Pteroylpolyglutamate hydrolase from human jejunal brush borders. Purification and characterization

Pteroylpolyglutamate hydrolase was solubilized with Triton X-100 from human jejunal mucosal brush borders and purified approximately 5,000-fold using organomercurial affinity chromatography, DEAE-cellulose chromatography, and gel filtration. The apparent molecular weight of the purified enzyme in the Triton micelle was estimated as 700,000 using Bio-Gel A-1.5m gel filtration. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis followed by Coomassie stain demonstrated two polypeptide bands at 145,000 and 115,000 daltons. The purified enzyme had an isoelectric point of 7.2, was maximally active at pH 5.5, and was stable above pH 6.5 and at temperatures up to 65 degrees C for at least 90 min. Human jejunal brush-border pteroylpolyglutamate hydrolase is an exopeptidase which liberated [14C]Glu as the sole labeled product of PteGlu2[14C]Glue (where PteGlun represents pteroylpolyglutamate), failed to liberate a radioactive product from PteGlu2[14C]GluLeu2, and released all possible labeled PteGlun products during incubation with Pte[14C]GluGlu6 with the accumulation of Pte[14C]Glu. PteGlu2, PteGlu3, and PteGlu7 were substrates, each with Km = 0.6 microM, whereas PteGlu was a weak inhibitor of the hydrolysis of PteGlu3 with Ki = 20 microM. Components of the pteroyl moiety, Glu, and short chain Glun in alpha or gamma linkages were not inhibitory. The enzyme was activated by Zn2+ or Co2+. The properties of brush-border pteroylpolyglutamate hydrolase are different from those described for the soluble intracellular pteroylpolyglutamate hydrolase in other species and in human mucosa, yet are consistent with previous data on the process of hydrolysis of PteGlun in the intact human intestine.     

Transient-state kinetics of phosphoenzyme transformation in the rabbit skeletal sarcoplasmic reticulum calcium-dependent adenosine triphosphatase reaction. Two distinct modes of ADP and K+ regulation

We have observed two modes each of ADP and K+ regulation of phosphoenzyme (EP) intermediates formed in the early phase of skeletal sarcoplasmic reticulum hydrolysis of ATP at 20 degrees C, using, for the first time, a five-syringe quench flow apparatus for transient-state kinetic measurements. The total acid-stable EP formed for 20.5 and 116 ms in the K+ medium appears to be composed of either two monomers in rapid equilibrium, E1P in equilibrium E'1P, or a dimer of the two subunits, PE1E'1P. The ADP-sensitive E1P may form an acid-labile ADP X E1P (or ATP X E1) complex rapidly, giving ATP as a consequence of acid quenching. The ADP may also induce decomposition of the ADP-reactive E'1P. Monomeric and dimeric mechanisms are introduced to account for the hyperbolic relation between the rate constant of the ADP-induced E'1P decomposition and [ADP], consistent with the fact that the E'1P may also give ATP in the presence of ADP. As to the K+ effects, the K+, which is bound to the unphosphorylated enzyme and possibly becomes occluded during EP formation, may either facilitate the one-to-one E1P in equilibrium E'1P equilibrium or maintain the dimeric functional unit. The subsequent forward transformation of the E'1P to the ADP-insensitive K+-sensitive E'2P, possibly the rate-determining step for the catalytic cycle, is found to be K+ independent. The major effect of the K+ in the medium is its catalytic cleavage of the E'2P, which is detected as the missing EP under these conditions. When K+ is not involved in the EP formation, the forward sequential transformation E1P----E'1P----E'2P----E2P or PE1E'1P----PE'2E2P is apparent in the time range from 20.5 to 116 ms after EP formation, and the E'2P may accumulate in the K+ devoid medium and be detected as the major component of the total acid-stable EP. The Mg2+-sensitive E2P represents the EP missing in the medium containing no ADP and K+.     

Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS- 1

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.     

Reorganization of alpha-actinin and vinculin induced by a phorbol ester in living cells

We have used fluorescent analogue cytochemistry, image intensification, and digital image processing to examine the redistribution of alpha-actinin and vinculin in living cultured African green monkey kidney (BSC-1) cells treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Before treatment, microinjected alpha-actinin shows characteristic distribution along stress fibers and at adhesion plaques; vinculin is localized predominantly at adhesion plaques. Soon after the addition of TPA, highly dynamic membrane ruffles begin to form. These incorporate a large amount of alpha-actinin but little vinculin. Alpha-actinin is subsequently depleted, more or less uniformly, from stress fibers. Disrupted stress fibers often fragment into aggregates and move into the perinuclear region. Careful analyses of fluorescence intensity distribution indicate that alpha-actinin is depleted more rapidly from adhesion plaques than from stress fibers. Furthermore, the depletion of alpha-actinin from adhesion plaques is also faster than either the depletion of vinculin or the disappearance of focal contacts. These observations indicate that TPA may initiate disruption of stress fibers by interfering with a link between alpha-actinin and vinculin, causing alpha-actinin to be preferentially depleted from adhesion plaques.     

