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Biomedical subjects

H C Li

Publications and source records attributed to H C Li.

At least 19 recordsLinked to original sources
PubMed1990

[Retinoblastoma gene in human esophageal cancer].

Retinoblastoma gene (Rb) is a tumor suppressor gene. In 21 esophageal cancer and 43 pericancerous non-tumor samples obtained from patients who had undergone surgery for esophageal cancer in Linxian County, a high incidence area of esophageal cancer, Rb gene was studied. DNAs were extracted from the above esophageal tissues, then were separately digested with Hind III, EcoRI, BamHI, PstI, MspI, HpaII and subjected to Southern analysis. The Southern blots were sequentially probed with two fragments of Rb cDAN clone: 0.9 kb and 3.8 kb. Of 43 adjacent non-tumor tissues examined, 15 were found to have structural anomaly (34.9%). Seven of 21 esophageal cancers also showed structural anomaly (33.3%). In order to study the cause of Rb gene deletion, esophageal carcinoma of human fetal origin induced by N-methyl-N-benzylnitrosamine (NMBzA) in nude mice were similarly examined and found to be deleted of Rb gene. Thus, deletional inactivation of Rb gene may play an important role in the pathogenesis of esophageal cancer in this high-risk area. N-nitrosamine may play a causative role in deletion of Rb gene. The above data for the first time demonstrate structural anomaly of Rb gene in esophageal cancer and confirm that chemical carcinogens, such as N-nitrosamines, can delete the antioncogene.

Blotting, Southern↗
PubMed1989

Regional distribution of calcium/calmodulin-dependent phosphatase activity of calcineurin in rat brain.

The regional distribution of calcineurin activity (measured using p-nitrophenyl-phosphate which detects the phospho-tyrosylphosphatase activity of calcineurin) shows that the striatum, hippocampus and cerebral cortex contains high calcineurin activity. Within the striatum, calcineurin activity does not appear to be present in dopaminergic terminals, since lesions of the nigro-striatal dopaminergic pathway (which reduce striatal dopamine levels by 97%) had no effect on calcineurin activity. On the other hand, kainic acid, which destroys neurons whose perikarya are in the striatum, reduced calcineurin activity by 86% indicating that calcineurin activity is localized in striatal intrinsic neurons. Calcineurin apparently does not exist in glia, since glial cells actually proliferate in kainic acid lesioned striatal tissues.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1987

Ca2+/calmodulin-dependent phosphoprotein phosphatase activity of calcineurin in rat striatum: effect of kainic acid lesions.

Calcineurin, a Ca2+ and calmodulin (CM)-dependent phosphatase, has been shown to be present in high concentrations in the striatum. Using inhibitor-1(phosphorylated by cAMP-dependent protein kinase) as a substrate, we found Ca2+/CM-dependent phosphatase (calcineurin) to be more than 2-fold higher than non-Ca2+/CM-dependent phosphatase in the rat striatum. In order to determine the cellular localization of calcineurin, striatal kainic acid injections were used to destroy neurons whose cell bodies are present at the site of injection. Glutamic acid decarboxylase (GAD) activity was measured as an indicator of destruction of striatal GABA-ergic neurons. After intrastriatal injection of 1 and 2 ug of kainic acid, there was a significant decrease of both calcineurin and GAD. However, injection of 0.5 ug kainic acid into the striatum reduced GAD activity by 81%, but had no effect on calcineurin phosphatase activity. Thus calcineurin does not appear to be equally distributed among all types of striatal neurons, but rather may be concentrated in neurons that are less sensitive to kainic acid than the GABA-ergic neuron.

Animals↗
PubMed1986

Purification, subunit composition and regulatory properties of the ATP X Mg2+-dependent form of type I phosphoprotein phosphatase from bovine heart.

