Molecular basis for the special properties of proteins and enzymes from Halobacterium marismortui

Abstract Three proteins from Halobacterium marismortui , malate dehydrogenase (hMDH), glutamate dehydrogenase (hGDH) and ferredoxin (hFD) were purified and characterized with respect to their molecular masses, amino acid composition and, for hFD only, primary structure. Striking features of halophilic proteins are: the high excess of acidic over basic residues; acidic clusters in the sequence. Low-salt concentration causes inactivation and changes in structural parameters of hMDH and hGDH. Reactivation of hMDH involves long-lived stable intermediates. The salt concentration optimum of enzymic activity is independent of salt nature. The high capacity of halophilic proteins to retain water and salt is due to unique molecular properties, studied by physico-chemical techniques.     

Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation

Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups.     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Isolation and characterization of a human interleukin 2 gene

An interleukin 2 (IL-2) gene was isolated from a Charon 4A human gene library. Electron microscopic examination of 15 heteroduplexes formed between the genomic DNAs and the IL-2 cDNAs demonstrated that the size of the IL-2 gene is about 5.1 +/- 0.5 kb and that there are at least two introns in this gene. Nucleotide sequence of the 5' flanking region of the IL-2 gene showed a homology with that of the corresponding region of the human immune interferon gene.     

Partial purification,subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides (M_r = 30000 and 28000) in a reaction center complex of M_r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.     

Light-driven proton transport of bacteriorhodopsin incorporated into long-term stable liposomes of a polymerizable sulfolipid

The chromoprotein bacteriorhodopsin from Halobacterium halobium has been incorporated into liposomes made of a fully synthetic, polymerizable lipid. Bacteriorhodopsin is found to be active in these polymer liposomes. The advantage in the use of such polymer systems concerning long-term stability in comparison with liposomes made of natural lipid is demonstrated.     

Sexual differentiation of rat liver carbonic anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.     

Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate-Early Genes c-fos and c-jun in Rat Cerebral Cortex

Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate-early genes c-fos and c-jun, also referred to as nuclear proto-oncogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c-fos and c-jun to an acute, "test" seizure was examined. Four hours after a single ECS, the induction of c-fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c-fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to "reset" the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time-dependent decrease in the expression of c-fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8-10 days of treatment, at which time the induction of c-fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c-jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8-10 days) ECS block the induction of c-fos may differ.     

The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

Differential Expression of mRNA and Protein Encoding Retinal and Pineal S-Antigen During the Light/Dark Cycle

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.