Organellar Calcium Buffers

Ca²⁺ is an important intracellular messenger affecting many diverse processes. In eukaryotic cells, Ca²⁺ storage is achieved within specific intracellular organelles, especially the endoplasmic/sarcoplasmic reticulum, in which Ca²⁺ is buffered by specific proteins known as Ca²⁺ buffers. Ca²⁺ buffers are a diverse group of proteins, varying in their affinities and capacities for Ca²⁺, but they typically also carry out other functions within the cell. The wide range of organelles containing Ca²⁺ and the evidence supporting cross-talk between these organelles suggest the existence of a dynamic network of organellar Ca²⁺ signaling, mediated by a variety of organellar Ca²⁺ buffers.     

A new mutant class of soybean lacks urease in leaves but not in leaf-derived callus or in roots

Summary We reported earlier the recovery of two classes of soybean urease mutants in soybean (Glycine max L. Merr. cv. Williams). Class I mutants lack the embryo-specific urease while class II mutants lack the activities of both urease isozymes, the embryo-specific and the ubiquitous urease, the latter found in all tissues examined. We report here the recovery of a true-breeding mutant, aj3, which represents the third phenotypic class: normal levels of embryo-specific urease and little or no ubiquitous urease. Unlike class II mutant plants which lack urease in all tissue, aj3 lacks urease activity only in leaves (ca. 2% normal activity); its roots have near normal urease activity. Callus derived from leaves of aj3 has 14% to 40% the urease activity of Williams 82 callus. This partial reduction in urease activity in aj3 callus is sufficient to reduce growth with urea as sole nitrogen source and to confer resistance to 50 mM urea added to callus maintenance medium. Leaves of aj3 produce more than 40 times the urease antigen expected from their urease activity. The aj3 trait is due to a single recessive lesion which is not allelic with lesions at theEu2, Eu3 (class II) orEu1 (class I) loci. We designate the aj3 genotype aseu4/eu4.     

Calcium storage in nonmuscle tissues: is the retina special?

It is now well established that calreticulin is a high capacity Ca(2+)-binding protein which is a major Ca2+ storage protein of the lumen of endoplasmic reticulum membranes in a wide variety of tissues with the exception of skeletal and cardiac muscles. However, in nervous tissue, confusion exists regarding the nature of the intracellular Ca2+ stores, as the organelle responsible for Ca2+ storage has been identified as the endoplasmic reticulum by some investigators and as the specialized organelle, calciosome by others. Calreticulin, calsequestrin, and calsequestrin-like proteins have all been, on different occasions, reported to be present in calciosomes. Cerebral and cerebellar tissues, moreover, have been shown to contain somewhat different systems of Ca(2+)-buffering proteins. In the present paper we discuss evidence that the Ca2+ storage systems of the retina may prove to be more complex than those of other neuronal tissues. Biochemical and immunocytochemical evidence indicates the presence of either an isoform of calreticulin or another protein that is antigenically similar to calreticulin, but of slightly higher molecular weight, in the endoplasmic reticulum of both neurons and Müller glia from rabbit neural retina. However, as retinal neurons express Purkinje cell markers, one may expect to observe the presence of calsequestrin in these cells as well. Secondly, antibodies against the onchocercal RAL-1 antigen recognize a protein sharing 62-65% amino acid sequence identity with calreticulin. The anti-RAL-1 antibodies show specificity for the retina. Whether or not the RAL-1 antigen is an active part of the Ca2+ storage systems of the retina remains to be verified.(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation and immunolocalization of iron-binding compounds produced by Gloeophyllum trabeum

Summary Low molecular weight iron-binding compounds are produced by the brown-rot fungus Gloeophyllum trabeum. These chelators may function in scavenging transition metals for fungal metabolism and extracellular enzyme production. Because of the low molecular mass of the chelate-metal complex (below 1000 Da), and the oxidizing potential of the bound transition metals, certain chelating compounds could also play a role in the early stages of cellulose depolymerization by brown-rot fungi. High-affinity iron-binding compounds were isolated and partially purified from both liquid cultures of the brown-rot Gloeophyllum trabeum and from infected wood. Chelating compounds purified by thin-layer chromatography were used to prepare specific antibodies. These antibodies were shown to detect the chelator in infected wood and liquid fungal cultures by enzyme-linked immunosorbent assay and could be used in immunotransmission electron microscopy to visualize the high-affinity iron-binding compounds in situ. Elucidating the physiological roles of fungal chelate-metal complexes and determining their function in lignocellulose depolymerization will help us to better understand the mechanism of wood biodegradation.Publication no. 1549 Maine Agricultural Experiment Station Offprint requests to: J. Jellison     