The ATP X Mg2+-dependent phosphoprotein phosphatase has been purified from bovine heart to near-homogeneity. It is a heterodimer (75 kDa) consisting of a catalytic (C) subunit (40 kDa) and a regulatory (R) subunit (35 kDa). The R subunit, which is identical to inhibitor-2, is transiently phosphorylated during activation of the enzyme catalyzed by phosphatase-1 kinase (FA). Maximal activation requires preincubation of the phosphatase with FA and ATP X Mg2+. However, relatively low yet definitively demonstrable basal activity can be expressed by Mg2+ alone (ranging from 3% to 10% of the FA X ATP X Mg activity, depending on the degree of endogenous proteolytic damage of the phosphatase during purification), but not by either FA or ATP alone. Limited trypsinization results in a rapid and total degradation of the R subunit and partial degradation of the 40-kDa C subunit to active proteins of 35-38 kDa. The resulting 'nicked' C subunit of 35-38 kDa is no longer dependent on FA for activation and can be fully activated by Mg2+ (or Mn2+) alone. Endogenous proteolytic damage of the R subunit also results in an increase of activity that can be expressed by M2+ alone with a concomitant decrease of the FA-dependent activation. Although Mn2+ is slightly more effective than Mg2+ in expressing the holoenzyme basal activity, the activation by Mn2+ is only about 60% of that of Mg2+ when FA and ATP are also present. In the activation by adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), Co2+ is the most effective cofactor. The activation by ATP[gamma S] X Co2+ is more than 50% of that by ATP X Mg2+. The present studies indicate that Mg2+ is the natural divalent cation for the FA-catalyzed activation in which Mg2+ plays two distinctly different roles: it forms Mg2+ X ATP which serves as a substrate for the kinase; it acts as an essential cofactor for the catalytic function of the phosphatase. The discrepancies between the results obtained by this and other laboratories with respect to the effectiveness of Mg2+ and ATP[gamma S] in the activation of the phosphatase are discussed.

Adenosine Triphosphate↗
PubMed1985

On the mechanism of regulation of type I phosphoprotein phosphatase from bovine heart. Regulation by a novel intracyclic activation-deactivation mechanism via transient phosphorylation of the regulatory subunit by phosphatase-1 kinase (FA).

Adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) can substitute for ATP in the activation of the ATP X Mg2+-dependent form of bovine heart type I protein phosphatase (Mr = 75,000) catalyzed by phosphatase-1 kinase (FA). ATP gamma S activates the enzyme to a lower level than ATP, but it phosphorylates the regulatory (R)-subunit to a much higher extent. An [35S]phosphatase-1 [( 35S]E-P) has been isolated, identified, and shown to be a key intermediate in the activation reaction. Treatment of [35S]E-P with dimethyl suberimidate results in cross-linking of the Mr = 34,000 [35S]R-subunit with the Mr = 40,000 catalytic (C)-subunit to form a Mr = 75,000 species, indicating that phosphorylation is not accompanied by dissociation of the holoenzyme. The catalytically active form (Ea) is not the phosphorylated enzyme intermediate. Instead, Ea is directly produced from the intermediate by a Mg2+-dependent, intramolecular autodephosphorylation reaction. The isolated Ea derived from [35S]E-P or from ATP-activated phosphatase-1 has the same half-life (23 min at 30 degrees C). It spontaneously deactivates, via an intramolecular process, to a resting state (Er) which can be fully reactivated by FA X ATP X Mg2+. The deactivation of Ea can be accelerated by chelators, PPi greater than ATP X Mg2+ blocks the PPi effect. Limited trypsinization selectively digests the R-subunit and the resulting C-subunit is Mg2+-dependent. Based on the present data, a novel intracyclic activation-deactivation mechanism via transient phosphorylation of the R-subunit is proposed for regulation of phosphatase-1. (formula; see text).

Adenosine Triphosphate↗
PubMed1985

Activation of bovine heart ATP-MG2+-dependent phosphoprotein phosphatase: isolation of a phosphoenzyme intermediate and its conversion to the active form via a Mg2+-dependent autodephosphorylation reaction.

The ATP-Mg2+-dependent protein phosphatase, a holoenzyme form of type I protein phosphatase (phosphatase-1) requires the action of phosphatase-1 kinase (FA) for activation. The enzyme (75 kDa) purified from bovine heart consists of a catalytic (C) and a regulatory (R) subunit of 40 kDa and 34 kDa, respectively, and activation is associated with phosphorylation of the R-sub-unit. A procedure has been developed for isolation of [32P]phosphatase-1 ( [32P]E-P) in non-denatured form. In the absence of divalent cation, [32P]E-P is catalytically inactive and the phosphorylation is stable. Addition of Mg2+ triggers autodephosphorylation of [32P]E-P with concomitant generation of phosphorylase phosphatase activity. The autodephosphorylation/activation process is dependent on Mg2+ concentration. The KA value for Mg2+ is 0.6 mM. The phosphorylase phosphatase activity generated from the release of 1 mol. 32P is 1.1 X 10(12) units which is equivalent to 15,000 units per mg enzyme protein. The present findings provide direct evidence that the phosphorylated phosphatase-1 is not the active form (Ea). Instead, Ea is directly produced from the intermediate by a Mg2+-dependent autodephosphorylation reaction.

