Diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase activities of red cells: comparative electrophoretic study

Erythrocyte diphosphoglycerate mutase (EC 2.7.5.4.) and 2,3-diphosphoglycerate phosphatase (EC 3.1.3.13.) activities of normal human adults, and DPG mutase deficient subject as well as of several animal species were subjected to electrophoretic study on starch gel. In U.V. light 2,3-diphosphoglycerate phosphatase activity was revealed as a band of fluorescence decrease on a fluorescent background, by the oxydation of NADH, whereas diphosphoglycerate mutase appeared as a fluorescent zone. It was found that the electrophoretic pattern of both DPG mutase and 2,3-DPG phosphatase activities was different from one species to the other, but that, in each species, 2,3-DPG phosphatase activity showed the same electrophoretic pattern as DPG mutase activity.     

Chemical and physiological changes in fungi during autolysis

A comparative study was done on some of the chemical changes occurring during autolysis of cultures ofAspergillus flavus in both physiologically acid and alkaline media. The mycelium ofA. flavus lost during autolysis 44 % of its maximum dry weight in the physiologically alkaline medium, whereas this loss was apparently nil in the physiologically acid medium. Nitrogen containing compounds seemed not to be affected by autolysis either in the physiologically acid or alkaline media. The disappearance of P-containing compounds in mycelium ofA. flavus autolysed in both conditions (NO ₃ ^– and NH ₄ ⁺ as N source) amounted to 64 % in the alkaline autolysis and to nearly 77 % in the acid autolysis. The results we have obtained for the acid autolysis strongly suggest that very little activity is shown by autolytic enzymes in the interval 10–133 days of incubation, when measuring autolysis by the loss in mycelial dry weight.

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Zusammenfassung Eine vergleichende Untersuchung war unternommen an einigen der chemischen Veränderungen, die während der Autolyse der Kulturen vonAspergillus flavus in physiologischen sauren und alkalischen Medien vorkommen. Die Myzelien vonA. flavus haben während der Autolyse 44 % ihres größten Trockengewichtes in physiologisch alkalischem Medium verloren, während dieser Verlust in physiologisch saurem Medium anscheinend Null gewesen ist. Stickstoff enthaltende Substanzen erschienen während der Autolyse weder in physiologisch saueren noch in alkalischen Medien beeinflußt zu sein. Das Verschwinden von P-enthaltenden Substanzen in Myzelien vonA. flavus in Autolyse unter beiden Bedingungen (NO ₃ ^– und NH ₄ ⁺ als Stickstoffquelle) erreichte 64 % in alkalischer Autolyse und beinahe 77 % in der saueren Autolyse. Die Ergebnisse, die wir in der saueren Autolyse erhalten haben legen es sehr nahe, daß autolytische Enzyme eine sehr geringe Aktivität in der Zeitspanne von 10–133 Tagen der Inkubazion zeigen, wenn die Autolyse an dem Verlust des mycelialen Trockengewichtes gemessen wird.

     

Poly- and Monoclonal Antibodies Against Recombinant Rat Brain Calbindin D-28K Were Produced to Map Its Selective Distribution in the Central Nervous System

Many processes in the CNS depend on calcium. The calcium signal is transduced into an intracellular response via Ca2(+)-binding proteins, including calbindin D-28K. In many laboratories, polyclonal antibodies against chicken intestinal calbindin D-28K have been used to study its localization in the brain (normal and degenerated) of various species, including humans, but some of these antisera cross-reacted with other proteins, including calretinin. We purified recombinant rat brain calbindin D-28K to raise antisera in rabbits and purified a recombinant rat-chicken calbindin D-28K hybrid protein to immunize mice for the generation of monoclonal antibodies. These antisera were highly specific for calbindin D-28K, as demonstrated by two-dimensional Western blotting analysis. Immunohistochemical analyses combined with in situ hybridization studies demonstrated that calbindin D-28K in the Purkinje cells of the cerebellum is independent of vitamin D. The antibodies described here will be important tools for studying the regulation of expression of calbindin D-28K and its biological function in the brain and in the PNS.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

Catabolite repression of the synthesis of inducible polygalacturonase and pectinesterase byAspergillus niger sp.

Several recent reports have described large numbers of monoclonal antibodies that cross-react with toxins A and B ofClostridium difficile; this suggests that the toxins share major epitopes. Our results show that monoclonal antibodies (MAb) against other antigens bind nonspecifically to both toxins. Therefore, we believe that the cross-reacting MAb bind by this manner and not by a true immune reaction.     

The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.35-kilobase cDNA clone isolated from a human pituitary library and identified as secretogranin II by various criteria showed that human presecretogranin II is a 617-residue polypeptide containing an NH2-terminal located signal peptide. Secretogranin II lacks the disulfide-bonded loop structure near the NH2 terminus which is conserved in chromogranin A and chromogranin B (secretogranin I), two other widespread constituents of neuroendocrine secretory granules, but like the latter two proteins contains (i) an -E-N/S-L-X-A/D-X-D/E-X-E-L- motif and (ii) multiple potential dibasic cleavage sites for the generation of smaller, perhaps biologically active peptides. Another structural feature that secretogranin II shares with chromogranin A and chromogranin B (secretogranin I) is the abundance of acidic residues all along the polypeptide chain whose negative charge must somehow be neutralized to allow condensation and packaging of the protein into secretory granules. Experiments with purified secretogranin II showed that in the presence of 10 mM calcium at pH 5.2, conditions characteristic of the milieu of neuroendocrine secretory granules, this protein formed aggregates. Immunoglobulin G, a secretory protein that in vivo is not packaged into secretory granules, did not form aggregates under these in vitro conditions and was excluded from the secretogranin II aggregates. Very little aggregation of secretogranin II was observed in the absence of calcium at pH 5.2 or in the presence of calcium at neutral pH. In vivo, ammonium chloride, which is known to neutralize the pH of acidic intracellular compartments, inhibited the packaging of newly synthesized secretogranin II into secretory granules. Our results suggest that the low pH- and calcium-induced aggregation of secretogranin II may be important for the organization of the secretory granule matrix and raise the possibility that aggregation of secretogranin II may be involved in its sorting to secretory granules.     

An antibody against secretogranin I (chromogranin B) is packaged into secretory granules

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.