Further characterization and amino acid sequence of m-type thioredoxins from spinach chloroplasts

The complete primary structure of m-type thioredoxin from spinach chloroplasts has been sequenced by conventional sequencing including fragmentation, Edman degradation and carboxypeptidase digestion. As already reported [Tsugita, A., Maeda, K. & Schürmann, P. (1983) Biochem. Biophys. Res. Commun. 115, 1-7] these thioredoxins contain the same active-site sequence as thioredoxins from other sources. Based on the amino acid sequence thioredoxin mc contains 103 residues, has a relative molecular mass of 11425 and a molar absorption coefficient at 280 nm of 19 300 M-1 cm-1. The spinach thioredoxin mc has an overall homology of 44% with the thioredoxin from Escherichia coli mainly due to differences in the N-terminal and C-terminal regions.     

Immunological studies on the interaction of polyadenylic acid and human liver ribonuclease

The effect of polyadenylic acid, a potent inhibitor of mammalian and bacterial RNAses, on the binding of human liver RNAse to its antibody was studied. To do this, a human liver RNAse antibody was immobilized on Sepharose 4B. Examination of the ability of the enzyme to bind to the immobilized anti-RNAse in the presence or absence of polyadenylic acid indicated that enzyme-antibody binding was more sensitive to the presence of polyadenylic acid than was enzyme activity. Furthermore, the effect of polyadenylic acid on enzyme-antibody binding was specific since neither polycytidylic acid nor polyuridylic acid had much effect on the antigenicity of the enzyme. The metal cation, Mg2+, and the polyamine, spermidine, but not putrescine, readily reversed the effects of polyadenylic acid on enzyme-antibody binding.     

Mitochondrial polyadenylic acid-containing RNA: localization and characterization

The RNA from the mitochondrial fraction of animal cells contains a polyadenylic acid sequence, approximately 55 nucleotides in length, which migrates at about 4 S in gel electrophoresis and which is attached to high molecular weight RNA. The experiments reported here indicate that: (a) the 4 S poly(A) sequence is found only in the mitochondrial fraction; (b) the RNA containing 4 S poly(A) is located within structures (presumably mitochondria) which protect it from pancreatic ribonuclease; (c) no RNA containing the longer poly(A) of nuclear origin appears to be located in mitochondria; (d) the 4 S poly(A), but not the longer poly(A), is attached to RNA which hybridizes to mitochondrial DNA; and (e) this poly(A) sequence is located at the 3′ end of the RNA molecule.The poly(A)-containing RNA can be isolated by affinity to oligodeoxyribothymidylic acid cellulose and resolved into approximately eight distinct species by acrylamide gel electrophoresis. These may correspond to individual mitochondrial messenger RNA molecules.     

Isolation of RNA containing a polyadenylic acid sequence by chromatography on unmodified cellulose]

A new modification of the procedure of the isolation of polyA-containing RNAs is worked out, which makes possible to isolate this RNA fraction free of considerable contamination with rRNA. The administration of 0.0001 M EDTA-Na2 provides the absence of RNA aggregation and prevents non-specific RNA binding on cellulose columns, which takes place when more high EDTA-Na2 concentrations in elution solutions are applied. Under these conditions synthetic polyA in model experiments practically completely binds with cellulose in a broad range of concentrations. It permits to use the procedure described for the preparative isolation of RNA fractions, containing polyA sequences.     

A rat beta-interferon-induced mRNA: sequence characterization.

Polymerase chain reaction amplification of a cDNA derived from rat aortic smooth muscle cells, using sequences from conserved regions of the intramembrane domains of adrenergic receptors as primers, yielded the clone, rat8. This clone possesses a high degree of sequence similarity to a series of human interferon (IFN)-inducible genes. The rat8 sequence is 70% similar to that derived from the human alpha-IFN-induced gene, 9-27; there is 66% similarity between the deduced amino acid sequences encoded by the rat and the human genes. The rat homologue hybridizes with many bands in Southern analysis of rat DNA, suggesting that it is a member of a large multigene family.     

Kinetic studies on the depolymerization of polyadenylic acid by ribonuclease A.

