Martin Flavin

Inhibition of dynein ATPase by vanadate, and its possible use as a probe for the role of dynein in cytoplasmic motility

Vanadate selectively inhibited dynein ATPase, 10−7M causing 50% inhibition under favorable conditions. Actomyosin ATPase was inhibited only by up to a thousand times higher concentration. In both cases vanadate inhibition was not competitive with ATP. Reversal by catecholamines was correlated with reduction of vanadate. The motility of demembranated sea urchin or mammalian sperm was arrested by vanadate concentrations similar to those which inhibited dynein ATPase; a thousand times higher concentration was needed to paralyze live sperm. The possible utility of vanadate sensitivity as a probe for dynein involvement in non-axonemal motile systems was explored with respect to brain ATPase associated with tubulin obtained by cycles of assembly, and ATPases associated with mitotic apparatus isolated from sea urchin embryos.

An enzyme tyrosylating α-tubulin and its role in microtubule assembly

Summary Alpha tubulin is shown to be the only polypeptide in brain extracts which undergoes C-terminal addition of a tyrosine residue. Tubulin purified by cycles of in vitro polymerization can be used as substrate to assay and purify the tyrosine ligase. Tubulin dimer is a substrate for the enzyme and axonemal doublet microtubules are not. Both tyrosylated and untyrosylated dimers can polymerize in vitro .

A structural difference between cytoplasmic and membrane-bound tubulin of brain

Tubulin tyrosine ligase catalyzes a unique posttr~slation~ rnodi~cat~on of the a: chain of tubulin, whereby, in an ATP-dependent reversible reaction, a tyrosine residue is added through peptide linkage to the cY-carboxyl of C-terminal glutamate [l-4]. The enzyme has now been purified 300-fold and is active enough to ensure maximal tyrosylation of any tubulin preparation [S] and thus enables one to determine the fraction of tubulin susceptible to tyrosylation in a particular sample. It has been observed in our laboratory that every cytoplasmic tubulin preparation tested so far (>20), can be considered to be composed of three species: one which has pre-existing tyrosine; one which can accept further tyrosine in vitro; and one which appears not to be a substrate for ligase [S]. To investigate the function of this post-translational modification, we have begun to compare the state of tyrosylation of tubulin isolated from different sources. In the present communication we wish to report that a membrane-bound fraction of brain tub&n, purified by vinblastine precipitation from a detergent extract, can be tyrosylated similarly to the brain cytoplasmic tubulin, but differs strikingly from the latter, by the complete absence of pre-existing tyrosine. In the course of this investigation we have also been able to obtain membrane tubulin which is 35% pure. This is the purest preparation of native membrane tubulin reported so far.

Enzymatic synthesis of homocysteine or methionine directly from o-succinyl-homoserine

The bacterial biosynthesis of methionine has previously been shown to involved formation of cystathionine from cysteine and O-succinyl-hormoserine, catalyzed by cystathionine γ-synthase, followed by cleavage of cystathionine to yield homocysteine. This report presents evidence that hydrogen sulfide can replace cysteine as a substrate for cystathionine γ-synthase, yielding homocysteine directly. The maximum velocity of homocysteine formation is about half that of cystathionine formation, but the Km for hydrogen sulfide is 50 times higher than that for cysteine. The leakiness of Salmonella mutants blocked in the conversion of cystathionine to homocysteine suggests that direct synthesis of homocysteine from H2S may take place in the cell, although only to a limited extent. Cystathionine γ-synthase has also been shown to catalyze formation of methionine directly from O-saccinyl homoserine and methyl mercaptan. This reaction is presumed to have no role in the utilization of inorganic sulfur compounds for methionine biosynthesis, but may provide an explanation for the ability of S-methyl cysteine to support the growth of methionine auxotrophs of some microorganisms.

Reclinomonas americana N. G., N. Sp., a new freshwater heterotrophic flagellate.

ABSTRACT. A new heterotrophic flagellate has been discovered from sites in Maryland, Michigan and Wyoming. The flagellate resides within a lorica constructed of a meshwork of intertwined fibrils with the outer surface invested with nail‐shaped spines. The organism “reclines” within the lorica with its ventral aspect directed upward, and has two heterodynamic flagella, neither of which bears mastigonemes. One flagellum is directed upward and the other is arched over the ventral aspect of the body. Ingestion of bacteria takes place at the left posterior half of the cell. The organism is anchored to the lorica on the right posterior side by a series of regularly spaced cytoplasmic bridges and at the left anterior of the cell by a cytoplasmic appendage similar to the “languette cytoplasmique” found in some bicosoecids. The right side of the cell is raised into a flattened lip with the outer margin reinforced by a ribbon of microtubules. The new flagellate has mitochondria with tubular cristae and lacks a Golgi. A new genus is created to accommodate both the new flagellate described herein and Histiona campanula Penard. A new family is proposed to include the new genus and Histiona.