Adenosine Triphosphate↗
PubMed1985

A major phosphotyrosyl-protein phosphatase from bovine heart is associated with a low-molecular-weight acid phosphatase.

The phosphotyrosyl [Tyr(P)]-immunoglobulin G (IgG) phosphatase activity in the extracts of bovine heart, bovine brain, human kidney, and rabbit liver can be separated by DEAE-cellulose at neutral pH into two fractions. The unbound fraction exhibits a higher activity at acidic than neutral pH while the reverse is true for the bound fraction. Of all tissues examined, the Tyr(P)-IgG phosphatase activity in the unbound fraction measured at pH 5.0 is higher than that in the bound fraction measured at pH 7.2. The acid Tyr(P)-IgG phosphatase activity has been extensively purified from bovine heart. It copurified with an acid phosphatase activity (p-nitrophenyl phosphate (PNPP) as a substrate) throughout the purification procedure. These two activities coelute from various ion-exchange and gel filtration chromatographies and comigrate on polyacrylamide gel electrophoresis, indicating that they reside on the same protein molecule. The phosphatase has a Mr = 15,000 by gel filtration and exhibits an optimum between pH 5.0 and 6.0 when either Tyr(P)-IgG-casein or PNPP is the substrate. It is highly specific for Tyr(P)-protein with little activities toward phosphoseryl [Ser(P)]- or phosphothreonyl [Thr(P)]-protein. The enzyme activities toward Tyr(P)-casein and PNPP are strongly inhibited by microM molybdate and vanadate but insensitive to inhibition by L(+)-tartrate, NaF, or Zn2+. The molecular and catalytic properties of the acid Tyr(P)-protein phosphatase purified from bovine heart are very similar to those of the low-molecular-weight acid phosphatases of Mr = 14,000 previously identified and purified from the cytosolic fraction of human liver, placenta, and other animal tissues.

Acid Phosphatase↗
PubMed1984

Activation of brain calcineurin towards proteins containing Thr(P) and Ser(P) by Ca2+, calmodulin, Mg2+ and transition metal ions.

Using Thr(P)-inhibitor-1 and Ser(P)-casein as substrates, studies on the activation of calcineurin purified from bovine brain have been carried out. The phosphatase requires the synergistic action of Ca2+, calmodulin and another divalent cation (Mg2+, Mn2+, Co2+ or Ni2+, but not Zn2+) for full expression of its activity. Ca2+ and Ca2+ X calmodulin act as allosteric activators to transform the phosphatase to a relaxed conformation, while Mg2+ acts solely as a cofactor for the catalytic action of the enzyme. In addition to their function as cofactors for catalysis, transition metal ions can also substitute for Ca2+ as allosteric activators. Ca2+ and calmodulin exert their activating effects mainly by increasing the Vm of the phosphatase reaction with little effect on the Km values for the substrates or on the KA values for the divalent cation cofactors. The predominant factor in dictating the catalytic properties of calcineurin is the divalent cation cofactor. For example, with Mg2+ as a cofactor, the phosphatase exhibits an optimum around pH 8.0-8.5; while with a transition metal ion as a cofactor, the optimum is around pH 7.0-7.5, regardless of whether Thr(P)-inhibitor-1 or Ser(P)-casein serves as a substrate, in the absence or the presence of Ca2+ X calmodulin.

Animals↗
PubMed1984

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+.

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.

Animals↗
PubMed1984

Characterization of phosphotyrosyl-protein phosphatase activity associated with calcineurin.

Calcineurin purified from bovine brain is shown to possess phosphotyrosyl -protein phosphatase activity towards proteins phosphorylated by the epidermal growth factor receptor/kinase. The phosphatase activity is augmented by Ca2+/calmodulin or divalent cation (Ni2+ greater than Mn2+ greater than Mg2+ greater than Co2+). In the simultaneous presence of all three effectors, the enzymatic activity is synergistically increased. Ca2+/calmodulin activates the Mg2+-supported activity by decreasing the Km value for phosphotyrosyl -casein from 2.2 to 0.6 microM, and increasing the Vmax from 0.4 to 4.6 nmol/min/mg. These results represent the first demonstration that calcineurin can dephosphorylate phosphotyrosyl -proteins and suggest a novel mechanism of activation of this enzyme.

Animals↗
PubMed1984

A phosphotyrosyl-protein phosphatase activity associated with acid phosphatase from human prostate gland.