Inhibition of Na+/Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7

Monoclonal antibodies 44D7 and 4F2 inhibited specifically the Na+-dependent Ca2+ fluxes characteristic of the Na+/Ca2+ exchanger in cardiac and skeletal muscle sarcolemmal vesicles. Preincubation of membrane vesicles with monoclonal antibody 44D7 inhibited 90% of the Na+-dependent Ca2+ uptake measured in the first 10 s of the reaction and 50% of that measured after 60 s. Ca2+/calmodulin-dependent ATPase activity and ATP-dependent Ca2+ uptake by sarcolemmal vesicles were not affected by monoclonal antibody 44D7 whereas the Na+-dependent release of accumulated Ca2+ was inhibited. In the presence of the 44D7 antigen isolated from human kidney, monoclonal antibody 44D7 could no longer inhibit Na+-dependent Ca2+ fluxes. The distribution of 4F2 antigenic activity in the isolated muscle membrane fractions correlated with that of Na+/Ca2+ exchanger activity; cardiac and skeletal muscle sarcolemmal vesicles expressed higher levels of the antigen than skeletal muscle transverse tubule membrane, while no antigen could be detected in sarcoplasmic reticulum membranes. Our results suggest that monoclonal antibodies 44D7 and 4F2 interact either directly with the Na+/Ca2+ exchanger molecules or with some other protein(s) responsible for the regulation of this activity in the heart and skeletal muscle.     

Observations of Barophilic Microbial Activity in Samples of Sediment and Intercepted Particulates from the Demerara Abyssal Plain

To better understand the ecological significance of pressure effects on bacteria in the abyssobenthic boundary layer, experimental suspensions of sediments and sinking particulates were prepared from samples collected in boxcore and bottom-moored sediment traps at two stations (depth, 4,470 and 4,850m) in the Demerara abyssal plain off the coast of Brazil. Replicate samples were incubated shipboard at 3°C and at both atmospheric and deep-sea pressures (440 or 480 atm [4.46 × 10⁴ or 4.86 × 10⁴ kPa]) following the addition of [¹⁴C]glutamic acid (<10 μg liter⁻¹) or yeast extract (0.025%) and the antibiotic nalidixic acid (0.002%). In seven of the eight samples supplemented with isotope, a barophilic microbial response was detected, i.e., substrate incorporation and respiration were greater under in situ pressure than at 1 atm (101.3 kPa). In the remaining sample, prepared from a sediment trap warmed to 24°C before recovery, pressure was observed to inhibit substrate utilization. Total bacterial counts by epifluorescence microscopy decreased with depth in each sediment core, as did utilization of glutamic acid. Significant percentages of the total bacterial populations in cold sediment trap samples (but not the prewarmed one or any boxcore sample) were abnormally enlarged and orange fluorescing after incubation with yeast extract and nalidixic acid under deep-sea conditions. Results indicated that in the deep sea, barophilic bacteria play a predominant role in the turnover of naturally low levels of glutamic acid, and the potential for intense microbial activity upon nutrient enrichment is more likely to occur in association with recently settled particulates, especially fecal pellets, than in buried sediments.     

Targeted Selection Experiments and Enzyme Polymorphism: Negative Evidence for Octanoate Selection at the G6PD Locus in DROSOPHILA MELANOGASTER

Published studies have reported significant selection with respect to the G6pd locus for Drosophila melanogaster reared on Na-octanoate food. We have reexamined the selective effects of Na-octanoate on egg to adult viability with respect to the G6pd polymorphism using specially constructed X chromosomes. Four experiments were carried out using different 6Pgd backgrounds in two recombinant sets of chromosomes segregating for the G6pd locus but constructed so as to minimize variation over most of the X chromosome. In addition, two measures of viability were used, and the size of the experiments and their associated degrees of freedom are approximately double those reported in the former studies. Our results find no evidence for differential selection on G6pd genotypes (males and females) by Na-octanoate and, therefore, do not corroborate the positive results of selection reported by other investigators. The reasons for our different results are discussed.     

Identification and localization of calreticulin in plant cells

Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in⁴⁵Ca²⁺ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca²⁺ binding and storage.Abbreviations ER endoplasmic reticulum - SR sarcoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid