Christopher I. Pogson

Evidence that griseofulvin binds to a microtubule associated protein.

We have recently shown that the anti-mitotic drug, griseofulvin, inhibits assembly of brain microtubules in vitro [l] . Similar data have been presented by Weber et al. [2] . Our earlier experiments indicated that griseofulvin inhibits microtubule assembly by preventing the association between tubulin and the microtubule associated proteins (MAPS) which normally co-purify with tubulin [3] and promote assembly of purified tubulin dimer [4] . We now present evidence that griseofulvin binds specifically to MAPS and not to tubulin dimer at concentrations of griseofulvin which inhibits microtubule assembly.

A comparison of tubulins from mammalian brain and Physarumpolycephalum using SDS—polyacrylamide gel electrophoresis and peptide mapping

IBM isolated from rn~rn~~ brain consists of two equimdar protein species designated (Y- and /?-tubulins. Conventionally cw-tubulin is the slower, and P-tubulin the faster migrating species during sodium dodecyl sulphate-polyacrylamide gel electrophoresis [ 11. Tubulin has been considered to be a highly conserved protein ]2,3]; however, we now show differences between tubulins from mammalian brain and myxamoebae of the slime mould Physarum poZycephaZum from which microtubule proteins have been purified by an assembly procedure [4]. Evidence is baaed on differences in migration of these tubulins on SDS- and urea-SDS-PAGE, and peptide mapping, and demonstrates that Physarum tubulin consists of an ol-tubulin which is similar to brain /3-tubulin, and a P-tubulin which is dissimilar to both brain subunits. In addition, the analogous proteins from Physarum and brain demonstrate micro heterogeneity during urea-SDS-PAGE. The migration of the Physarum P-tubulin is altered during urea-SDS-PAGE such as to cause the order of migration of the myxamoebal tubulins to be reversed with respect to that during SDS-PAGE. In addition the importance of the source of SDS in resolving tubu~ subunit proteins during PAGE is highlighted. Abbreviations: EGTA, ethylene glycol-bis@aminoethyl ether) tetra acetic acid; PAGE, polyac~~rn~e gel electrophoresis; PfPES,pi~r~~e-~~~is(2~th~e~~pho~c acid); SDS, sodium dodecyl sulphate

In vitro assembly of microtubule proteins from myxamoebae of Physarum polycephalum

Abstract Microtubule protein of >95% purity has been isolated by self-assembly from concentrated cell extracts of myxamoebae of Physarum polycephalum . Ninety-eight percent of the amoebal microtubule protein was tubulin. Both a and β subunits of amoebal tubulin were different from neurotubulin α and β subunits, but very similar to those of Tetrahymena ciliary tubulin. The non-tubulin components, which co-purified with tubulin through three assembly cycles, were essential to microtubule formation and contained several polypeptides including some of apparent molecular weights 49000, 57000 and 59000. Purified amoebal microtubule protein formed microtubules on warming in the absence of glycerol which were cold- and Ca 2+ -labile. In vitro, microtubule assembly was inhibited by vinblastine, benzimidazole derivatives and griseofulvin, but not by 10 −4 M colchicine. Amoebal tubulin had a much lower affinity than neurotubulin for colchicine.

Inhibition by griseofulvin of microtubule assembly in vitro

The antimitotic drug, griseofulvin, has been shown to arrest cell division at a point close to metaphase in a variety of cells [1]. The microtubules in griseofulvinblocked HeLa cells appear to have a normal morphology and orientation within the spindle [2] and it has been suggested that griseofulvin inhibits some aspect of microtubule function rather than microtubule assembly [3]. Using a crude extract of brain, Wilson and Bryan [1 ] were unable to inhibit in vitro microtubule assembly, as measured by electron microscopy, with griseofulvin at a concentration known to inhibit mitosis (60/aM). We report here that griseofulvin at concentration between 20 and 200/IM does inhibit in vitro assembly of purified microtubule protein from brain as measured by turbidimetry, electron microscopy and a sedimentation method. Inhibition of assembly by griseofulvin is distinctly different from inhibition by colchicine. Disc gel electrophoretic analysis indicates that microtubule protein polymerized in the presence of griseofulvin is depleted in the high molecular weight (HMW) components which normally copurify with tubulin [4,5].

The metabolic integrity of hepatocytes in sustained incubations

Over the past few years, suspensions of isolated hepatocytes have become accepted as important systems for studying hepatic metabolism and its control [ 1) . Several methods that yield hepatocyte suspensions physiologically similar to intact liver have been published [2-41 . Although this similarity is apparent immediately after isolation, incubation of hepatocytes at 37°C in simple salt solutions is accompanied by substantial deterioration after 2-3 h [2,5] . Thus, although the preparation is suitable for investigation of shortterm events [6,7] , study of long-term processes under these conditions will be of limited validity. Various workers have attempted to increase hepatocyte viability by incubating cells under culture conditions [8,9] . During colony formation, however, many parenchymal cells are lost completely and, although cells in culture maintain some of the properties of hepatocytes in intact liver, many functions are altered [ 10,l l] . Many of these studies do not apply sufficiently stringent biochemical tests to assess cellular viability throughout culturing. A system for the preparation of biochemically viable non-dividing parenchymal cells and their maintenance for incubations of 8-12 h. would prove a valuable tool in the study of hepatic anabolism. Complex physiological mixtures of amino acids, vitamins and serum have advantages over KrebsHenseleit buffers for polysome aggregation [ 121 , albumin synthesis [ 131 and inhibition of ribonuclease activity following hepatocyte isolation [ 141 . This paper details the biochemical stability of isolated parenchymal hepatocytes incubated in minimum essential medium (Eagle) plus foetal calf serum and

