivity to different inhibitors demonstrate that the 86-kDa polypeptide is a phosphoesterase, forming a catalytic intermediate. On the other hand, the comparative slow rate of turnover of the polypeptides of higher molecular mass (130, 120, 110, and 100 kDa) suggests that the bound AMP could play a regulatory rather than a catalytic role. Using the nonhydrolyzable ATP analogue [alpha, beta-methylene]ATP and dilution experiments with Triton X-100-solubilized membranes, it has been possible to identify the 130-kDa adenylylated polypeptide as a possible target of an adenylylating system. These polypeptides, except the 86-kDa phosphoesterase, are affected in their electrophoretic mobility in the absence of beta-mercaptoethanol. An intercatenary disulfide bond(s) appear(s) to link the polypeptide(s) of 120 kDa and/or 110 kDa in a dimeric structure of apparent molecular mass of 240 kDa. All five polypeptides labeled with [alpha-32P]ATP are glycoproteins bound to the cell plasma membrane.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex

Digestion of HeLa cell nuclei with micrococcal nuclease or deoxyribonuclease I (DNase I) released the 86-kilodalton-70-kilodalton (kDa) protein complex in particles sedimenting at approximately 10 S in sucrose density gradients. Immunoaffinity-purified 32P-labeled complexes contained 86- and 70-kDa polypeptides with phosphorylated serine residues and DNA fragments, of which the largest was 110 base pairs long. Digestion of nick-translated nuclei with micrococcal nuclease released 32P-labeled 10S particles that were immunoaffinity-purified; they contained labeled 110-base-pair DNA fragments. The micrococcal nuclease digests were analyzed by two-dimensional electrophoresis, which separated nucleosomes in the first dimension and the associated proteins in the second. Western blots of the separated proteins showed that the 86-kDa-70-kDa complex was associated with the mono-, di-, and trinucleosomes. A more extensive electrophoretic separation revealed that the 10S particle from nick-translated nuclei migrated with a subfraction of the mononucleosomes that lacked H1 histones. These results suggest that the 10S particle which contains the 86-kDa-70-kDa complex is associated with an unfolded nucleosome that is present in DNase I sensitive regions.     

Secretion of proteins by cultured bovine oviducts collected from estrus through early diestrus

To characterize in vitro protein secretion by the oviduct throughout early stages of the estrous cycle, 16 cows received a luteolytic dose of PGF2 alpha and were randomly assigned to be killed on day (D) 0, 2, 5, or 8 after subsequent estrus. Explant cultures of oviducts (ampulla and isthmus) were incubated for 24 h at 39 degrees C in a modified Eagle's minimal essential medium supplemented with 50 microCi L-[4,5-3H]leucine. Oviductal secretion of de novo synthesized protein measured by incorporation of [3H]leucine into nondialyzable radioactivity in culture supernatants was greatest at D 0 and declined thereafter. Incorporation of [3H]leucine into TCA-precipitable macromolecules in tissue homogenates was also greatest at D 0. Analysis of culture supernatants by one-dimensional polyacrylamide gel electrophoresis revealed four major bands of radiolabeled proteins at greater than 97 kDa, 85-97 kDa, 55 kDa, and 30 kDa. Analysis of individual polypeptides resolved by two-dimensional polyacrylamide gel electrophoresis indicated that five of 32 individual polypeptides examined were secreted at significantly greater rates at estrus than at other times examined. One of these five polypeptides, a 97-kDa peptide with an apparent pI of 5.0, was the major secretory product at estrus and accounted for 18% of total radioactivity recovered from two-dimensional gels. Two of 32 polypeptides examined were secreted at significantly greater rates by explants of the oviduct contralateral to the side of ovulation. In summary, estrus is associated with an elevation in total protein secretion by the bovine oviduct. This increase is due to selective amplification of secretion of several but not all secretory proteins.     

Molecular characterization of two novel crystal protein genes from Bacillus thuringiensis subsp. thompsoni.

