Spinach ferredoxin is a calcium-binding protein

Spinach-leaf ferredoxin was identified as a calcium-binding protein by ⁴⁵Ca autoradiography on nitrocellulose membranes and with the cationic carbocyanine dye 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2-d]thiazolium bromide (0g220187771678/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">stains-all0g220187771678/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0">). Binding of ⁴⁵Ca was observed at pH 6.8 and pH 7.8 and in the presence of 5 mM and 20 mM MgCl₂. At the higher MgCl₂ concentration the Ca²⁺-binding capacity is reduced. Only micromolar concentrations of LaCl₃, however, are required to achieve a similar effect. Both the oxidized and reduced forms of ferredoxin bind calcium.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - 0g220187771678/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">stains-all0g220187771678/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> 1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naptho[1,2-d]thiazolium bromide     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-conglycinin, encoded by a multigene family. 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-Conglycinin consists of three subunits; 0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">0/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">, 0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">, and 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">. A genomic clone for a 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit of 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit expressed in seeds of petunia and tobacco was recognized by anti-0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit were produced. There was approximately a twofold variation in the accumulation of the 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit protein in the mature seeds of transgenic petunia plants, each containing a single 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit gene. However, the level of protein accumulation in mature seeds and the amount of 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit mRNA in developing seeds was not correlated. Accumulation of the 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit protein in transgenic seeds was less than the 0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">0/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-subunit protein that accumulated in transgenic petunia seeds containing a single 0/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">0/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-subunit gene and less than the amount of the 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit in mature soybean seeds which contain 8013;13 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit genes. In transgenic tobacco plants, the accumulation of the 0/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

C-banding polymorphism and the analysis of nucleolar activity in Dasypyrum villosum (L.) Candargy,its added chromosomes to hexaploid wheat and the amphiploid Triticum dicoccum — D. villosum

Summary C-banding patterns and nucleolar activity were analyzed in Dasypyrum villosum, its added chromosomes to hexaploid wheat and the hexaploid amphiploid Triticum dicoccum-D. villosum. Two different populations of the allogamous species D. villosum (2n= 14, VV) from Greece and Italy were analyzed showing a similar polymorphism for C-banding pattern. Six of the seven addition lines were identified by their characteristic C-banding pattern. No polymorphism between both members of each added alien chromosome was found. Furthermore, nucleolar activity and competition were studied by using silver staining procedure. In D. villosum only one chromosome pair, A, was found to be responsible for organizing nucleoli. The results obtained in the amphiploid and in the addition lines demonstrate that nucleolar activity is restricted to SAT-chromosomes 1B and 6B of wheat, while those of D. villosum remain inactive.     

Patch-clamp study of isolated taste receptor cells of the frog

Summary Taste discs were dissected from the tongue ofR. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110mm KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was 013;65.2 mV and the slope resistance 150 to 750 M014711541244r/xxlarge937.gif" alt="OHgr" align="BASELINE" BORDER="0">. Pulse-depolarization from a holding voltage of 013;80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at 013;25 mV and was half-maximal at 013;8 mV. Steady-state inactivation was half-maximal at 013;67 mV and complete at 013;50 mV. Peak Na current averaged 013;0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5mm) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5mm) and 4-aminopyridine (1mm) did not block, but 5mm Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted theI _K(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10mm Ca caused a reversible 5-to 10-mV depolarization in the current-clamp mode. Quinine (0.1mm, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5mm in the external solution or 0.5 014711541244r/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">m in the pipette) caused reversible depolarization (to 013;40 to 013;20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP. Inclusion of ATP-014711541244r/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-S in the pipette caused slow depolarization to 013;40 to 013;20 mV, due to partial blockage of K currents. Subsequently, cAMP was without effect. The remaining K currents were blockable by Ba. These results suggest that cAMP initiates phosphorylation of one set of K channels to a nonconducting conformation.     

