Isolation of plaice (Pleuronectes platessa) alpha1-microglobulin: conservation of structure and chromophore.
A cDNA coding for plaice (Pleuronectes platessa) alpha1-microglobulin (Leaver et al., 1994, Comp. Biochem. Physiol. 108B, 275-281) was expressed and purified from baculovirus-infected insect cells. Specific monoclonal antibodies were then prepared and used to isolate the protein from plaice liver and serum. Mature 28.5 kDa alpha1-microglobulin was found in both liver and serum. The protein consisted of an 184 amino acid peptide with a complex N-glycan in position Asn123, one intrachain disulfide bridge and a yellow-brown chromophore. Physicochemical characterization indicated a globular shape with a frictional ratio of 1.37, electrophoretic charge-heterogeneity and antiparallel beta-sheet structure. A smaller, incompletely glycosylated, yellow-brown alpha1-microglobulin as well as a 45 kDa precursor protein were also found in liver. The chromophore was found to be linked to alpha1-microglobulin intracellularly. Recombinant plaice alpha1-microglobulin isolated from insect cells had the same N-terminal sequence, globular shape and yellow-brown color as mature alpha1-microglobulin, but carried a smaller, fucosylated, non-sialylated N-glycan in the Asn123 position. The concentration of alpha1-microglobulin in plaice serum was 20 mg/l and it was found both as a 28.5 kDa component and as high molecular weight components. Thus, the size, shape, charge and color of plaice alpha1-microglobulin were similar to mammalian alpha1-microglobulin, indicating a high degree of structural conservation between fish and human alpha1-microglobulin. The monoclonal antibodies against plaice alpha1-microglobulin cross-reacted with human alpha1-microglobulin, emphasizing the structural similarity. (+info)
Recent and rapid amplification of the sperm basic nuclear protein genes in winter flounder.
The high molecular weight basic nuclear proteins (HMrBNPs), which are tightly bound to sperm chromatin in winter flounder, are made up of imperfect reiterations of simple peptide sequences that contain phosphorylatable DNA-binding motifs. Genomic Southern blots hybridized with probes to the coding and non-coding regions of HMrBNP mRNA showed that HMrBNP sequences form a complex multi-gene family. Previously, one gene (2B) was used to establish an evolutionary link between histone H1 and the HMrBNPs. Further examination of this complex, multi-gene family has now revealed that the majority of the HMrBNP genes are linked as 4.5 kb direct tandem repeats that each contain a 2.8 kb coding region and a 1.7 kb intergenic region (IR). These findings, combined with the cloning of the IR, established that the tandemly repeated genes lack introns and code for the abundant 3 kb HMrBNP mRNAs that produce the prominent 110 kDa HMrBNP. Southern blotting of DNAs from other righteye flounder species showed that HMrBNP multi-gene families were present in closely related species, though with substantial differences in restriction patterns and band intensities, but were not detected in more distantly related flounders. These observations are consistent with recent and rapid elaboration of the HMrBNP gene family. (+info)
Stoichiometry and Na+ binding cooperativity of rat and flounder renal type II Na+-Pi cotransporters.
The stoichiometry of the rat and flounder isoforms of the renal type II sodium-phosphate (Na+-Pi) cotransporter was determined directly by simultaneous measurements of phosphate (Pi)-induced inward current and uptake of radiolabeled Pi and Na+ in Xenopus laevis oocytes expressing the cotransporters. There was a direct correlation between the Pi-induced inward charge and Pi uptake into the oocytes; the slope indicated that one net inward charge was transported per Pi. There was also a direct correlation between the Pi-induced inward charge and Na+ influx; the slope indicated that the influx of three Na+ ions resulted in one net inward charge. This behavior was similar for both isoforms. We conclude that for both Na+-Pi cotransporter isoforms the Na+:Pi stoichiometry is 3:1 and that divalent Pi is the transported substrate. Steady-state activation of the currents showed that the Hill coefficients for Pi were unity for both isoforms, whereas for Na+, they were 1.8 (flounder) and 2.5 (rat). Therefore, despite significant differences in the apparent Na+ binding cooperativity, the estimated Na+:Pi stoichiometry was the same for both isoforms. (+info)
Protein kinase C activators induce membrane retrieval of type II Na+-phosphate cotransporters expressed in Xenopus oocytes.
1. The rate of inorganic phosphate (Pi) reabsorption in the mammalian kidney is determined by the amount of type II sodium-coupled inorganic phosphate (Na+-Pi) cotransport protein present in the brush border membrane. Under physiological conditions, parathyroid hormone (PTH) leads to an inhibition of Na+-Pi cotransport activity, most probably mediated by the protein kinase A (PKA) and/or C (PKC) pathways. 2. In this study, PKC-induced inhibition of type II Na+-Pi cotransport activity was characterized in Xenopus laevis oocytes using electrophysiological and immunodetection techniques. Transport function was quantified in terms of Pi-activated current. 3. Oocytes expressing the type IIa rat renal, type IIb flounder renal or type IIb mouse intestinal Na+-Pi cotransporters lost > 50 % of Pi-activated transport function when exposed to the PKC activators DOG (1,2-dioctanoyl-sn-glycerol) or PMA (phorbol 12-myristate 13-acetate). DOG-induced inhibition was partially reduced with the PKC inhibitors staurosporine and bisindolylmaleimide I. Oocytes exposed to the inactive phorbol ester 4alpha-PDD (4alpha-phorbol 12,13-didecanoate) showed no significant loss of cotransporter function. 4. Oocytes expressing the rat renal Na+-SO42- cotransporter alone, or coexpressing this with the type IIa rat renal Na+-Pi cotransporter, showed no downregulation of SO42--activated cotransport activity by DOG. 5. Steady-state and presteady-state voltage-dependent kinetics of type II Na+-Pi cotransporter function were unaffected by DOG. 6. DOG induced a decrease in membrane capacitance which indicated a reduction in membrane area, thereby providing evidence for PKC-mediated endocytosis. 7. Immunocytochemical studies showed a redistribution of type II Na+-Pi cotransporters from the oolemma to the submembrane region after DOG treatment. Surface biotinylation confirmed a DOG-induced internalization of the transport protein. 8. These findings document a specific retrieval of exogenous type II Na+-Pi cotransporters induced by activation of a PKC pathway in the Xenopus oocyte. (+info)
Structure and expression of the highly repetitive histone H1-related sperm chromatin proteins from winter flounder.
In the late stages of spermatogenesis, winter flounder produce a family of high molecular mass (80-200 kDa) basic nuclear proteins (HMrBNPs) that combine with the normal complement of histones to produce condensed sperm chromatin with an increased nucleosomal repeat length. The HMrBNPs have a biased amino-acid composition in which Arg, Ser, Lys and Pro are abundant because of their presence in many simple peptide repeats. The organization of these repeats was deduced by cDNA cloning. The predominant repeating units are related 26- and 30-amino-acid sequences that in turn are linked by 6-amino-acid spacers to form 58- and 62-amino-acid repeats. Subsets of these repeats are also present, such as a dispersed 20-amino-acid repeat and a tandem array of nine heptapeptides at the C-terminus. The HMrBNPs appear to have evolved from an extreme H1 variant that has an N-terminal tail of HMrBNP-like sequence linked to an H1 globular region. Based on sequences of the most abundant HMrBNP cDNAs, and the lack of hybridization between HMrBNP mRNAs and a DNA probe for the H1 globular region, the latter domain appears to have been lost during expansion and amplification of the HMrBNP-like repeats. Transcripts of the HMrBNP and H1 variant genes are present in testis RNAs only during the mid-spermatid stage of spermatogenesis, at the same time that HMrBNPs in their highly phosphorylated form first appear in the nucleus. Judging by the lack of a lag between HMrBNP mRNA synthesis and translation, the mRNAs for these highly basic proteins are not stored for any length of time. Instead, the deposition of HMrBNPs onto DNA, which coincides with the major reorganization and silencing of the chromatin, may be controlled by dephosphorylation. (+info)
NO2- uptake and HCO3- excretion in the intestine of the European flounder (Platichthys flesus).
