Effect of 5-Phosphoribosyl-1-pyrophosphate on Crystalline Quinolinate Phosphoribosyltransferase Activities from Hog Kidney and Hog Liver

The pH profiles of crystalline quinolinate phosphoribosyltransferase (EC 2.4.2.19) activities from hog kidney and hog liver were found to vary according to 5-phosphoribosyl-1-pyrophosphate concentration. Both the kidney and liver enzyme activities were inhibited by 5-phosphoribosyl-1-pyrophosphate at an alkaline pH and physiological pH (pH 7.4) but not at an acidic pH. The inhibition by 5-phosphoribosyl-1-pyrophosphate was competitive for quinolinic acid. In the presence of 30% glycerol, both the kidney and liver enzyme activities were inhibited by 5-phosphoribosyl-1-pyrophosphate, even at an acidic pH.     

Ontogeny of enzymatic activities in fed and fasting turbot, Scophthalmus maximus L.

The presence and localization of acid and alkaline phosphatase, non-specific proteases, aminopeptidase, amylase, non-specific esterase and lipase was investigated by histoenzymologic methods in fed and fasting turbot from day 1 to day 40 post-hatching and compared with published data. Alkaline phosphatase and aminopeptidase activities were delected at day 1 in the distal region of the developing digestive tube. At day 3 (opening of the mouth) aminopeptidase and alkaline phosphatase activities were found all along the intestine. Sites of non-specific esterase and protease activities became apparent in the digestive tract at days 2 and 3 respectively. Amylase was present in the exocrine pancreas at day 3 and in the lumen of the intestine at day 4. Acid phosphatase was active in the cellular structure surrounding the yolk stores and in the lipid droplets at day 1 and in the intestinal epithelium at day 3. Lipase was found at day 15 when the larvae metamorphose into juveniles.
All the investigated enzymes were detected in fasting animals, except for lipase. However, the intensities of the enzymatic activities were weaker in the fasting specimens relative to the fed specimens between days 7 and 10.     

Phosphate utilization and constitutive synthesis of phosphatases in Thermoactinomyces vulgaris Tsilinsky.

Thermoactinomyces vulgaris utilized both organic and inorganic phosphates with equal efficiency for its growth. The specific activities of the thermophilic acid and alkaline phosphatases were found to be maximum at 1 mM concentration of each phosphate source. All the phosphatase isoenzymes (three alkaline and one acidic) were observed irrespective of the substrate source and concentration, suggesting constitutive synthesis of the enzymes. During growth and differentiation, both acid and alkaline phosphatases exhibited uniformly stable patterns of isoenzymes with slight variations in their specific activities.     

RIBONUCLEASE ACTIVITIES IN VARIOUS CLASSES OF NUCLEI FROM BOVINE CEREBRUM

Abstract— Nuclei of adult bovine cerebrum were separated by dissection of grey matter from white matter and discontinuous sucrose gradient centrifugation, into neuron, astrocyte-enriched and oligodendrocyte fractions. The activities of alkaline and acidic RNAses were determined in the different nuclei types. The acidic and alkaline RNAse activities of the neuronal nuclei were much higher than those of the glial nuclei. In each type of nuclei the acidic RNAse activity was lower than the measured alkaline RNAse activity.     

Synthesis of 1-O-(2′,4′-Dichlorophenoxyacetyl)-d-glucopyranose

1-O-Glucosyl esters of 2,4-dichlorophenoxyacetic acid (2,4-D) were easily prepared from 4,6-O-benzylideneglucose. The configuration of 1-O-ester linkage was affected by pH at the end of the reaction, that is, β-type was a major product at a neutral or acidic condition and α-type at an alkaline condition. Both of the anomers showed the same biological activities as sodium salt of 2,4-D.     

Acidic and alkaline bottom ash and composted manure blends as a soil amendment

Potential water quality impacts associated with using bottom ash (BA) and composted dairy manure (CM) as a soil amendment were evaluated in this study. Two column studies were conducted to evaluate three blends of acidic BA and CM (BA:CM, v/v) namely, B1_ac (95:5), B2_ac (90:10), and B3_ac (80:20) and three blends of alkaline BA and CM (BA:CM, v/v), namely, B1_al (95:5), B2_al (90:10), and B3_al (80:20) under constant head water table conditions. Samples from standing water (top) and leachate (bottom) were collected at weekly intervals until day 49 to evaluate the effects of different blend ratios and elapsed time on standing water and leachate chemical and physical properties. A higher CM content in both acidic and alkaline blends resulted in higher leachate concentrations for solids and nutrients tested in this study. Alkaline blends had higher standing water and leachate nutrients concentration compared to acidic blends. After day 28, standing water total dissolved solids (TDS) concentrations for all acidic blends was below the USEPA drinking water standard however, TDS value for alkaline blend was always below the standard. Similar trends were also observed for NO₃–N and phosphorus (P) concentrations for both blends. Based on these findings, it was concluded that acidic and alkaline blends B1_ac, B1_al, B2_ac and B2_al may be considered as a soil amendment material.     