Using [32P]P-Tyr-IgG and [32P]P-Tyr-casein phosphorylated by pp60v-src as substrates, studies on the phosphotyrosyl-protein phosphatase activity in human prostate gland indicate that it is associated with prostatic acid phosphatase. Evidence to support this conclusion include the following: (a) these two enzymatic activities co-purify to apparent homogeneity; (b) they co-migrated on polyacrylamide gel electrophoresis, ion-exchange and gel filtration chromatographies; (c) the exhibit identical thermostability; and (d) the phosphotyrosyl-protein phosphatase activity is sensitive to inhibition by p-nitrophenyl phosphate and by several classical inhibitors of prostatic acid phosphatase including L(+)-tartrate, molybdate, vanadate and NaF. The purified enzyme exhibits high specificity towards phosphotyrosyl-proteins with little activity towards several phosphoseryl-proteins and phosphothreonyl-proteins examined. The present findings indicate that prostatic acid phosphatase may function in vivo as a phosphotyrosyl-protein phosphatase.

Acid Phosphatase↗
PubMed1983

Multiple forms of phosphotyrosyl- and phosphoseryl-protein phosphatase from cardiac muscle: partial purification and characterization of an EDTA-stimulated phosphotyrosyl-protein phosphatase.

Chromatography of cardiac muscle and brain extracts on DEAE-cellulose resolved phosphotyrosyl-protein phosphatase activity into three fractions, termed Y-1, Y-2, and Y-3. These were eluted at 0.05, 0.15, and 0.3 M KCl, representing about 33, 55, and 12%, respectively, of the enzymatic activity recovered from the resin. Comparative studies demonstrated that the properties of phosphatases Y-1, Y-2, and Y-3 were distinctly different from those of previously identified phosphoseryl-protein phosphatases-1, -2, -3, and -4. Phosphatases Y-1, Y-2, and Y-3 were stimulated by EDTA, and exhibited optimal activity at neutral pH. These properties were different from those of the two minor phosphotyrosyl-protein phosphatase activities associated with phosphoseryl-protein phosphatases-3, and -4, which were divalent cation dependent, and exhibited optimal activity at alkaline pH. Further purification of phosphatase Y-2 from bovine heart has been carried out. The enzyme had a Mr = 65,000 (Stokes radius = 3.8 nm; S020,W = 4.1). Its activity was stimulated by 5- to 10-fold in the presence of EDTA (Ka = 15 microM) and was strongly inhibited by micromolar concentrations of vanadate. Phosphatase Y-2 was highly specific for phosphotyrosyl-IgG and -casein, and showed little activity toward phosphoseryl-casein, -phosphorylase a, phosphothreonyl-inhibitor-1, and p-nitrophenyl phosphate. The present studies indicate that phosphotyrosyl-protein phosphatase activity in animal tissues exists in multiple forms. The major active species are specific for phosphotyrosyl proteins, and represent enzymes different from the known phosphoseryl-protein phosphatases and p-nitrophenyl phosphatases.

Animals↗
PubMed1983

Characterization of a phosphotyrosyl protein phosphatase activity associated with a phosphoseryl protein phosphatase of Mr = 95,000 from bovine heart.

A cytosolic phosphoprotein phosphatase of Mr = 95,000 purified from bovine cardiac muscle, which contains a catalytic subunit of Mr = 35,000, is known to be associated with a Mg2+-activated p-nitrophenyl phosphatase activity. We have found that the enzyme preparation is also active toward phosphotyrosyl-IgG and -casein phosphorylated by pp60v-src, the transforming gene product of Rous sarcoma virus. The properties of this phosphotyrosyl protein phosphatase activity closely resemble those of the p-nitrophenyl phosphatase activity but sharply differ from those of the phosphorylase phosphatase activity. Comparative studies of the activities of the Mr = 95,000 phosphatase, bovine kidney alkaline phosphatase, and ATP X Mg-dependent phosphatase toward phosphoseryl, phosphothreonyl, and phosphotyrosyl proteins and p-nitrophenyl phosphate under various conditions have been carried out. The results indicate that the Mr = 95,000 enzyme exhibits higher activity toward phosphoseryl and phosphothreonyl proteins than toward phosphotyrosyl proteins, while the kidney alkaline phosphatase preferentially dephosphorylates phosphotyrosyl proteins. ATP X Mg-dependent phosphatase is inactive toward phosphotyrosyl proteins.

Animals↗