Tapan K. Biswas

Characterization of β-galactosidases from the germinating seeds of Vigna sinensis

Abstract Four forms of β-galactosidase from the germinating seeds of Vigna sinensis were separated and partially purified by ammonium sulphate precipitation, ion exchange chromatography (DE-52) and gel filtration to more than 50% purity as judged by PAGE. The pH and temperature optima, stability, M r , kinetic parameters and energy of activation of each enzyme have been determined. The four forms differed in their M , s and ionic charges.

USAGE OF NON-CANONICAL PROMOTER SEQUENCE BY THE YEAST MITOCHONDRIAL RNA POLYMERASE

Prior work has demonstrated that a conserved nonanucleotide [5-TATAAGTAA(+2)] promoter sequence is used by the mitochondrial [mt]1 RNA polymerase in Saccharomyces cerevisiae. However, the highly AT-rich yeast mt genome carries many other promoter-like sequences, but only a fraction of them are involved in gene-specific transcription. To examine the sequence variability of this nonanucleotide promoter motif, single or multiple nt substitutions were introduced into the canonical promoter sequence. The transcriptional activity of these altered promoter sequences was examined under the in-vitro reaction conditions. The results presented here determined that several variant promoter sequences (i. e. TAAAAGTAA, TATAAGAAA, TATAAGTAG, TATAAGAAG, TATAAGAGA, TATAAGGGA, TATAAGTGG, TAAAAGTAG) were efficiently used by the mtRNA polymerase. However, a single (i.e. AATAAGTAA, TTTAAGTAA, TATTAGTAA, TATAACTAA, TATAAGGAA, TATAAGTAT) or multiple (TATAGGAAA, TAAAAGGAA, TATAGGGAA, TAAAGGAAA, TAAAGGGAA) nt substitution(s) in other locations drastically reduced mt promoter function. Interestingly, some of these poorly or partially active promoter variants (i.e. TATAAGGAA, TATAAGTAT, TATAAGTCA) became fully functional in the presence of sequence-specific dinucleotide primer. Since dinucleotide primer bypasses the first phosphodiester bond formation in transcription, it is suggested that the -1T-->G, +1A-->C and +2A-->T mutations affect mt transcription at the level of initiation rather than polymerase binding.

The Specific Amino Acid Sequence between Helices 7 and 8 Influences the Binding Specificity of Human Apolipoprotein A-I for High Density Lipoprotein (HDL) Subclasses A POTENTIAL FOR HDL PREFERENTIAL GENERATION

Humans have two major high density lipoprotein (HDL) sub-fractions, HDL2 and HDL3, whereas mice have a monodisperse HDL profile. Epidemiological evidence has suggested that HDL2 is more atheroprotective; however, currently there is no direct experimental evidence to support this postulate. The amino acid sequence of apoA-I is a primary determinant of HDL subclass formation. The majority of the α-helical repeats in human apoA-I are proline-punctuated. A notable exception is the boundary between helices 7 and 8, which is located in the transitional segment between the stable N-terminal domain and the C-terminal hydrophobic domain. In this study we ask whether the substitution of a proline-containing sequence (PCS) separating other helices in human apoA-I for the non-proline-containing sequence (NPCS) between helices 7 and 8 (residues 184–190) influences HDL subclass association. The human apoA-I mutant with PCS2 replacing NPCS preferentially bound to HDL2. In contrast, the mutant where PCS3 replaced NPCS preferentially associated with HDL3. Thus, the specific amino acid sequence between helices 7 and 8 influences HDL subclass association. The wild-type and mutant proteins exhibited similar physicochemical properties except that the two mutants displayed greater lipid-associated stability versus wild-type human apoA-I. These results focus new attention on the influence of the boundary between helices 7 and 8 on the properties of apoA-I. The expression of these mutants in mice may result in the preferential generation of HDL2 or HDL3 and allow us to examine experimentally the anti-atherogenicity of the HDL subclasses.

Temperature sensitive pet mutants in yeast Saccharomyces cerevisiae that lose mitochondrial RNA

SummaryThis is a description of a new class of temperature sensitive pet mutants in Saccharomyces cereviase that lose all or part of their mitochondrial RNA at the restrictive temperature. These mutants fall into 8 different complementation groups, mna1 to mna8, and 2 different classes based on their phenotype. Class I mutations, mna1-1 through mna5-1, cause complete or partial loss of mitochondrial RNA at the restrictive temperature. The mutation, mna1-1, is especially interesting since it causes a loss of both mitochondrial DNA and RNA when the mutant is grown on a fermentable carbon source at the restrictive temperature. However, when this mutant is grown at the permissive temperature on a non-fermentable carbon source then shifted to the restrictive temperature, only the mitochondrial RNA is lost. This indicates that the primary cause for the pet phenotype is due to the loss of mitochondrial RNA and not DNA. Class II mutations, mna6-1 through man8-1, cause complete loss of the 14S rRNA after growth at the restrictive temperature in a fermentable carbon source. This loss appears to be specific for the 14S rRNA, since all other transcripts probed by Northern analysis are normal.

