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A Przybyla

Publications and source records attributed to A Przybyla.

15 recordsLinked to original sources
PubMed1990

Primary structure of the thermostable formyltetrahydrofolate synthetase from Clostridium thermoaceticum.

The complete nucleotide sequence of the Clostridium thermoaceticum formyltetrahydrofolate synthetase (FTHFS) was determined and the primary structure of the protein predicted. The gene was 1680 nucleotides long, encoding a protein of 559 amino acid residues with a calculated subunit molecular weight of 59,983. The initiation codon was UUG, with a probable ribosome binding site 11 bases upstream. A putative ATP binding domain was identified. Two Cys residues likely to be involved in subunit aggregation were tentatively identified. No characterization of the tetrahydrofolate (THF) binding domain was possible on the basis of the sequence. A high level of amino acid sequence conservation between the C. thermoaceticum FTHFS and the published sequences of C. acidiurici FTHFS and the FTHFS domains of the Saccharomyces cerevisiae C1-THF synthases was found. Of the 556 residues shared between the two clostridial sequences, 66.4% are identical. If conservative substitutions are allowed, this percentage rises to 75%. Over 47% of the residues shared between the C. thermoaceticum FTHFS and the yeast C1-THF synthases are identical, 57.4% if conservative substitutions are allowed. Hydrophobicity profiles of the C. acidiurici and C. thermoaceticum enzymes were very similar and did not support the idea that large hydrophobic domains play an important role in thermostabilizing the C. thermoaceticum FTHFS.

Amino Acid Sequence↗
PubMed1988

Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase.

Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592. The clone (CRL47) contained a 9.5 kb EcoRI fragment of C. thermoaceticum DNA ligated into pBR322. It produced catalytically active, thermostable FTHFS, that was not found in E. coli SK1592 containing native pBR322. The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C. thermoaceticum FTHFS. The specific activities (mumol.min-1.mg-1) of FTHFS in cell free extracts of CRL47 were 28-89 when assayed at 50 degrees C and pH 8. This was from 3-10-fold higher than in C. thermoaceticum extracts. FTHFS was purified to homogeneity from CRL47. The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C. thermoaceticum.

Cloning, Molecular↗
PubMed1988

Expression of the 31-kD stress protein in rat myoblasts and hepatocytes.

Rat tissue cultures cells respond to stress by inducing the synthesis of about 20 proteins, including two low-molecular-weight species of about 31 kD and 27 kD. We have cloned a cDNA for the 31-kD protein. This protein is induced in myoblasts and hepatoma cells in response to a 43 degrees C heat shock, or exposure to sodium arsenite or cadmium chloride salts. Furthermore, this protein is superinduced in hepatoma cells conditioned to grow in cadmium and zinc salts when they are exposed to a standard sodium arsenite stress. Induction of the gene encoding the 31-kD protein has been characterized as follows: (i) Transcripts accumulate maximally with similar kinetics when myoblasts are induced with either heat shock or sodium arsenite; (ii) accumulation of transcripts decays to preinduction levels within 4 hr of a heat shock, but requires more than 8 days after sodium arsenite stress; (iii) basal levels of transcript are reduced when myoblasts are cultured in the presence of steroid hormones; and (iv) stress induction is virtually abolished once myoblasts have differentiated.

Animals↗
PubMed1987

Molecular cloning of human cathepsin G: structural similarity to mast cell and cytotoxic T lymphocyte proteinases.

Human cathepsin G is a serine proteinase with chymotrypsin-like specificity found in both polymorphonuclear leukocytes (neutrophils) and the U937 leukemic cell line. Utilizing RNA from the latter, we have constructed a cDNA library in lambda gt11 and isolated a clone which apparently codes for the complete amino acid sequence of this enzyme. Analysis of the sequence reveals homology with rat mast cell proteinase II (47%) but a greater degree of identity (56%) with a product of activated mouse cytotoxic T lymphocytes. The close relationship between the three proteins indicates similarities in substrate specificity and in biosynthesis which we predict involves removal of a two amino acid activation peptide during or just before packaging into their respective storage granules.

Amino Acid Sequence↗
PubMed1984

Nuclear localization and phosphorylation of three 25-kilodalton rat stress proteins.

The nuclear localization and phosphorylation of three 25-kilodalton rat myoblast stress proteins were examined. Data obtained in these analyses led to the following conclusions: (i) all three proteins become localized in the nucleus of stressed cells, (ii) two of the proteins are modified by phosphorylation, and (iii) phosphorylation occurs exclusively on serine residues.

Animals↗
PubMed1983

Phosphorylation pattern of a 25 Kdalton stress protein from rat myoblasts.

Phosphorylation of a 25 Kdalton nuclear stress protein from rat muscle was examined by two-dimensional gel electrophoresis and one-dimensional peptide mapping. These studies show that three 25 Kdalton stress proteins found in stressed rat myoblasts are actually the same protein with charge variation brought about by multiple phosphorylations. Furthermore, the predominant charge variant of 25 Kdalton protein found in cells is dependent on the intensity of the stress applied to cells.

