Determination of amino acid sequences of two subunits in sarcosine oxidase from Corynebacterium sp. U-96.

The primary structures of the C and D subunits of sarcosine oxidase from Corynebacterium sp. U-96 were determined by sequencing the peptide fragments derived from their enzymatic digestions. The C and D subunits were shown to be composed of 199 and 92 residues, respectively. Each amino acid sequence showed a high homology with the sequence of the corresponding subunit from Corynebacterium sp. P-1. However, there were some differences between these two species, that is, four N-terminal residues were truncated in the C subunit, but six C-terminal residues were truncated in the D subunit. The D subunit contained three cysteine residues, but no disulfide bonds are in the subunit. Overall sequences of both subunit showed no homology with any other protein in the data base.

Chemical modification of L-phenylalanine oxidase from Pseudomonas sp. P-501 by phenylglyoxal. Identification of one essential arginyl residue.

L-Phenylalanine oxidase from Pseudomonas sp. P-501 was irreversibly inactivated by the arginine-specific reagents, phenylglyoxal (PGO) and p-hydroxyphenylglyoxal (HPG). The inactivation by PGO and HPG follows pseudo-first-order kinetics with second-order rate constants of 10.6 and 15.1 M-1.min-1, respectively, and a single arginyl residue was modified specifically. The effective protection by substrate L-phenylalanine against the inactivation by these reagents strongly suggests that the arginyl residue is located in the substrate binding site. SDS/PAGE analysis of the enzyme modified with [14C]PGO revealed that the arginyl residue was in the beta subunit of the enzyme. The fragment containing the 14C-labeled arginyl residue was purified from the enzymatic digests of the labeled beta subunit by HPLC and sequenced. The modification of Arg-35 in the beta subunit was identified. The sequence around Arg-35 shows homology to the corresponding regions of tryptophan-2-monooxygenases.

A catalytic antibody that accelerates the hydrolysis of carbonate esters. Prediction of the binding-site structure of the substrate.

Monoclonal antibodies catalyzing the hydrolysis of p-nitrophenyl alkyl carbonate were obtained using p-nitrophenyl phosphonate as hapten. One of the antibodies, 4A1, has a relatively high activity for the substrate having a bulky group. To determine the amino acid residues related to the binding of the bulky group, we determined the amino acid sequences of VL and VH regions of 4A1 by the cycle sequencing method, built the three-dimensional structure of the V regions, labeled 4A1 with [14C]phenyl glyoxal in the presence and absence of I-1 or I-13, and analyzed the labeled incubation mixture with SDS-PAGE. From these results, the possibility that Arg-H28 of the heavy chain is involved in binding the bulky group of the substrate is discussed.

Delayed appearance of the catalytic activity by immunization of a rabbit compared with the hapten binding.

Polyclonal antibodies catalyzing the hydrolysis of carbonate ester were generated by immunizing a rabbit with hapten(4-nitrophenyl phosphate II) conjugated to keyhole limpet hemocyanin. The hydrolytic activity of IgG purified from antisera exhibited plateu one month later than the simple hapten-binding. The affinity of IgG with substrate increased even after the hapten-binding reached plateu. These suggest a strategy to generate good polyclonal catalytic antibodies and the day to fuse spleen cell with myeloma cell to get good monoclonal catalytic antibodies.

New subunit in L-phenylalanine oxidase from Pseudomonas sp. P-501 and the primary structure.

L-phenylalanine oxidase from Pseudomonas sp. P-501 was shown by isoelectric focusing and HPLC experiments to consist of two kinds of nonidentical subunits. The newly identified subunit is designated as alpha, and the larger subunit, which has been reported previously, as beta. The apparent molecular weight of alpha subunit was estimated to be 8200 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From the molecular mass of each subunits and their contents in the enzyme, the enzyme was shown to be composed to two alpha and two beta subunits. The amino acid sequence analysis of alpha subunit showed that the subunit consists of 92 amino acid residues and contains considerably hydrophobic arrangements and the candidate for the common sequence characteristic of the AMP binding in the FAD binding domain.

Chemical modification of pig liver initiation factor eIF-2 with N-ethylmaleimide. Amino acid sequences around the N-ethylmaleimide-reactive sulfhydryl groups and the effect of GDP on the modification.

The activity of eukaryotic initiation factor eIF-2 as to the formation of the ternary complex, eIF-2 GTP Met-tRNA(f), is inhibited by N-ethylmaleimide. Our preparation of pig liver eIF-2 contained alpha and gamma subunits and was inhibited by more than 90% by N-ethylmaleimide. Using our eIF-2, we determined the sequences around the N-ethylmaleimide-reactive sulfhydryl groups, studied the effect of GDP on the sulfhydryl modification and that of NEM on the [3H]GDP binding, and examined the protective effect of GTP against the inhibition of ternary complex formation by N-ethylmaleimide. Both subunits of native eIF-2 contained [14C]N-ethylmaleimide-reactive sulfhydryl groups. One N-ethylmaleimide-reactive sulfhydryl group was in the alpha subunit and 4 were in the gamma subunit. The sequence of the peptide of the alpha subunit was determined to be: Ala-Gly-Leu-Asn-Cys-Ser-Thr-Glu-Thr-Met-Pro-Ile. Two of the four [14C]N-ethylmaleimide-reactive sulfhydryl groups in the gamma subunit were highly reactive, their sequences being: Ile-Val-Leu-Thr-Asn-Pro-Val-Cys-Thr-Glu-Val-Gly-Glu-Lys (gamma 1); Ser-Cys-Gly-Ser-Ser-Thr-Pro-Asp-Glu-Phe-Pro-Thr-Asp-Ile-Pro-Gly-Thr-Lys (gamma 3a). Peptide gamma 3a contained the consensus sequence element (AspXaaXaaGly) of GTP-binding proteins. With preincubation of eIF-2 with GDP, the incorporation of [14C]N-ethylmaleimide into the gamma subunit was reduced to 40% of the control level, but the 14C-incorporation into the alpha subunit did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of stimulation of globin synthesis by adenosine-3',5'-monophosphate and guanosine 5'-triphosphate in a rabbit reticulocyte lysate system.

The inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2 alpha. High concentrations of cAMP (5-10 mM) and GTP (1-2 mM) stimulated the globin synthesis in hemin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide (NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined. As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2 alpha in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S]Met-tRNAf to the 40S ribosomal subunits, and promoted the dephosphorylation of eIF-2(alpha P), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H]GDP from the eIF-2(alpha P) X [3H]GDP complex and the formation of eIF-2(alpha P) X eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2 alpha and promoted both the release of GDP from eIF-2 and the formation of eIF-2(alpha P) X eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2 alpha. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2 alpha and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.

Specific phosphorylation of pig liver initiation factor eIF-2 by the N-ethylmaleimide-treated hemin-controlled translational inhibitor.

The specific phosphorylation of pig liver initiation factor 2(eIF-2) by the N-ethylmaleimide (NEM)-treated hemin-controlled translational inhibitor (HCI) from rabbit reticulocytes was investigated. The inhibitor phosphorylated the serine residue of the alpha subunit of eIF-2 (eIF-2 alpha) and 1 mol of phosphate was incorporated into 1 mol of eIF-2 alpha by the inhibitor on maximal phosphorylation, even when eIF-2 was pretreated with alkaline phosphatase prior to phosphorylation. The 32P-labeled eIF-2 alpha was subjected to tryptic digestion and the tryptic digest was analyzed by two-dimensional peptide mapping on a cellulose thin-layer sheet. After 94 h digestion, the autoradiograph of the peptide map showed a single 32P-labeled band with a molecular weight of approximately 1,200. These findings suggest that one specific serine residue of pig liver eIF-2 alpha was phosphorylated by the NEM-treated HCI.

Poly(dT) inhibition of globin synthesis in the rabbit reticulocyte lysate system. Reversal of the inhibition by poly(dT)-binding protein.

The mechanism of inhibition of globin synthesis by poly(dT) was studied in the rabbit reticulocyte lysate system. When the lysate was incubated with [14C]poly(dT), poly(dT) was found to bind with the native 40S ribosomal subunit and the "supernatant factor." But the binding to the native 40S ribosomal subunits was not directly related to the poly(dT) inhibition. Ribosomal subunits were prepared from rabbit reticulocytes and tested for their binding with poly(dT) and their effect on the poly(dT) inhibition. Poly(dT) was found to bind with the derived 40S ribosomal subunit, but not with the derived 60S subunit, and the poly(dT) inhibition was slightly reversed by the derived 40S ribosomal subunit. Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of the 80S/Met-tRNAf complex was inhibited by poly(dT) and the inhibition was greater at high concentration of KOAc. However, the formation of the 40S/Met-tRNAf complex was inhibited to the same extent at 70 mM and 200 mM KOAc in the presence of GMPPCP. A factor (TF) that reverses the poly(dT) inhibition was partially purified from the KCl-wash of rabbit reticulocyte ribosomes by ammonium sulfate fractionation, and Sephadex G-150 and DEAE-cellulose column chromatographies. From the Sephadex G-150 and DEAE-cellulose column chromatography of TF, the molecular weight of TF was estimated to be 81,000-102,000. TF reversed the poly(dT) inhibition of 80S/[3H]mRNA/Met-tRNAf complex or that of 40S/[3H]mRNA/Met-tRNAf complex. TF bound to [14C]poly(dT) or 3H-labeled globin mRNA. SDS/polyacrylamide slab gel electrophoresis of the complexes between the factor and [14C]poly(dT) or [3H]mRNA showed common polypeptide bands of 22,500, 25,000, and 49,000 daltons. These data can be explained by assuming that poly(dT) binds to a factor which is required for the binding of 40S/Met-tRNAf complex with mRNA to form inactive complexes, and thus inhibits globin synthesis. The relationship between the poly(dT)-binding protein and known initiation factors is discussed.

Effect of concentration of potassium salts on the activity of the N-ethylmaleimide-treated hemin-controlled translational inhibitor.

The effect of various concentrations of potassium salts on the activity of the N-ethylmaleimide(NEM)-treated hemin-controlled translational inhibitor (HCI) was investigated using the rabbit reticulocyte lysate system. The NEM-treated HCI was found to be less active for the inhibition of globin synthesis at higher concentrations of potassium salts. In addition, the binding of [35S]Met-tRNAf to the 40S ribosomal subunits was inhibited more profoundly at 100 mM than at 240 mM KOAc. The phosphorylation of the NEM-treated HCI and eukaryotic initiation factor, eIF-2, was studied at various concentrations of KOAc. The NEM-treated HCI was phosphorylated to an almost constant extent at 20 to 300 mM K+, but the phosphorylation of eIF-2 alpha by the inhibitor decreased with increasing concentration of KOAc. It was also found that the incubation of the 32P-labeled inhibitor with eIF-2 did not produce the 32P-labeled eIF-2. In the light of these data, a possible relationship between the phosphorylation of the inhibitor or eIF-2 alpha and the inhibition of globin synthesis by the inhibitor is discussed.