Succinic Ester and Amide of Homoserine: Some Spontaneous and Enzymatic Reactions

O-Succinylhomoserine and N-succinylhomoserine have been synthesized. The first is rapidly transformed into the second by alkali. In acid, the second undergoes ring closure to the lactone, rather than the reverse acyl transfer. Neither supports the growth of methionine auxotrophs of Neurospora or Salmonella. However, bacterial extracts rapidly catalyze formation of a compound, chromatographically identical with cystathionine, from cysteine and O-succinylhomoserine. In the absence of cysteine the O-succinylhomoserine is converted to α-ketobutyrate. Both these reactions are absent from the same Salmonella mutant, and therefore are probably catalyzed by a single enzyme which is needed for methionine synthesis. Both reactions require pyridoxal phosphate. N-succinylhomoserine does not undergo either reaction.

The derepression and function of enzymes of reverse trans-sulfuration in neurospora

Abstract Sulfur transfer from homocysteine to cysteine is mediated by 2 enzymes, cystathionine β-synthase and γ-cystathionase, which are present in animals and fungi, but absent from bacterial species so far examined. It has now been found that when nutrient sulfur limits the growth of Neurospora, the level of γ-cystathionase is increased 30-fold. Derepression is not coordinate, since the level of cystathionine β-synthase is not changed. β-Cystathionase is also unaffected. Non-coordinate derepression suggests that γ-cystathionase might have a more general function in mobilizing sulfur; the enzyme is shown able to rapidly decompose a wide variety of β- or γ-substituted amino acids besides cystathionine. The derepression was shown not to be brought about by starvation in general, and may therefore result from depletion of a low molecular weight, sulfur-containing corepressor

[22] Tyrosine incorporation in tubulin

Publisher Summary This chapter illustrates tyrosine incorporation in tubulin. Tubulin tyrosinolation refers to the tRNA-independent addition of tyrosine to the C-terminal glutamate of its α chain. It was first characterized in Caputtos laboratory, as the fortuitous result of a control experiment carried out while studying developmental fluctuations in tRNAs from brain. The assay method for tubulin-tyrosine ligase is based on the rate at which labeled tyrosine is fixed into a trichloroacetic acid-insoluble form in the presence of purified microtubule protein. A unit of enzyme is the amount transferring 1 nmol of tyrosine to tubulin in 1 min, under the assay conditions. Specific activity is defined as units per milligram of protein. All purification procedures are carried out at 0-4°. The pH optima are observed at 8 and 6.5, and 8.5 μM ATP or 30 μM L-tyrosine give half maximal activity. Tubulin dimer appears to be the only substrate. The enzyme is homogeneous by SDS-PAGE, and the native molecular weight appears to be 37,000 by HPLC gel filtration.

An Apparent Paradox in the Occurrence, and the In Vivo Turnover, of C-Terminal Tyrosine in Membrane-Bound Tubulin of Brain

: Tubulin tyrosine ligase catalyzes the reversible addition of tyrosine to the C‐terminus of tubulin α chains. By using ligase and carboxypeptidase A in conjunction, we have previously shown that brain cytoplasmic tubulin exists in three forms: 15–40% already has C‐terminal tyrosine, another 10‐30% can accept additional tyrosine, and about one‐half is an uncharacterized species which is not a ligase substrate. A membrane‐bound fraction of brain tubulin, purified by vinblastine precipitation from a detergent extract, has been found to differ by the complete absence of preexisting tyrosine. The membrane fraction from which tubulin was extracted also contained masked forms of both ligase and a distinct detyrosylating enzyme, which can be released by detergent extraction. The turnover of α‐chain C‐terminal tyrosine in vivo was studied by incubating brain mince with labeled tyrosine, or injecting it intracerebrally, under conditions where protein synthesis was inhibited. Tyrosine appeared to turn over to about the same extent in membrane‐bound, as in soluble, tubulin. This apparently paradoxical result was not due to ATPase in the membrane fraction, which might have allowed ligase‐catalyzed exchange between free and fixed tyrosine. Authentic [14C]tyrosylated tubulin added to the brain membrane fraction was not detyrosylated or subject to endoprotease digestion during subsequent procedures to isolate tubulin. The unexpected finding that tubulin tyrosylated at the C‐terminal in vivo appears to be in the “non‐substrate” fraction points toward a possible resolution of the paradox.

Cyclic nucleotide- and Ca2+-independent phosphorylation of tubulin and microtubule-associated protein-2 by glycogen synthase (casein) kinase-1.

MAP-2 and tubulin are both shown to be substrates for glycogen synthase (casein) kinase-1 (CK-1). Greater than 40 mol 32P is incorporated into MAP-2 by CK-1 compared to only 14 mol 32P observed when cyclic AMP-dependent protein kinase (A-kinase) is the catalyst. Peptide mapping shows that CK-1 and A-kinase recognize a few common sites; the majority of the sites phosphorylated on MAP-2 by CK-1 are quite distinct. Up to 4 mol 32P can be incorporated into the tubulin dimer by CK-1 compared to only 0.9 mol 32P by A-kinase. The preferred substrate for both kinases is beta-tubulin.