Structural analysis of homologous repeated domains in alpha-actinin and spectrin

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.     

Complete nucleotide sequence of the Escherichia coli gdhA gene

The DNA sequence of the gdhA gene of Escherichia coli K12, which encodes the 447 amino acid polypeptide subunit of NADP-specific glutamate dehydrogenase, is presented. The deduced protein sequence is strongly homologous to the corresponding enzyme of the eukaryotic fungus Neurospora crassa. The upstream DNA sequence includes several overlapping promoter consensus sequences. The downstream DNA sequence contains inverted repeats, predicted as forming long stable stem-loop structures in RNA, homologous to those found in several enterobacterial intergenic regions.     

Effect of food on the growth of carcase, liver and ovary in female Gasterosteus aculeatus L.

The effect of ration size on the growth of the carcase, liver and ovary in female three-spined sticklebacks, Gasterosteus aculeatus L., was studied experimentally at monthly intervals throughout the annual reproductive cycle. Temperature and photoperiod during each experiment corresponded to the average, natural conditions for that month.
Growth of the carcase was sensitive to ration in all months. At a given ration, the growth rate of the carcase was relatively constant. The water content of the carcase was inversely correlated with ration size. Growth of the liver was also sensitive to ration, but the sensitivity varied in different phases of the reproductive cycle. High rates of weight loss occurred in the liver under starvation, but at maximum rations the liver showed high rates of weight gain particularly in the post-spawning period. Growth of the ovary was relatively insensitive to ration but directly reflected the changes occurring naturally in the reproductive cycle.
It is suggested that the liver acts as a buffer between the carcase and the ovary and this helps to insulate the ovary from the immediate effects of fluctuations in food supply.
The growth rates of the carcase and liver may provide useful indices of the rate of food consumption in natural populations. Although, if the liver is used, its growth has to be interpreted in the context of the annual reproductive cycle.     

Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca2+-binding sites.

2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 micro m, respectively, measured at pH 7.2). The Ca2+-ATPase is also more potently inhibited at lower pH (IC50 = 70 micro m for SERCA1A at pH 6). 2-APB decreases the affinity for Ca2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca2+-ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca2+ to their binding sites.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. The tryptic peptides.

The NADP-specific glutamate dehydrogenase of Neurospora crassa was digested with trypsin, and peptides accounting for 441 out of the 452 residues of the polypeptide chain were isolated and substantially sequenced. Additional experimental detail has been deposited as Supplementary Publication SUP 50052 (11 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem J. (1975) 145, 5.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptic and chymotryptic peptides and the complete sequence.

Peptic and chymotryptic peptides were isolated form the NADP-specific glutamate dehydrogenase of Neurospora crassa and substantially sequenced. Out of 452 residues in the polypeptide chain, 265 were recovered in the peptic and 427 in the chymotryptic peptides. Together with the tryptic peptides [Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 749-755], these establish the complete sequence of the chain, including the acid and amide assignments, except for seven places where overlaps are inadequate. These remaining alignments are deduced from information on the CNBr fragments obtained in another laboratory [Blumenthal, K. M., Moon, K. & Smith, E. L. (1975), J. Biol. Chem. 250, 3644-3654]. Further information has been deposited as Supplementary Publication SUP 50054 (17 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1975) 145, 5.     

The inter-spawning interval of the female Three-spined stickleback, Gasterosteus aculeatus

Female Three-spined sticklebacks spawn several times in a breeding season, at intervals of a few days. The changes that take place in females during the interval between successive spawnings were analysed. The average time between spawnings was 87.5 hours. Between spawnings females increased in weight by between 15 and 30%. The bulk of this weight increase was lost when the eggs were spawned. The increase in weight was because of an increase in weight of the ovaries, which showed an increase in both dry matter and water content. After spawning the ovaries formed 8 to 9% of the total body weight, and this increased to up to 24% just prior to the next spawning. The ovaries were maximally hydrated immediately after spawning. There were no significant changes in the calorific value of the ovaries, but the calorific value of the rest of the body was a minimum just after spawning. It was estimated that between spawnings females were making a net gain of energy at an average rate of 2 cal/hour. After spawning, courtship behaviour by the females waned, but recovered over the last half of the interval between spawnings. The inter-spawning interval of the Three-spined stickleback provides a useful situation in which physiological and nutritional aspects of reproduction in a teleost fish can be studied.     

Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers.

The am1 and am3 mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase show complementation activity in hybrid hexamers. A freeze-thaw hybridization method was used to construct hybrids from purified enzymes and the products were separated into species of different monomer ratio by affinity chromatography. Hexamers with am1:am3 ratios of 1:5, 2:4, 3:3, 4:2 and 5:1 were all recovered as resolved or partially resolved peaks in quantities approximating to a binomial distribution. Reassociation of monomers during the hybridization process was random, except for some differential loss of am3 protein by precipitation and an apparent absence of reassociated am1 homohexamers. Complementation activity was shown by hybrids of all five monomer ratios, owing to activation of am3 monomers by conformational constraints arising from the intrinsically inactive am1 monomers. The activating effect of such constraints was greatest in hexamers containing only a single am1 monomer and least in the 5 am1:1am3 species. When fully activated by L-glutamate all am3 monomers were equivalent in intrinsic catalytic activity, irrespective of the number of am1 monomers per hexamer.