Studies were conducted on the depolymerization of polyadenylic acid (poly (A)) by RNAse A (EC 3.1.4.22) depending on the pH (pH 5-8). The results showed that depending on the pH, the ratio Vmax/Km was analogous to that described earlier for nucleoside-2', 3'-cyclophosphates and dinucleoside phosphates. This indicates that depolymerization of poly (A), transesterification and hydrolysis of specific substrates is achieved by the same ionizing groups of the enzyme with pKa 5.4 and pKb 6.4. The rate of degradation of poly (A) is also influenced by the state of adenine ionization, the protonation of which leads to the formation of a double helical poly (A), and does not serve as a substrate for RNAse A. The low rate for the depolymerization of poly (A) in the presence of RNAse A is related to a decrease in the turnover number of the enzyme, and an increase in the molecular weight of the enzyme (RNAse dimer), leads to a decrease in the Km, and does not effect Vmax. This indicates that the rate of depolymerization of polynucleotides is determined by orientation of factors. On the basis of the comparison of the resultant kinetic data, and the structure of the enzyme inhibitory complexes of RNAse S, which were studied by the method of x-ray structural analysis, a conclusion was reached on the kinetic characteristics of RNAse A specificity with respect to polymeric substrates, which is determined by the orinetation of the ribose phosphate relative to the catalytic groups of the active site.     

Newly formed mRNA lacking polyadenylic acid enters the cytoplasm and the polyribosomes but has a shorter half-life in the absence of polyadenylic acid.

Labeled adenovirus type 2 nuclear RNA molecules from cells treated with 3'-deoxyadenosine (3'dA) were earlier reported to lack polyadenylic acid [poly(A)], but to be correctly spliced in the nucleus (M. Zeevi et al., Cell 26:39-46, 1981). We have now found that the shortened mRNA molecules, lacking poly(A), can also be found in the cytoplasm of 3'dA-treated cells in association with the polyribosomes. In addition, the accumulation of labeled, nuclear adenovirus-specific RNA complementary to early regions 1a, 1b, and 2 of the adenovirus genome was approximately equal in 3'dA-treated and control cells. At the initial appearance of newly labeled adenovirus type 2 RNA (10 min) in the cytoplasm, there was one-half as much labeled RNA in 3'dA-treated cells as in the control. However, control cells accumulated additional mRNA in the cytoplasm very rapidly in the first 40 min of labeling, whereas the 3'dA-treated cells did not. Therefore, it appears that the correctly spliced, poly(A)- mRNA molecules that are labeled in the presence of 3'dA can be transported from the nucleus with nearly the same frequency and the same exit time as in control cells and can be translated in the cytoplasm but have a much shorter half-life than the poly(A)+ mRNA molecules from control infected cells. From these results it is suggested that the role of poly(A) may be entirely to increase the longevity of cytoplasmic mRNA.     

Further characterization and structural studies on human placenta lectin

The properties of a previously purified beta-galactoside-binding lectin of human placenta were studied in detail. Isoelectric focusing gave multiple bands around pH 4.9, although the lectin preparation was homogenous in SDS-polyacrylamide gel electrophoresis. High-performance gel chromatography suggested that the lectin exists mainly as the monomer and that a small fraction forms a dimer. From all the criteria examined, human placenta lectin resembles one of the chick lectins obtained from embryonic skin or adult intestine (subunit molecular weight: 14,000). The lectin was inactivated by thiol-modifying reagents, p-chloromercuribenzoic acid and N-ethylmaleimide. Reduced and carboxymethylated lectin contained five carboxymethylated cysteines per subunit, and five free thiol groups were titrated by using 5,5'-dithio-bis(2-nitrobenzoic acid). Preliminary sequence analysis showed the presence of a region highly homologous to the corresponding region of the chick lectin (13 identical residues out of 18 from number 70 to 87 of the chick lectin), suggesting a close evolutionary relation between these lectins and the importance of this conserved region in the function of the lectins.     

Further characterization of collagen galactosyltransferase from chick embryos.