Animals↗
PubMed1983

Arsenate induces stress proteins in cultured rat myoblasts.

The induction of stress proteins was examined in rat myoblast cultures by two-dimensional gel electrophoresis. Data obtained by this analysis led to the following observations. (a) Arsenate, which behaves as a phosphate analogue in cellular phosphate-transfer reactions, stresses cultured rat cells and induces the synthesis of a unique set of proteins. (b) Most of the proteins synthesized after the addition of arsenate are identical to proteins synthesized in rat myoblasts in response to heat shock or arsenite stress. (c) However, both arsenic salts induce the synthesis of two unique proteins not induced by heat shock. (d) Five 25-30-kdalton stress proteins of rat cells do not contain methionine residues. (e) A majority of the proteins synthesized in stressed myogenic cells are also induced by stress in other rat cells such as hepatoma cells, pituitary tumor cells, and fibroblasts. The 25-30-kdalton stress-related proteins identified in myogenic cells, on the other hand, are induced in fibroblasts but not hepatoma or pituitary cells.

Animals↗
PubMed1981

Optimal conditions for cell-free synthesis of elastin.

Optimal conditions for the translation of elastin mRNA1 in a mRNA-dependent rabbit reticulocyte lysate were determined. Using total RNA isolated from embryonic chick aortae as the source of exogenous RNA, the concentrations of various components present in the translation assay were varied and the effect on elastin synthesis quantitated by immunoprecipitation. Components examined included: magnesium acetate, potassium chloride, spermidine, creatine phosphate, ATP, and GTP. In addition, it was found that heating of the RNA prior to translation significantly enhanced total protein synthesis, elastin synthesis, and the synthesis of proteins possessing molecular weights of greater than 80,000.

Cell-Free System↗
PubMed1980

Translation of chick aortic elastin messenger ribonucleic acid. Comparison to elastin synthesis in chick aorta organ culture.

Studies were undertaken to define the molecular size of the elastin primary gene product. Translation of chick aortic messenger ribonucleic acid (mRNA) in an mRNA-dependent reticulocyte lysate resulted in the synthesis of two major proteins of 70 000 and 73 000 molecular weights. Both proteins were shown to be soluble forms of elastin by isotope incorporation, immunoprecipitation, collagenase and cyanogen bromide sensitivity, and two-dimensional gel electrophoresis. The 70 000-dalton protein behaves similarly to authentic tropoeleastin in sodium dodecyl sulfate gel electrophoresis. There was no evidence for a high molecular weight form of soluble elastin, although procollagen chains were indirectly identified among the aortic mRNA-directed translation products. The same molecular size proteins were also seen in organ cultures of chick embryonic aortas labeled with [3H]valine. However, the 73 000-dalton protein was not extractable in a neutral salt buffer but was found only if the aortas were extracted with urea in the presence of reducing and alkylating reagents. The results from these studies suggest that elastin is first synthesized as two distinct polypeptide chains which differ slightly in size and overall charge. The possibility that these two proteins may associate posttranslationally to form a dimer prior to secretion is postulated to explain the existence of a putative proelastin molecule seen in other systems.

Animals↗
PubMed1977

Messenger RNA for myosin polypeptides: isolation from single myogenic cell cultures.

Messenger RNA which stimilates the synthesis of myosin heavy chain in a reticulocyte lysate has been isolated from single myogenic cell cultures. Specific myosin polypeptides have been identified by immunoprecipitation with an antibody made to purified adult chicken skeletal muscle myosin. This mRNA binds to oligo(dT)-cellulose, and an active fraction from sucrose gradients migrates as 26S on formamide-polyacrylamide gels. The relative amount of this RNA increases dramatically at the time of terminal differentiation.

Cell Differentiation↗
PubMed1974

Myosin heavy chain messenger RNA from myogenic cell cultures.

The appearance of messenger RNA for myosin heavy chains in chick-embryo myogenic cell cultures was investigated. Total polyribosomes were isolated from cultures at various times of development and were purified in sucrose step gradients. These polysomes were either extracted with phenol or were treated with puromycin. The ribonucleoprotein particles and ribosomal subunits released by puromycin were fractionated on sucrose gradients. RNA from polysomes or from puromycin-dissociated subunits was fractionated on oligo(dT)-cellulose columns, and the bound and unbound RNA was assayed for activity of myosin heavy chain messenger RNA in a rabbit reticulocyte cell-free system. RNA stimulating myosin heavy-chain synthesis was found predominantly in the unbound fractions of the oligo(dT)-cellulose columns. After puromycin treatment of polysomes, the myosin heavy chain messenger RNA, which sediments at 18-26 S, was associated with a ribonucleoprotein particle sedimenting between 30 and 40 S. Myosin heavy chain messenger RNA was obtained from cultures containing well-developed myotubes and from cultures undergoing myogenic cell fusion. This messenger RNA was not detectable in early, unfused cultures, or in later cultures in which myogenic cell fusion had been prevented by treatment with ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These experiments demonstrate that messenger RNA for myosin heavy chains becomes associated with ribosomes only after myogenic cell fusion has begun.

Acetates↗