Non-dependence on native structure of pig liver pyruvate kinase when used as a substrate for cyclic 3',5'-AMP-stimulated protein kinase.

Alkali-inactivated pig liver pyruvate kinase, type L, and a cyanogen bromide fragment from the same enzyme were shown to be phosphorylated by (³²P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase. In both cases the rate of phosphorylation was higher than with the native enzyme. Pyruvate kinases types A and M were not phosphorylated under the same conditions. From the ³²P-labelled cyanogen bromide fragment (³²P)phosphorylserine was isolated. The electrophoretic pattern of (³²P)phosphopeptides obtained on partial acid hydrolysis of the fragment indicated that the phosphorylated site of the fragment was identical with that of the native pyruvate kinase.     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.     

Regulation of Sertoli cell cyclic adenosine 3':5' monophosphate phosphodiesterase activity by follicle stimulating hormone and dibutyrl cyclic AMP

Total phosphodiesterase activity was measured in Sertoli cell culture after exposure to isobutyl-methyl-xanthine, dibutyryl cyclic AMP and FSH. After 24 hr of incubation both FSH and dibutyryl cAMP caused a significant increase in total phosphodiesterase activity of Sertoli cell homogenates (control: 66 ± 16 pmoles/min/mg protein; FSH: 291 ± 25 pmoles/min/mg protein; dibutyryl cAMP: 630 ± 70 pmoles/min/mg protein). FSH stimulation was potentiated by isobutyl-methyl-xanthine. Both in the presence and absence of xanthine, the induction of phosphodiesterase was dependent on the FSH concentration, with maximal stimulation achieved with 0.5–1.0 μg FSH/ml. The induction of phosphodiesterase activity by hormone was abolished by cycloheximide treatment. The data suggest that FSH regulates phosphodiesterase activity via changes of cAMP levels in Sertoli cell in culture.     

A difference in the affinity labeling of Ca2+, Mg2+-activated ATPases of normal and unc A strains of Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate

The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca²⁺, Mg²⁺-activated ATPase of $\text{Escherichia}\text{coli}$. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an $\text{unc A}$ mutant of $\text{E.}\text{coli}$ was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.     

Catabolism of capped (3'-5')- and (2'-5')-adenylates in rat liver nuclei

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.     

我国两株登革2型病毒5′和3′端非编码区序列测定及二级结构分析

 为测定我国两株临床症状、乳鼠神经毒力不同的登革 2型病毒流行株 5′和 3′端非编码区序列 (untranslated region,UTR) ,分析二级结构差异与毒力变化的关系 ,分别从 D2 - 0 4、D2 - 44株感染的 C6/ 36细胞及鼠脑中提取总 RNA.以该 RNA为模板 ,利用 RACE法 ,分别扩增了 D2 - 0 4、D2 -44株的 5′和 3′末端 c DNA片段 .将其分别与 p GEM- T载体连接得到重组质粒 ,测定上述 c DNA插入片段的序列 .用 RNAdraw软件预测 D2 - 0 4、D2 - 44株 5′和 3′端非编码区的二级结构 .D2 - 0 4、D2 -44株 5′端和 3′端非编码区分别有 96和 454个核苷酸 .其中 5′非编码区 59位 C(D2 - 0 4 )→T(D2 -44 ) ,使 D2 - 44二级结构稳定性下降 ;3′端非编码区有 1 5个核苷酸不同 ,其中 T(355)→ A,T(32 6)→ G引起了所在位置二级结构自由能变化 ,且分别位于两个保守序列区 (conserved sequence,CS)CS1、CS2 A.这些位点变化可能与毒力有关 .     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

Guanosine 3',5'-monophosphate and the action of alkylating agents.

The intracellular level of guanosine 3',5'-monophosphate (cGMP) has been measured in Walker carcinoma cells in tissue culture after treatment with various alkylating agents. At concentrations which caused a rise in the level of adenosine 3',5'-monophosphate (cAMP) chlorambucil and 5-(1-aziridinyl)-2,4-dinitrobenzamide (CB 1954) produced only a small (35%) elevation of cGMP, while merophan had no such effect. This suggests that any effect of cAMP will not be outweighed by an equivalent rise in cGMP. Sepcific cytosolic binding of cGMP decreased with increasing resistance of Walker cells to alkylating agents, while the dissociation constant, KD, for binding increased. This was also observed with cAMP binding which suggests that the same protein in responsible for binding both nucleotides.     

Inhibition of ADP-ribosylation of histone by diadenosine 5', 5"' -p (1), p(4)-tetraphosphate

Diadenosine 5′, 5?-p¹, p⁴-tetraphosphate (Ap4A) strongly inhibited ADP-ribosylation reaction of histone by purified bovine thymus poly(ADP-ribose) polymerase. This compound showed a relatively weak inhibitory effect on Mg²⁺-dependent, enzyme-bound poly(ADP-ribose) synthesis. Among various adenine nucleotides tested, including several diadenosine nucleotides with varying phosphate chain length, Ap4A was the most effective inhibitor of the histone-modification reaction. Ap5A and Ap6A showed slightly lower inhibitory effect than Ap4A. Kinetic analysis of the inhibitor (Ap4A) with varying concentration of substrate (NAD⁺) revealed that this compound is a “mixed type inhibitor”, with a Ki value of 5.1 μM.     

3',5' Cyclic nucleotide phosphodiesterase from human platelets: effect of heat upon the multiple forms and their interconversion

A 33,000 g supernatant from human platelets showed a biphasic heat inactivation curve at 45, 50 and 55 degrees C of the cAMP and cGMP phosphodiesterase. This could suggest the presence of two differently heat sensitive phosphodiesterases. However, a preparation heated for 30 min at 55 degrees C, where only the apparently thermostable form of the enzyme remained, still displayed the same characteristics as the starting material, i.e. two apparent Km values for cAMP, a cAMP specific activity lower at low protein concentration (less than 50 micrograms/ml) than at high protein concentration(greater than 100 micrograms/ml), and three peaks of activity upon linear sucrose density gradient. Moreover, a biphasic inactivation curve was again observed after a second heat treatment. These results demonstrated that the heat effect is not a simple protein denaturation of one of two independent species. A study at different temperatures of the profile of the cAMP phosphodiesterase upon sucrose gradient demonstrated that the dissociated form was predominant at high temperature whereas lower temperature favored the associated form. During heat treatment, the dissociated form is at first denatured and this leads to a shift in the equilibrium between the associated and dissociated forms of the phosphodiesterase in favor of the dissociated form. From the overall results, one can draw a model for phosphodiesterase regulation by dissociation-reassociation.     

The effect of hormones on synthesis of the region linking chondroitin sulfate to protein

1. 1. Particulate fractions of costal cartilage from young rats are capable of catalyzing the formation of the first two monosaccharide units of the chondroitin sulfate-protein linkage region.

2. 2. Hormonal imbalance has been shown to influence the activity of the glycosyltransferases responsible for the sequential transfer of xylose and galactose from UDPxylose and UDPgalactose, respectively, in the formation of the linkage region.

3. 3. The activity of xylosyltransferase was found to be decreased in costal cartilage of diabetic, thyroidectomized and hypophysectomized rats, but not in rats injected with either testosterone or hydrocortisone. In the latter two treatment groups, galactosyltransferase activity was decreased only in the group receiving hydrocorsitone.

4. 4. The combined results of this and previous studies suggest that decreased levels of chondroitin sulfate in diabetic, thyroidectomized and hypophysectomized animals are due to interference in the synthesis of the linkage region of the proteoglycan at the xylosyltransferase level whereas hydrocortisone acts primarily at the level of the galactosyltransferase.