A Phorbol Ester-Sensitive Kinase Catalyzes the Phosphorylation of P0 Glycoprotein in Myelin

The proposed structural protein of peripheral nerve myelin, P0, has been shown to have several covalent modifications. In addition to being glycosylated, sulfated, and acylated, P0 is phosphorylated, with the intracellular site of this latter addition being in question. By employing nerve injury models that exhibit different levels of P0 biosynthesis in the absence and presence of myelin assembly, we have examined the cellular location of P0 phosphorylation. It is demonstrated that there is comparable P0 phosphorylation in both normal and crush-injured adult rat sciatic nerves, although the level of biosynthesis of P0 differs between these myelin maintaining and actively myelinating nerve models, respectively. The glycoprotein does not appear to be phosphorylated readily in the transected adult sciatic nerve, a preparation in which P0 biosynthesis is observed but that lacks myelin membrane. These observations suggest that the modification is not associated with the biosynthesis or maturation of P0 in the endoplasmic reticulum or Golgi, but that it instead occurs after myelin assembly. That P0 phosphorylation occurs in the normal nerve even when translation is inhibited by cycloheximide treatment lends further support to this conclusion. P0 is shown to be phosphorylated on one or more serine residues, with all or most of the phosphate group(s) being labile as evidenced by pulse-chase analysis. Addition of a biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate or 4 beta-phorbol 12,13-dibutyrate, substantially increases the extent of [32P]orthophosphate incorporation into the glycoprotein of normal and crushed nerve but not transected nerve. Biologically inactive 4 alpha-phorbol 12,13-didecanoate has no effect on P0 phosphorylation. Similarly, the addition of the cyclic AMP analog 8-bromo-cyclic AMP causes no appreciable changes in P0 labeling. These findings indicate that the phorbol ester-sensitive enzyme, protein kinase C, may be responsible for the phosphorylation of P0 within the myelin membrane.     

Selection of Lactobacillus acidophilus strains for use in “acidophilus products”

Recent studies by DNA-DNA hybridization revealed that strains now designated as L. acidophilus, can be divided into several groups and only one group should be classified as L. acidophilus. We studied several phenotypic characteristics in representative strains from the six DNA-homology groups of L. acidophilus. No group specific pattern was observed among the strains for fermentation of eight carbohydrates, growth at 15 and 45°C, resistance to 0.2% oxgall, lysis by lysozyme or sensitivity to 17 antibiotics. However, some differences among groups were observed in 0">-galactosidase (0">-gal) activity and surface layer (s-layer) protein. Strains in B1 do not have a s-layer or 0">-gal while B2 strains also lack a s-layer but do possess 0">-gal. All strains in groups A1, A2, A3 and A4, capable of growing in lactose, have 0">-gal activity and also have a s-layer composed of protein subunits of different molecular weights (MW). Strains in A1 homology group have a s-layer with 46 Kd protein subunits while strains in other A groups have s-layer protein subunits that varied in MW within each group. On the basis of these two traits several isolates of unknown homology groups have been tentatively placed in A1, B1 or B2 groups. L. acidophilus from A1 group showed strain variation in 0">-gal specific activity and rate of acid production and growth. For use in dietary adjuncts, L. acidophilus strains should be selected for these three and other desirable traits. They should be maintained and grown in media containing lactose.     

A gene (Bmn) controllingβ-mannosidase activity in the mouse is located in the distal part of chromosome 3

A gene (Bmn) with a major effect on 037372l85461/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-mannosidase activity in kidney and liver of the house mouse was revealed by assay with the synthetic substratep-nitrophenyl-037372l85461/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-d-mannoside. Activity is low in DBA/2J and CSB mice and high in C57BL/6J mice. By the use of the BXD series of recombinant inbred strains and by crosses between C57BL and CSB, it was possible to map the gene to the distal part of chromosome 3 by demonstration of linkage to a gene for cadmium resistance,cdm, as well as to theAdh-3 locus.This work was supported by Swedish Natural Science Research Council Project B-BU 2992-108.     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase. The loss of liver lysosomal 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase activity was shown by immunotitration to be due to a decrease in the number of 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver 0hl233u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of 035412/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by 035412/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-chymotrypsin. Limited 035412/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, 035412/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid 0515u7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 30515u7/xxlarge8758.gif" alt="ratio" align="BASELINE" BORDER="0">1. While the oligomannoside-type chains show some heterogeneity in size (Man_5013;8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-0515u7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">(2013;6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of 0515u7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339013;50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, 0515u7/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-N-acetylhexosaminidase, 0515u7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-l-fucosidase and 0515u7/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.