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Schistosoma mansoni. A potential target for chemotherapy

Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities are essential for the supply of guanine nucleotides in Schistosoma mansoni schistosomules. In crude extracts of adult S. mansoni, these two activities co-elute in size exclusion, ion exchange, and chromatofocusing chromatography and exhibit similar stabilities to heat treatment, suggesting that they are associated in one enzyme protein hypoxanthine-guanine phosphoribosyltransferase. This enzyme has been purified by a combination of heat treatment at 85 degrees C and chromatofocusing chromatography with elution at an apparent pI of 5.27 +/- 0.15. Pore gradient electrophoresis of the native enzyme followed by subsequent activity staining demonstrate an enzyme molecular weight of 105,000. The activity staining pattern remains the same whether hypoxanthine or guanine is used as the substrate, further supporting the existence of a single protein, hypoxanthine-guanine phosphoribosyltransferase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein results in a single protein band with a subunit molecular weight estimate of 64,000, suggesting that the native enzyme is a dimer. Preliminary kinetic studies showed that the purified hypoxanthine-guanine phosphoribosyltransferase reacted with guanine at a rate twice as fast as it did with hypoxanthine, but it did not act on xanthine at all. A full-length mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase cDNA clone pHPT5 and a plasmid pSV2-gpt containing the xanthine-guanine phosphoribosyltransferase gene for Escherichia coli were utilized as probes on Southern blots of S. mansoni DNA digests, and no significant hybridization was found under relatively relaxed conditions. Polyclonal antibodies made against human erythrocyte hypoxanthine-guanine phosphoribosyltransferase and E. coli xanthine-guanine phosphoribosyltransferase were tested in enzyme-linked immunosorbent assays of S. mansoni protein extracts, and no detectable cross-reacting protein was found. S. mansoni hypoxanthine-guanine phosphoribosyltransferase thus may bear rather limited homology to mammalian hypoxanthine-guanine phosphoribosyltransferase or bacterial xanthine-guanine phosphoribosyltransferase and could be an attractive target for antischistosomal chemotherapeutic drug design.     

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.     

Increased 5-Hydroxytryptamine and Norepinephrine Release from Rat Brain Slices by the Red Sea Flatfish

The effect of the Red Sea flatfish toxin pardaxin was examined on K+-evoked and on basal release of either [3H]norepinephrine or [3H]5-hydroxytryptamine from preloaded rat cortical slices. The K+-induced release of the neurotransmitters was stimulated in a dose-related manner at concentrations ranging from 0.5 to 4 micrograms/ml. Basal release of the two transmitters was elevated to a lesser extent. Although the stimulation of evoked release was approximately equivalent for the two neurotransmitters, the response to 5-hydroxytryptamine was reversible whereas that of norepinephrine was not washed by 20 min of superfusion. The mechanisms involved in producing these actions of pardaxin are not known; however, they may be mediated by changes in electrolyte fluxes across the neuronal membranes.     

狗的无氧阈与骨骼肌某些特征在锻炼、脱锻炼动态变化中的关系

本实验在体力锻炼与脱锻炼过程中观察了五条成年雄性杂种狗的无氧阈(AT)、骨骼肌毛细血管密度(cap/mm~2)、毛细血管数与肌纤维数之比(C:F)、肌纤维组成——慢肌纤维百分比(％ST)以及毛细血管弥散距离(弥散距离)的变化。结果表明:锻炼前AT与cap/mm~2、C:F,％ST及弥散距离均分别有显著相关。锻炼后第五周,AT、cap/mm~2、C:F和％ST分别增加了40.9％、12.2％、22.9％和2.4％,弥散距离降低6.3％,其中只有AT的增加有显著意义,锻炼十周后,AT增加68.2％(P<0.001)、cap/mm~2增加37.8％(P<0.05)、C:F增加78.1％(P<0.001),弥散距离降低17.0％(P<0.01)而％ST无显著变化。停止锻炼后第五周,AT等各指标都有降低的趋势,但无显著意义,停止锻炼后第12周,AT、cap/mm~2,C:F,％ST分别降低了18.9％、10.9％、10.5％、2.9％,弥散距离增加5.1％,其中除AT的降低有显著意义外,其余指标的变化均无显著意义。与锻炼前相比,这些指标的变化。除％ST外,仍有显著变化。在锻炼与脱锻炼的过程中,AT与cap/mm~2、C:F、弥散距离在各期或整个过程中均呈显著相关,但与％ST相关不显著。实验结果提示,骨骼肌的毛细血管供应状况是决定无氧阈的重要因素之一。     

梭曼等胆碱酯酶抑制剂对猫膈神经单纤维电活动的影响

在麻醉猫,经推动脉注入梭曼、VX,沙林及乙酰甲胆碱引起呼吸中枢严重抑制的剂量分别为0.5—1、3、15、2001μg/头;但在无麻醉、箭毒麻痹、人工呼吸并用药物保护循环的清醒猫,VX用量要增加十多倍,沙林用量增加2～8倍,棱曼用量不变。在严重抑制剂量的给药早期,梭曼使34.8％动物较早地出现膈神经单纤维放电加强,其每次吸气放电的冲动频率由20～30Hz增至50～80Hz,冲动个数由15～25个/每次放电增至40～60个/每次放电,兴奋持续短、迅速转入抑制且不易自动恢复;VX和乙酰甲胆碱使100％动物出现显著的放电加强,其冲动频率由20～30Hz增至70～130Hz、冲动个数由15～25个/每次放电增至60～80个/第次放电,兴奋持续时间较长、转入抑制慢但自动恢复较快;沙林使76.9％动物出现放电加强,其他表现类似VX。三种胆碱酯酶抑制剂和乙酰甲胆碱共使33/52根单纤维放电发生时相变化。结果表明:梭曼对呼吸中枢作用最强、沙林次之、VX最弱且更似乙酰甲胆碱。