TYROSINE AMINOTRANSFERASE AS THE RATE-LIMITING STEP FOR TYROSINE CATABOLISM IN ISOLATED RAT LIVER CELLS

In a number of species, hepatic tyrosine aminotransferase (TAT) activity increases rapidly in response to stress occassioned by nutritional or hormonal imbalance [l-5]. To a large extent the activity changes observed are attributable to direct hormonal effects on the rates of TAT synthesis or degradation in the liver [6-91. TAT is the first enzyme in the catabolic pathway and is subject to numerous hormonal control mechanisms; it has therefore been proposed as the rate limiting enzyme for hepatic tyrosine catabolism [lo]. There is, however, no direct evidence for this. From measurements of the rate of metabolism of specifically labelled tyrosine in liver cell suspensions, we have shown that TAT is indeed rate limiting for tyrosine breakdown within the intact cell. Comparison of enzyme activities in extracts with the rates of flux through TAT in whole cells under various conditions indicates that the enzyme is subject to regulation by several distinct factors in vivo.

A simple and rapid assay for tyrosine aminotransferase.

The activity of hepatic tyrosine aminotransferase (TAT; L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1 S) is increased by several hormonal [l-7] and nutritional [6-91 agents. This fact, together with the short half-life in vivo, has contributed to TAT being chosen by many workers as a model for studies of hepatic enzyme turnover, both in vivo and in vitro. TAT activity, particularly in ‘fed control’ conditions, is quite low, so that appreciable amounts of sample are required for spectrophotometric procedures [lo-141 . The elaboration of techniques for handling isolated hepatocytes and liver-derived tumour cell lines dictates a parallel development of more sensitive assay systems. Spectrophotometric assays are prone to high blanks when used with crude extracts. Greater sensitivity may be achieved with radioisotopic methods, although these require physical separation of substrate and product; ion-exchange chromatography [ 15 171 and extraction in organic solvents [l&19] have been used effectively, although these are cumbersome procedures when numbers of samples are involved. In this paper we present a method using L-[side ~hain-2,3-~H] tyrosine, which is simple, rapid, convenient, cheap and applicable to measurements in large numbers of crude extracts.

Microtubule proteins in the yeast, Saccharomyces cerevisiae

Microtubule proteins, particularly tubulin, have been isolated and characterised from a range of higher eukaryote tissues [ 1,2] and cultured cells [3]. In virtually all the higher eukaryotes the main protein tub&n occurs in its native state as a heterodimer. The two constituent subunits, cr and 0 tubulin, of -55 000 mol. wt, can be separated on SDS-polyacrylamide gel electrophoresis. The microtubules and microtubule proteins of non-flagellated lower eukaryotes have received very little attention. However, organisms such as the yeast Saccharomyces cerevisiae have great potential as systems for the study of microtubule function. Studies with these organisms are hampered by the low tubulin concentration in cell extracts [4]. Tubulin from various cell types will copolymerise with brain microtubules. However, it is known that cell extracts inhibit the assembly of microtubules in vitro [5,6]. We have markedly reduced the inhibitory effects of yeast cell extracts by using RNase and DNase treatment, proteolysis inhibitors and a strain of yeast which posesses very reduced protease activity. Using the above precautions with brain microtubule protein depleted in microtubule-associated proteins we have been able to identify radiolabelled yeast proteins

Preparation and characterization of isolated parenchymal cells from guinea pig liver.

Summary1.A method is described for the preparation of isolated cells from guinea pig liver. This involves perfusionin situ, in the non-physiological direction, with collagenase.2.The cell yield was 20-30%, comparable with those from the livers of other species.3.The ratio of lactate dehydrogenase to glutamate dehydrogenase in the cells was similar to thatin vivo, indicating that there was negligible leakage of cytoplasmic enzymes.4.The concentrations of K+ and adenine nucleotides were initially lower than in the perfused liver; normal values were obtained on incubation, particularly in the presence of substrate.5.The L-lactate : pyruvate ratio is 16 : 1, close to established values. The totalβ-hydroxybutyrate : acetoacetate ratio indicates that the mitochondrial redox state is more oxidised than in the perfused liver, but the intracellular ratio is similar to that of the intact liver.6.Rates of gluconeogenesis and ureogenesis, are within the physiological range. Maximal gluconeogenesis from L-lactate was preceded by a lag period. L-lysine stimulated glucose production from L-lactate but did not abolish the lag phase.7.The effects of aminooxyacetate and octanoate on L-lactate gluconeogenesis were similar to those in the perfused liver.

Control of enzyme activities in rat liver by tryptophan and its metabolites.

Abstract Tryptophan administration increases the activities of tyrosine and tryptophan transaminases, tryptophan dioxygenase, histidase, serine dehydratase and phosphoenolpyruvate carboxykinase in the livers of fed rats; these increases are blocked by cycloheximide. In fasted rats, the changes are similar except that histidase is unaffected. In diabetic rat liver, only tryptophan dioxygenase and in adrenalectomized rat liver, tyrosine and tryptophan aminotransferases, tryptophan dioxygenase and phosphoenolpyruvate carboxyki- nase activities are increased after tryptophan. Low doses of both tryptamine and 5-hydroxytryptamine, in the presence of pargyline, increase the activities of tyrosine and tryptophan aminotransferases and phosphoenol- pyruvate carboxykinase; these effects are distinct from those of the parent aminoacid. The variation in responses indicates that a number of mechanisms may be involved. Tryptophan is, however, directly effective in increasing tyrosine aminotransferase activity in isolated liver cells.