Requirement of different mitochondrial targeting sequences of the yeast mitochondrial transcription factor Mtf1p when synthesized in alternative translation systems

Mitochondrial (mt) translocation of the nuclearly encoded mt transcription factor Mtf1p appears to occur independent of a cleavable presequence, mt receptor, mt membrane potential or ATP [Biswas and Getz (2002) J. Biol. Chem. 277, 45704-45714]. To understand further the import strategy of Mtf1p, we investigated the import of the wild-type and N-terminal-truncated Mtf1p mutants synthesized in two different in vitro translation systems. These Mtf1p derivatives were generated either in the RRL (rabbit reticulocyte lysate) or in the WGE (wheat germ extract) translation system. Under the in vitro import conditions, the RRL-synthesized full-length Mtf1p but not the N-terminal-truncated Mtf1p product was efficiently imported into mitochondria, suggesting that the N-terminal sequence is important for its import. On the other hand, when these Mtf1p products were generated in the WGE system, surprisingly, the N-terminal-truncated products, but not the full-length protein, were effectively translocated into mitochondria. Despite these differences between the translation systems, in both cases, import occurs at a low temperature and has no requirement for a trypsin-sensitive mt receptor, mt membrane potential or ATP hydrolysis. Together, these observations suggest that, in the presence of certain cytoplasmic factors (derived from either RRL or WGE), Mtf1p is capable of using alternative import signals present in different regions of the protein. This appears to be the first example of usage of different targeting sequences for the transport of a single mt protein into the mt matrix.

Purification of acid phosphatase I from germinating seeds of Vigna sinensis

Acid phosphatase I (AP-I) is the major isoform of Vigna acid phosphatase. It is constitutively expressed in seed cotyledons during germination. AP-I was separated from other isoforms and purified to homogeneity by three simple purification steps; (NH4)2SO4 precipitation, and phosphocellulose and DEAE-cellulose column chromatography. The activity of AP-I was not affected by 1 mM Mg2+, Mn2+, Ca2+, Co2+ or Pb2+, but severely inhibited by 1 mM Cu2+, Fe3+, Hg2+, Mo6+ or Zn2+. AP-I has both phosphatase and pyrophosphatase activities, and is highly stable even at 50 degrees.

Expression of the mitochondrial RNase P RNA subunit-encoding gene from a variant promoter sequence in Saccharomyces cerevisiae

Ribonuclease P (RNase P) is a common tRNA processing enzyme that removes the 5 leader sequence of precursor tRNAs. This activity is identified in yeast mitochondria as a separate enzyme from the nuclear RNase P. Like other RNase P enzymes, the mitochondrial (mt) RNase P is also a ribonucleoprotein composed of both RNA and protein subunits. The RNA subunit is encoded by a mt gene and the protein subunit is supplied by a nuclear gene. Earlier studies described one active promoter (FP1) located 5 to the mt tRNA(fMet)-RNase P RNA-tRNA(Pro) gene cluster, so that the mitochondrially encoded RNA subunit was thought to be co-transcribed with two of its substrate tRNAs. However, the results of in vitro transcription and primer extension experiments presented here demonstrate that the mt RNase P RNA subunit-encoding gene (RPM1) is transcribed from a new promoter (SP)which is located between the tRNA(fMet) and RPM1 genes. The sequence [5-TATAAGAA(+1)] of the new promoter varies from the conserved promoter sequence [5-TATAAGTA(+1)], but is one of the sequences that is active in the in vitro transcription assay to determine the consensus promoter sequence [5-T A T/a A A/g/c G T/a/c N(+1)]. This result demonstrates that a naturally occurring variant promoter is used by RPM1. Identification of the novel SP promoter suggests that the synthesis of the mt RNase P RNA subunit might be uncoupled from the expression of upstream tRNA(fMet) gene, and that RPM1 might be independently transcribed in Saccharomyces cerevisiae.

Cationic form of β-galactosidase in the germinating seeds of Vigna sinensis (Linn) Savi☆

The cationic form of beta-galactosidase (EC 3.2.1.23) from the germinating seeds of Vigna sinensis has been separated from its other isoforms by DEAE-cellulose (DE-52) column chromatography and further purified by gel filtration and affinity chromatography. Polyacrylamide gel electrophoresis of the purified enzyme imparted a single protein band. The molecular mass of the enzyme as determined by Sephadex G-150 gel filtration is 58,800 Da. The optimum temperature and the optimum pH are 60 degrees C and 4.5, respectively. Most of the metal ions tested were inhibitory to the enzyme activity. The enzyme has Km for p-nitrophenyl beta-D-galactoside and o-nitrophenyl beta-D-galactoside of 0.56 and 2.0 mM, respectively. The Ki values of galactose and lactose are 2.4 and 70.0 mM, respectively. The energy of activation of PNPG for the enzyme is 10.3 kcal/mol.