Ion transport across isolated intestinal segments from the European flounder (Platichthys flesus) was studied with the primary aim of evaluating the mechanisms of nitrite (NO2-) uptake and HCO3- excretion. A double-radiolabelling technique was applied to monitor unidirectional Cl- and Na+ influx. Furthermore, net fluxes of NO2-, HCO3-, Cl-, Na+ and water were recorded. NO2- uptake was inhibited by mucosal application of bumetanide (10(-)4 mol l-1) but not DIDS (10(-)3 mol l-1), suggesting that NO2- is transported across the intestine via the Na+/K+/2Cl- cotransporter rather than via a Cl-/HCO3- exchanger. In addition to transport via the Na+/K+/2Cl- cotransporter, NO2- uptake may also occur through the Na+/Cl- cotransporter and by conductive transport. NO2- and Cl- influx rates seemed to reflect their mucosal concentrations, and NO2- did not influence unidirectional influx or net flux of Cl-. HCO3- efflux was significantly reduced in the presence of 10(-)3 mol l-1 DIDS in the mucosal solution. This may indicate the presence of an apical Cl-/HCO3- exchanger in the intestinal epithelium, which would not comply with the current model of HCO3- excretion in the intestine of marine teleost fish. An alternative model of HCO3- excretion across the intestinal epithelium is proposed. (+info)
Expression cloning and characterization of a novel sodium-dicarboxylate cotransporter from winter flounder kidney.
A cDNA coding for a Na+-dicarboxylate cotransporter, fNaDC-3, from winter flounder (Pseudopleuronectes americanus) kidney was isolated by functional expression in Xenopus laevis oocytes. The fNaDC-3 cDNA is 2384 nucleotides long and encodes a protein of 601 amino acids with a calculated molecular mass of 66.4 kDa. Secondary structure analysis predicts at least eight membrane-spanning domains. Transport of succinate by fNaDC-3 was sodium-dependent, could be inhibited by lithium, and evoked an inward current. The apparent affinity constant (Km) of fNaDC-3 for succinate of 30 microM resembles that of Na+-dicarboxylate transport in the basolateral membrane of mammalian renal proximal tubules. The substrates specific for the basolateral transporter, 2,3-dimethylsuccinate and cis-aconitate, not only inhibited succinate uptake but also evoked inward currents, proving that they are transported by fNaDC-3. Succinate transport via fNaDC-3 decreased by lowering pH, as did citrate transport, although much more moderately. These characteristics suggest that fNaDC-3 is a new type of Na+-dicarboxylate transporter that most likely corresponds to the Na+-dicarboxylate cotransporter in the basolateral membrane of mammalian renal proximal tubules. (+info)
Studies of a putative ice-binding motif in winter flounder skin-type anti-freeze polypeptide.
Winter flounder contains two distinct anti-freeze protein isoforms, which are the liver-type extracellular anti-freeze proteins and the skin-type intracellular anti-freeze protein. The skin-type anti-freeze proteins exhibit lower anti-freeze activities than the liver-type isoforms and this might be due to their lacking complete ice-binding motifs. One of the skin-type anti-freeze proteins, skin-type anti-freeze protein-3, does contain putative overlapping ice-binding motifs with the sequences '-K-DT-' and '-DT-K-'. Synthetic anti-freezes containing 0-3 repeats of the '-DT-K-' motif were tested for stability and activity. Loss of the single '-DT-K-' of skin-type anti-freeze protein-3 increases the anti-freeze activity and increasing the number of motifs to two or three lowers the activity. The decrease in activity with an increasing frequency of the motif correlates with a decrease in the helical content of these peptides at 0 degrees C. (+info)