PubMed Health⌕ Search

Biomedical subjects

J L Hargrove

Publications and source records attributed to J L Hargrove.

At least 37 records · Page 2Linked to original sources
PubMed1984

Organ specificity of glucocorticoid-sensitive tyrosine aminotransferase. Separation from aspartate aminotransferase isoenzymes.

In order to study whether hormone-sensitive tyrosine aminotransferase exists in tissues other than liver, we have devised means to separate the liver-specific enzyme from other enzymes that transaminate tyrosine and to distinguish between the authentic enzyme and the principal "pseudotyrosine aminotransferases," which are the isoenzymes of aspartate aminotransferase. We accomplish this by suppressing proteolysis of the authentic enzyme using a buffer of pH 8.0 containing 0.1 M potassium chloride; enzyme extracted from liver in this buffer migrates as a single peak during chromatography on hydroxylapatite and represents the undegraded native form. A much smaller peak of tyrosine aminotransferase activity elutes at higher ionic strength and corresponds to a mixture of mitochondrial aspartate aminotransferase and partially degraded tyrosine aminotransferase. Cytosolic aspartate aminotransferase, in contrast, adsorbs weakly to the hydroxylapatite column and transaminates tyrosine very poorly although it readily utilizes monoiodotyrosine. The aspartate aminotransferase isoenzymes separate completely from tyrosine aminotransferase during chromatography on DEAE-Sepharose CL-6B. By combining these techniques with the use of specific antibodies, we show that brain, heart, and kidney do not contain tyrosine aminotransferase. Furthermore, we locate both isoenzymes of aspartate aminotransferase on polyacrylamide gels and show that both react histochemically as tyrosine aminotransferases when monoiodotyrosine is used as substrate. Use of these techniques, therefore, permits unambiguous identification of tyrosine aminotransferase and its separation from the background of nonspecific transamination.

Animals↗
PubMed1983

Inhibition by sodium butyrate of enzyme induction by glucocorticoids and dibutyryl cyclic AMP. A role for the rapid form of histone acetylation.

We have found that butyrate selectively inhibits hormonal induction of a few specific proteins and messenger RNAs in hepatoma cells. The fatty acid salt reversibly abolishes induction of tyrosine aminotransferase by dexamethasone and dibutyryl cyclic AMP in HTC cells by inhibiting the production of tyrosine aminotransferase messenger RNA. Half-maximal inhibition of enzyme induction occurred in 0.9 mM butyrate. This effect is highly specific, since 4 h after the addition of butyrate to induced HTC cells, the relative abundance of only five messenger RNA species out of several hundred observable on two-dimensional gels of translational products is changed. Upon removal of the butyrate from cell cultures pretreated with dexamethasone, tyrosine aminotransferase activity begins to increase more rapidly than if dexamethasone is added to control cultures, indicating that part of the induction process occurs in the presence of butyrate. A dose-dependent reduction of fast histone acetylation by butyrate was demonstrated by treating cells with butyrate followed by a short pulse with [3H]acetate and chase in a high concentration of butyrate. The butyrate concentration test range over which rapid histone acetylation is inhibited is similar to that which inhibits enzyme induction to the same extent. In contrast, the slow form of histone acetylation is unaffected in the concentration range examined. The induction of tyrosine aminotransferase by dexamethasone is delayed in hypoacetylated cells. This lag is consistent with the time required to initiate the recovery of the fast form of histone acetylation after its transient disappearance (Covault, J., Perry, M., and Chalkley, R. (1982) J. Biol. Chem. 257, 13433-13440). We conclude that sodium butyrate interferes with the ability of dexamethasone and dibutyryl cyclic AMP to increase production of several specific species of messenger RNA in hepatoma cells. This effect correlates well with its ability to reduce rapid acetylation of histones in HTC cells; we discuss potential roles of rapid histone acetylation in modulating hormonal stimulation of transcription.

Acetylation↗
PubMed1983

Regulation of the synthesis of tyrosine aminotransferase: the relationship to mRNATAT.

The activity of the hepatic enzyme tyrosine aminotransferase (TAT) is the sum of many diverse regulatory factors. These include the developmental stage of the animal, the hormonal and nutritional environment of the animal (or tissue culture cell), other extrinsic and intrinsic regulatory cycles and factors (including cytoplasmic substances), and chromatin structure. Although TAT is subject to a number of post-translational modifications, alterations in catalytic activity always parallel changes in enzyme amount. In a few instances this is due to a selective change in TAT degradation, but most are due to changes in the rate of aminotransferase synthesis. Recent studies have shown that TAT synthesis is generally directly correlated with the activity, and presumably amount, of the mRNA that codes for tyrosine aminotransferase.

Adrenalectomy↗
PubMed1982

Inhibition of hepatoma cell growth by analogs of adenosine and cyclic AMP and the influence of enzymes in mammalian sera.

The following evidence suggests that inhibition of hepatoma cell (HTC) growth by cyclic nucleotides is an adenosine-like effect that is greatly modified by the type and treatment of serum used in the culture medium and is probably not mediated by cyclic AMP-dependent protein kinase: 1) Heating serum reduces its phosphodiesterase content, thereby slowing metabolism of cyclic AMP and reducing the inhibition of HTC cell growth by cyclic AMP; 2) Using medium that contains phosphodiesterase but lacks adenosine deaminase causes adenosine to accumulate from cyclic AMP and increases the toxicity of cyclic AMP; 3) Uridine or cytidine reverses the growth inhibition caused by adenosine, 5'-AMP or cyclic AMP; 4) adenosine, 5'-AMP and N6-(delta 2-isopentenyl) adenosine are more toxic for HTC cells than is cyclic AMP, and N6,O2-dibutyryl cyclic AMP is not toxic; and 5) N6,O2'-dibutyryl cyclic AMP inhibits growth of Reuber H35 cells, but uridine prevents this inhibition of growth. We conclude that most, if not all, of the inhibitory effects of cyclic AMP and N6,O2'-dibutyryl cyclic AMP on HTc and Reuber H35 hepatoma cell growth are due to the generation of toxic metabolites.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Physical properties, limited proteolysis, and acetylation of tyrosine aminotransferase from rat liver.