Optimum extraction of collagen galactosyltransferase activity from chick embryos required relatively high concentrations of detergent and salt. The activity was inhibited by concanavalin A, and the enzyme had a high affinity for columns of this lectin coupled to agarose; these results suggest the presence of carbohydrate units in the enzyme molecule. Collagen galactosyltransferase was highly labile, and only 1% of the originally bound enzyme activity could be eluted from the concanavalin A-agarose column with a buffer containing methyl glucoside and ethylene glycol. The purification of the activity over the original supernatant of chick embryo homogenate was 250-300-fold, with the optimum reaction conditions for the purified transferase differing somewhat from those for crude enzyme preparations. The reaction was inhibited by glucose-free basement-membrane collagen, UDP and galactosylhydroxylsine, and also by Co2+ and a number of compounds resembling UDP-galactose. Hydroxylysine was also a weak inhibitor. Immobilized hydroxylysine and UDP-glucuronic acid did not bind the collagen galactosyltransferase, but the enzyme was retarded in a column of UDP-galacturonic acid linked to agarose.     

Partial amino acid sequence of human thyroxine-binding globulin. Further evidence for a single polypeptide chain.

The NH₂-terminal amino acid of highly purified thyroxine-binding globulin has been identified by dansyl chloride, cyanate and Edman degradation methods. All three gave alanine as the only amino terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98 – 1.05 mole of alanine per mole of protein, respectively. These data further indicate that thyroxine-binding globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-Val-Thr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-Ala-(Ser)-Leu-Tyr- A computer search revealed no homology of the NH₂-terminal segment of thyroxine-binding globulin with human prealbumin. The NH₂-terminal portion of prealbumin contains part of the thyroxine binding site.     

Studies on the stability of tritium-labeled polyuridylic acid and polyadenylic acid

Results are presented on the stability of high specific activity [uridylate-5,6-³H]polyuridylic acid and [adenylate-2,8-³H]polyadenylic acid stored under various conditions. Polyacrylamide disc gel electrophoresis in the presence of SDS was used to assess qualitatively the change in molecular weight distribution of the polynucleotides stored under different conditions. Products stored for a period of months in ethanol; water solution [1:1, $\text{v}\text{v}$] were found to have a significantly slower rate of decomposition than polynucleotides stored in frozen aqueous solution or as lyophilized solid.     

Further characterization of the antithrombin-binding sequence in heparin

An octasaccharide with high affinity for antithrombin, isolated after partial deaminative cleavage of heparin and previously found to have the following predominant structure

Full-size image (6K)

, has been studied further. High-voltage, paper electrophoresis of the ³H-labelled disaccharides obtained by deamination with HNO₂ (pH 1.5) followed by reduction with Na[³H]BH₄ showed 25% of mono-O-sulfated components, in addition to l-iduronic acid(2-O-SO₃)-2,5-anhydro-d-[³H]mannitol(6-O-SO₃). The monosulfated disaccharides were identified by high-pressure, ion-exchange chromatography as l-iduronic acid(2-O-SO₃)-2,5-anhydro-d-[³H]mannitol, l-iduronic acid-2,5-anhydro-d-[³H]mannitol(6-O-SO₃), and d-glucuronic acid-2,5-anhydro-d-[³H]-mannitol(6-O-SO₃). These components originated from the reducing, terminal disaccharide residue (units 7 and 8), as indicated by selective labelling with Na[³H]BH₄. The structural variability within this region suggests that it is not part of the antithrombin-binding sequence. Neither enzymic removal of the non-sulfated l-iduronic acid unit 1 nor N-deacetylation (by hydrazinolysis) at unit 2 had any significant effect on the affinity of the octasaccharide for antithrombin. However, removal of the disaccharide corresponding to units 1 and 2, by selective deamination of the N-deacetylated octasaccharide, yielded a low-affinity hexasaccharide. In addition, a high-affinity deamination product was formed, presumably an octasaccharide containing a 6-sulfated 2-deoxy-2-C-formyl-d-pentofuranosyl unit due to ring contraction in unit 2. These results suggest that the 6-sulfate group in unit 2 may be involved in antithrombin binding. It is concluded that the antithrombin-binding site in heparin is represented by the pentasaccharide sequence extending from unit 2 to unit 6 of the octasaccharide studied.