An immunoblot assay reveals that bacteriophage T4 thymidylate synthase and dihydrofolate reductase are not virion proteins.

Numerous reports describe the phage T4 enzymes thymidylate synthase and dihydrofolate reductase as structural components of the baseplate. However, Y. Wang and C. K. Mathews (J. Virol. 63:4736-4743, 1989) reported that antisera against the respective recombinant enzymes failed to neutralize phage infectivity, in contrast to previous results. Moreover, a deletion mutant lacking the genes for these two enzymes adsorbed normally to host cells. Since these findings tended to undermine the idea of the two enzymes as structural proteins, we developed a quantitative immunoblot assay to resolve the issue directly. Our results show that both enzymes are present only as minor contaminants (< 0.05 copy per phage) and as such cannot be bona fide structural proteins.     

Ornithine Decarboxylase Activity and Edema Formation in Cerebral Ischemia of Conscious Gerbils

Abstract: General anesthetic agents often affect the biochemical and physiologic changes triggered by cerebral ischemia. This study examined the regional activities of ornithine decarboxylase (ODC) in gerbils subjected to 5 min of bilateral carotid occlusion without anesthesia. At 2, 4, and 6 h of reperfusion, significant ODC activity was observed in both the cortex and the hippocampus. Pretreatment with α-difluoromethylornithine (DFMO) significantly blocked the ODC activity at 2, 4, and 6 h. Significant edema formation was found at 2, 4, and 6 h. At 2 h, edema formation was unaffected by administration of DFMO. However, DFMO treatment reduced later edema formation at 4 and 6 h. These results demonstrate that ODC activity and edema formation are delayed in gerbils after the induction of transient ischemia even with the removal of anesthetic agents and their potentially protective effects. These findings suggest that ODC activity and its induction of delayed cerebral edema are specific to cerebral ischemia and not to an anesthetic effect. DFMO treatment reduced both the ODC activity and edema formation, indicating a role for polyamines in postischemic edema formation.     

The primary structure of Lactobacillus casei thymidylate synthetase. I. The isolation of cyanogen bromide peptides 1 through 5 and the complete amino acid sequence of CNBr 1, 2, 3, and 5.

Thymidylate synthetase from Lactobacillus casei was S-carboxymethylated and degraded by treatment with cyanogen bromide. Although the protein contains 6 methionine residues, only 5 cyanogen bromide peptides were obtained due to the presence of 1 methionine on the NH2 terminus and another adjacent to a threonine residue which was resistant to cleavage. The peptides were isolated by differential extraction, first with ammonium acetate, then pyridine acetate, and finally the residue was solubilized with 50% acetic acid. Each peptide was further purified to homogeneity by Bio-Gel chromatography. The size of the peptides from the amino to carboxyl end of the enzyme subunit was CNBr 1, 4,100; CNBr 2, 10,300; CNBr 3, 8,100; CNBr 4, 11,800; CNBr 5, 2,200. The sum of the amino acid residues of the peptides is equal to the sum of the residues in an enzyme subunit, indicating that all of the CNBr peptides have been isolated. The CNBr-resistant methionine was located in CNBr 2 and the 5-fluoro-2'-deoxyuridine 5'-monophosphate binding site in CNBr 4. The holoenzyme molecular weight, based on the residue weights of the amino acids in the two equivalent subunits, is equal to 73,176. The complete sequence of each of the CNBr peptides, except for CNBr 4, which is presented in the following paper, is described.     

Purification and properties of endo-beta-N-acetylglucosaminidase L from Streptomyces plicatus.

An enzyme, previously described as endo-beta-N-acetylglucosaminidase L (Tarentino, A.L., and Maley, F. (1974) J. Biol. Chem. 249, 811-817) because of its apparent specificity for Man(GlcNAc)2Asn, has been purified to homogeneity. The enzyme has now been found to hydrolyze (GlcNAc)3 to (GlcNAc)2 plus GlcNAc, and (GlcNAc)4 to 2(GlcNAc)2, at twice the rate observed for Man(GlcNAc)2Asn. Removal of the asparagine from the latter compound reduces the rate of hydrolysis by about 30-fold. Reduction of (GlcNAc)3 to GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc-ol eliminates this compound as a substrate for endo-beta-N-acetylglucosaminidase L. However, the reduction of (GlcNAc)4 does not affect its rate of hydrolysis. Endo-beta-N-acetylglucosaminidase L consists of a single polypeptide chain with a molecular weight of 49,500 +/- 400, which on isoelectric focusing separates into two closely migrating bands; a major with a pI of 4.25 and a minor one with a pI 4.20. Both bands possess similar enzyme activities and amino acid compositions, but differ slightly in their tryptic peptide maps.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.     

Studies on identifying the allosteric binding sites of deoxycytidylate deaminase

Thymidine triphosphate, a negative regulator of deoxycytidylate deaminase, was found to bind covalently to this enzyme on exposure to UV light at 254 nM. The rate of half-maximal fixation was extremely rapid, occurring within 30 s and probably attaining a maximum of about 1 mol of dTTP fixed/mol of enzyme subunit. In contrast to the case of ribonucleotide reductase (Ericksson, S., Caras, I. W., and Martin, D. W., Jr. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 81-85) where the fixation of dTTP inactivated this enzyme, the activity of the deaminase was unaffected. The bound nucleotide could be released on exposure to UV 254 nm light in the presence of dCTP or dTTP but not dATP or dGTP. The enzyme-fixed nucleotide was found to remain with the larger of the two peptides released as a result of CNBr treatment of the labeled enzyme. Studies are in progress to define the location of this nucleotide, which will be aided greatly by our recent clarification of the complete amino acid sequence of T2-deoxycytidylate deaminase.     

Demonstration of separate binding sites for the folate coenzymes and deoxynucleotides with inactivated Lactobacilluscasei thymidylate synthetase

In contrast to (+)5,10-methylenetetrahydropteroylmonoglutamate which does not bind to $\text{Lactobacillus}\text{casei}$ thymidylate synthetase, the corresponding tetraglutamate analog binds to a single site with a K_D = 2 × 10^?5 M. Alkylation of one of the enzyme's four cysteines with N-ethylmaleimide or iodoacetate prevented the binding of dUMP, but did not affect the binding of the pteroyltetraglutamate. Inactivation of the synthetase with carboxypeptidase A, however, prevented the binding of (+)5,10-methylenetetrahydropteroyltetraglutamate but not that of dUMP. The binding of (+)5,10-methylenetetrahydropteroyltetraglutamate to native enzyme was associated with the appearance of a positive circular dichroic band at 303 nm ([θ] = 7 × 10⁴ deg·cm²dmol^?1). The latter effect was not impaired by the inhibition of the enzyme with N-ethylmaleimide, whereas formation of the ternary complex, coenzyme-synthetase-FdUMP, was prevented by alkylation. These studies reveal that thymidylate synthetase can be inactivated in a manner that does not prevent the binding of the substrates individually.