The native and one of the modified forms of tyrosine aminotransferase were purified from rat liver and characterized. Several hydrodynamic properties of the native enzyme are: Stokes radius, 46 A; subunit isoelectric point, 5.6; sedimentation coefficient, 5.6 S, frictional ratio, 1.44; diffusion coefficient, 4.65 X 10(-7) cm2 s-1; extinction coefficient of a 1% solution (w:v) at 280 nm, 10.5 cm-1. The molecular weight of the dimeric protein is 110,500 as calculated from the Stokes radius and sedimentation coefficient. The subunit of the modified form is of lower molecular weight than the subunit of the native enzyme and has a pI of about 5.9. During isoelectric focusing, both forms of the enzyme separate into two components. The more acidic component that is resolved from the native enzyme is phosphorylated, but the other component is not. The amino acid composition of native tyrosine aminotransferase differs from values reported for mixtures of the three forms of this enzyme. Neither the native nor the modified forms of the enzyme possess a free alpha-amino group as judged by dansylation, nor can they be digested with leucine aminopeptidase, implying that the NH2-terminus is blocked. The possibility that tyrosine aminotransferase is acetylated was examined by translating poly(A)+RNA from hepatoma cells in a cell-free translational system in the presence and absence of inhibitors of protein acetylation. [35S]Tyrosine aminotransferase synthesized in the presence of the inhibitors has a more basic isoelectric point than the native enzyme as determined by isoelectric focusing, suggesting that the enzyme is acetylated either at the NH2-terminal or the epsilon-amino group of an internal lysine. When digested by either of two lysosomal proteases, tyrosine aminotransferase is cleaved to a smaller size. These data show that tyrosine aminotransferase is susceptible to several post-translational modifications.

Amino Acids↗
PubMed1978

Species differences in contractility of seminiferous tubules and tunica albuginea as related to sperm transport through the testis.

Frequency and depth of contractions and tonus--i.e., diameter of the tubule or shift of the baseline for the capsule--of both the seminiferous tubules and the tunica albuginea were quantified for rat, mouse, rabbit, hamster and ground squirrel tests. In the ground squirrel, contractility was measured prior to, during and after breeding. The rat testes had the strongest tubular contractions; those of the mouse, squirrel and rabbit exhibited intermediate values, and the hamster tests had the weakest tubular contractions. The rabbit tests exhibited the strongest, most well-developed rhythmical capsular contractions while the rat, mouse, hamster and squirrel tests showed no definite rhythmical contractions. Hamster tests apparently depend primarily upon fluid secretion by the seminiferous tubules for sperm transport through the tests to the epididymis, while the squirrel, rat and mouse tests utilize both fluid secretion and some tubular contractions. The rat apparently utilizes tubular contractions more so than the other rodents studied for sperm transport.

Animals↗
PubMed1976

Indomethacin induces rat uterine contractions in vitro and alters reactivity to calcium and acetylcholine.

The initial contractions of uteri in vitro from castrated, estrogen-treated rats, were markedly diminished following replacement with fresh bathing medium. Indomethacin and aspirin (10(-5) to 10(-4)M) strongly stimulated such quiescent preparations and reduced their subsequent responsiveness to Ca++. Reintroducing the initial bathing medium (which contained prostaglandin-like material), or adding prostaglandin F2alpha to the fresh medium, initiated uterine contractions and restored responsiveness to calcium ion. Injections of indomethacin into castrated, estrogen-treated rats reduced initial in vitro uterine motility, abolished production of prostaglandin-like compounds, and prevented either indomethacin, aspirin, or Ca++ from stimulating uterine contractions. Uterine responsiveness to acetylcholine in vitro was significantly reduced in rats pretreated with indomethacin.

Acetylcholine↗
PubMed1975

Rabbit testicular contractions: bimodal interaction of prostaglandin E1 with other agonists.

Contractions of rabbit testicular capsules in vitro and in vivo in response to prostaglandin F2a, epinephrine, and acetylcholine were measured in the presence of prostaglandin E1 (PGE1). Contractions induced by these agents were markedly inhibited at 10-7 M concentrations of PGE1 in both preparations. Even so, lower levels of PGE1 (10-8 or 10-9 M) potentiated epinephrine- or acetylcholine-induced stimulation of increased tonus in vovo. Epinephrine, PGE1, and PGF2a caused increased tonus and contractions in vitro and in vovo; however, acetylcholine stimulated the testicular capsule much more in vivo than in vitro. The control log dose-response curve for epinephrine stimulation showed a slight decrease in the upper concentration range. Propranolol, a beta-adrenergic blocking agent, more than overcame this decrease and also prevented a theophylline inhibition of epinephrine-induced contraction. Prostaglandin E1's bimodal effect gives it the ability to modulate testicular contractions by increasing stimulation by other agonists in vovo at low concentrations of PGE1 but inhibiting it at higher concentrations. Thus PGE1 is a potential regulator of testicular capsular motility and possibly sperm transport and hemodynamics.

Acetylcholine↗