Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver

Ramesh, K.; Rao, N. Appaji

doi:10.1042/bj1870623

Cited by 24 publications

(9 citation statements)

References 31 publications

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“…For example, all known eukaryotic SHMT enzymes (whether mitochondrial or cytosolic) migrate between 50 and 53 kDa on denaturing SDS-polyacrylamide gels and, like p50 (20), most have isoelectric points near 7.0 (20,42). Not surprisingly, each of these enzymes is purified by separation methods very similar to those employed here (33,(43)(44)(45). Furthermore, [ 3 H]mimosine-labeled p50 elutes from a nondenaturing gel filtration column as a multimer with an estimated size of 200 -250 kDa, 4 as does purified rabbit mSHMT (33,(43)(44)(45).…”

Section: Figmentioning

confidence: 99%

Mimosine Targets Serine Hydroxymethyltransferase

Lin

Falchetto

Mosca

et al. 1996

Journal of Biological Chemistry

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The plant amino acid, mimosine, is an extremely effective inhibitor of DNA replication in mammalian cells (Mosca, P. J., Dijkwel, P. A., and Hamlin, J. L. (1992) Mol. Cell. Biol. 12, 4375-4383). Mimosine appears to prevent the formation of replication forks at early-firing origins when delivered to mammalian cells approaching the G 1 /S boundary, and blocks DNA replication when added to S phase cells after a lag of ϳ2.5 h. We have shown previously that [ 3 H]mimosine can be specifically photocross-linked both in vivo and in vitro to a 50-kDa polypeptide (p50) in Chinese hamster ovary (CHO) cells. In the present study, six tryptic peptides (58 residues total) from p50 were sequenced by tandem mass spectrometry and their sequences were found to be at least 77.5% identical and 96.5% similar to sequences in rabbit mitochondrial serine hydroxymethyltransferase (mSHMT). This assignment was verified by precipitating the [ 3 H]mimosine-p50 complex with a polyclonal antibody to rabbit cSHMT. The 50-kDa cross-linked product was almost undetectable in a mimosine-resistant CHO cell line and in a CHO gly ؊ cell line that lacks mitochondrial, but not cytosolic, SHMT activity. The gly ؊ cell line is still sensitive to mimosine, suggesting that the drug may inhibit both the mitochondrial and the cytosolic forms. SHMT is involved in the penultimate step of thymidylate biosynthesis in mammalian cells and, as such, is a potential target for chemotherapy in the treatment of cancer.Our laboratory's interest is the regulation of DNA synthesis in mammalian cells and, in particular, the nature of origins of replication. Although it is known that mammalian DNA is replicated from bidirectional origins spaced ϳ100 kilobase pairs apart (1), the molecular mechanisms of this process remain elusive (see Ref. 2, for review).In the absence of a viable assay for identifying the genetic elements (replicators) that control initiation in mammalian cells, attention has been focussed on determining the positions at which replication initiates, which should lie close to replicators. This approach requires methods for obtaining cell populations in which initiation at a given origin is occurring at the same time. In a commonly used synchronization protocol, cells are first arrested in the G 0 (non-proliferating) compartment by nutritional or serum starvation, followed by release into an inhibitor of DNA synthesis (e.g. Refs. 3-5). The drug treatment is enforced for a time long enough to allow all cells in the population to arrive at the beginning of the S period (a time when at least some origins are sure to be firing); the drug is then removed, allowing cells to enter S in a semi-synchronous wave. Unfortunately, this protocol is not entirely satisfactory for examining initiation events at the beginning of S, because even the most efficacious replication inhibitors do not inhibit initiation per se; rather, they slow the rate of replication fork movement by affecting DNA polymerases (e.g. aphidicolin (6)) or by lowering deoxyribonucleotide pools (e.g. hy...

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Section: Figmentioning

confidence: 99%

Mimosine Targets Serine Hydroxymethyltransferase

Lin

Falchetto

Mosca

et al. 1996

Journal of Biological Chemistry

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“…Whereas the monkey liver enzyme (17) isolated in the absence of PLP had very little activity, the mung bean enzyme isolated in the presence and in the absence of PLP was fully active (Table I). Additional evidence for the non-involvement of PLP in the reaction catalyzed by mung bean SHMT was the absence of the characteristic PLP-Schifls base spectrum in the mung bean enzyme (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Upon dialysis of the L-cysteine-treated enzyme, the activity was fully regained in the case of mung bean SHMT, and the addition of PLP (1 mM) did not enhance the activity of the enzyme. Although the activity of sheep (12), monkey (17), and human liver (24) SHMT were also inhibited by Lcysteine, the activity was not regained upon dialysis but was restored fully upon the addition of PLP (1 mM) (Table II).…”

Section: Effect Of I-cysteine Treatment On the Enzyme Activitymentioning

confidence: 99%

“…Effect of L-cysteine on the activity of SHMT from mung bean seedlings, monkey (17), sheep (12), and human liver (24) is given in Table II. It is evident that the enzyme from all these sources was completely inhibited by L-cysteine (10 mM).…”

Section: Effect Of I-cysteine Treatment On the Enzyme Activitymentioning

confidence: 99%

“…Purification of Sheep, Monkey, and Human Liver SHMT The procedures described by Baskaran et al (1), Ramesh and Rao (17), and Vijayalakshmi (24) were used to prepare mammalian SHMT. All of these enzyme preparations were homogeneous (1,17,24).…”

Section: Purification Of Mung Bean Shmt In the Presence Of Plpmentioning

confidence: 99%

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Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5′-Phosphate-Dependent Enzyme

Sukanya

Vijaya²,

Savithri³

et al. 1991

Plant Physiol.

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Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5'-phosphate-containing enzyme, but the requirement of pyridoxal-5'-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of senne hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5'-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5'-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with L-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5'-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5'-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-D-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-Dseine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5'-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean seine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyndoxal-5'phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean seine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5'-phosphate that is present in the mammalian and bacterial enzymes.

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Glycine hydroxymethyltransferase

Springer Handbook of Enzymes

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Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver

Cited by 24 publications

References 31 publications

Mimosine Targets Serine Hydroxymethyltransferase

Mimosine Targets Serine Hydroxymethyltransferase

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5′-Phosphate-Dependent Enzyme

Glycine hydroxymethyltransferase

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