Two genes encoding the predominant polypeptides of Bacillus thuringiensis subsp. thompsoni cuboidal crystals were cloned in Escherichia coli and sequenced. The polypeptides have electrophoretic mobilities of 40 and 34 kDa, with the deduced amino acid sequences predicting molecular masses of 35,384 and 37,505 Da, respectively. No statistically significant similarities were detected between the 40- or 34-kDa crystal protein and any other characterized B. thuringiensis crystal protein, nor were they detected between the 40- and 34-kDa crystal proteins. A 100-MDa plasmid carries both crystal protein genes, which appear to be part of an operon, with the 40-kDa gene 64 nucleotides upstream of the 34-kDa gene. Both crystal proteins are synthesized in approximately the same amounts. Even though small compared with other crystal proteins, the 34-kDa crystal protein has insecticidal activity against lepidopteran larvae (Manduca sexta). The 40-kDa polypeptide appears to have no insecticidal activity, but it could have a role in crystal structure.     

Heat-stable proteins and abscisic Acid action in barley aleurone cells

[³⁵S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100°C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heatstable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered.     

In vivo and in vitro acylation of polypeptides in Vibrio harveyi: identification of proteins involved in aldehyde production for bioluminescence.

Incubation of soluble extracts from Vibrio harveyi with [3H]tetradecanoic acid (+ ATP) resulted in the acylation of several polypeptides, including proteins with molecular masses near 20 kilodaltons (kDa), and at least five polypeptides in the 30- to 60-kDa range. However, in growing cells pulse-labeled in vivo with [3H]tetradecanoic acid, only three of these polypeptides, with apparent molecular masses of 54, 42, and 32 kDa, were specifically labeled. When extracts were acylated with [3H] tetradecanoyl coenzyme A, on the other hand, only the 32-kDa polypeptide was labeled. When luciferase-containing dark mutants of V. harveyi were investigated, acylated 32-kDa polypeptide was not detected in a fatty acid-stimulated mutant, whereas the 42-kDa polypeptide appeared to be lacking in a mutant defective in aldehyde synthesis. Acylation of both of these polypeptides also increased specifically during induction of bioluminescence in V. harveyi. These results suggest that the role of the 32-kDa polypeptide is to supply free fatty acids, whereas the 42-kDa protein may be responsible for activation of fatty acids for their subsequent reduction to form the aldehyde substrates of the bioluminescent reaction.     

Identification of low molecular weight GTP-binding proteins and their sites of interaction in subcellular fractions from skeletal muscle

The presence of low molecular weight GTP-binding proteins was investigated in subcellular fractions from skeletal muscle. Skeletal muscle homogenate, transverse tubules, triads, sarcoplasmic reticulum membranes, and cytosol fractions were separated in sodium dodecyl sulfate-gel electrophoresis and blotted onto nitrocellulose. The presence of GTP-binding proteins was explored by incubation of these blots with [alpha-32P] GTP. GTP labeled two polypeptides of Mr = 23,000 and 29,000 in all the fractions examined. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 29-kDa polypeptide, although both were enriched in transverse tubule fractions. A GTP-binding polypeptide of 40 kDa was also enriched in transverse tubule preparations and identified as Gi alpha by immunostaining with anti-Gi alpha. Using a blot overlay approach and [alpha-32P]GTP-labeled cytosolic components, several polypeptides were identified that interact with the 23- and 29-kDa GTP-binding proteins. Among these components were polypeptides of Mr = 60,000, 47,000, 44,000, 42,000, and 38,000, which were mainly of cytosolic origin but also associated with triads and transverse tubule membranes. The 47-, 44-, 42-, and 38-kDa polypeptides were found to be structurally related to the glycolytic enzymes enolase, 3-phosphoglyceric phosphokinase, aldolase, and glycoeraldehyde-3-phosphate dehydrogenase, respectively. The purified glycolytic enzymes specifically bound the 23- and 29-kDa GTP-binding proteins under both denaturing and nondenaturing conditions. The association of the GTP-binding proteins with these polypeptides was resistant to detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), Triton X-100, and Tween. A 23-kDa GTP-binding protein purified from chromaffin cells bound to a 157-kDa polypeptide in triads and chromaffin cell membranes. The 157-kDa polypeptide was a minor component in these membranes and not related to the subunits of the dihydropyridine receptor. In view of the proposed function of low molecular weight GTP-binding proteins in processes such as membrane communication and secretion coupling, the association of these proteins with transverse tubules and triads in skeletal muscle is discussed in terms of a role in signal transmission.