Nuclear and cytoplasmic RNase-activity in regenerating mouse liver

Nuclear and cytoplasmic RNase activities at pH 5.0 and 7.6 were analyzed in regenerating mouse liver at 6, 12, 24, 48, and 72 h after partial hepatectomy. Two different nucleus-isolation methods were used, one in a EDTA-spermidine medium free from divalent cations, and one in a sucrose medium containing these ions. During regeneration, the cytoplasmic alkaline RNase activity in the sucrose medium was unchanged, but in the spermidine medium showed an increase toward the end of the period. Also the cytoplasmic acid RNase activity was unchanged in sucrose medium, whereas in the spermidine it slightly increased during regeneration. The nuclear alkaline RNase activity showed a notable peak 6 h after the operation and later decreased. Also the nuclear acid RNase activity displayed a similar marked peak 6 h after operation, then decreased, but remained high throughout the period. The nuclear RNase activities were about 1% of the corresponding cytoplasmic RNase activities. The absolute activities varied greatly according to the nucleus-isolation methods. In the controls, the absolute activity of nuclear alkaline RNase was slightly above (1.2 times) that of the corresponding acid activity after the spermidine method. After the sucrose method the nuclear alkaline activity was 2.7 times that of the acid activity. The absoluted activity of cytoplasmic alkaline RNase was slightly above (1.2 times) the acid activity after the spermidine method but after the sucrose method it was only 0.25 times that of the acid activity. In sham-operated animals, cytoplasmic acid and alkaline RNase activities generally were fairly similar to the normal value, but corresponding nuclear activities showed marked variations indicating an influence by anesthesia.     

Stimulation of canine kidney BBMV ATPase activity by acidic pH in the presence of Zn2+: an ATPase activity distinct from transport ATPases and alkaline phosphatase that may be an ecto-ATPase

Renal brush border membrane vesicles (BBMV) of the dog possess at least two ATPase activities. In the present study, we have examined the effect of pH, ions, and inhibitors on the activity of ATPase in BBMV. Two different sets of conditions were identified that produced stimulation of ATPase activity. A unique stimulation of BBMV ATPase activity occurred at acidic pH in the presence of 1 mM ZnCl2. In the absence of Zn2+, a second ATPase activity was stimulated by alkaline pH values with peak stimulation occurring between pH 8.5 and 9.0. The results suggest that the alkaline pH-stimulated hydrolysis of ATP probably represents the activity of BBMV alkaline phosphatase. The unique acidic pH + Zn2(+)-stimulated ATPase activity must represent the activity of a second protein other than the alkaline phosphatase, since purified alkaline phosphatase did not show this activity. The biochemical identity and physiological function of this renal BBMV ATPase activity remain to be determined, but it may be an ecto-ATPase.     

Trichomonas vaginalis: identification of soluble and membrane-associated phospholipase A1 and A2 activities with direct and indirect hemolytic effects

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthal's inhibitor. The greatest effect was observed with 80 microM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.     

Histochemical studies on the effect of thyroid hormone on alkaline and acid phosphatase activities in liver of fish and amphibia.

The Lata fishes (Ophicephalus punctatus) showed increased alkaline and acid phosphatase activities in liver after immersion for 15-30 days in thyroxine-containing medium (0.025 mug/ml). A single injection of thyroxine (1-2 mug/g of body weight) caused increased acid phosphatase activity in liver of Lata fish in comparison to the controls on the 5th day after experiment but the alkaline phosphatase activity remained unchanged. Both alkaline and acid phosphatases showed increased activities in liver of Lata fishes treated with a single injection of 4 mug of thyroxine per g of body weight on the 5th day. Immersion of Lata fishes in thiourea solution (1 mg/ml) for 15 days did not show any alteration in alkaline or acid phosphatase activities but these enzyme activities decreased after 30 days' immersion in thiourea solution in comparison to the controls. A seasonal variation of alkaline and acid phosphatase activities was observed in liver of Lata fishes. More alkaline phosphatase activity was found in liver of summer fishes than in winter fishes. The winter fishes showed more acid phosphatase activity than the summer fishes. Three consecutive injections of thyroxine (0.1 mug/g of body weight) to toads (Bufo melanostictus) caused increased alkaline and acid phosphatase activities in liver on the 5th day of the experiment, in comparison to the controls.     

Arbuscular mycorrhizal status and root phosphatase activities in vegetative <Emphasis Type="Italic">Carica papaya</Emphasis> L. varieties

The arbuscular mycorrhizal (AM) status and root phosphatase activities were studied in four vegetative Carica papaya L. varieties viz., CO-1, CO-2, Honey Dew and Washington. Standard techniques were used to ascertain information on spore density and species diversity of AM fungi. Although in case of estimation of root colonization and root phosphatase activities, the existing methods were slightly modified. Root colonization and spore density of AM fungi along with root phosphatase (acid and alkaline) activities varied significantly in four papaya varieties. The present study recorded higher acid root phosphatase activity when compared with alkaline root phosphatase activity under P-deficient, acidic soil conditions. The present study revealed that the root colonization of AM fungi influenced acid root phosphatase activity positively and significantly under P-deficient, acidic soil conditions. A total of 11 species of AM fungi belonging to five genera viz., Acaulospora, Dentiscutata, Gigaspora, Glomus and Racocetra were recovered from the rhizosphere of four papaya varieties.     

pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase and trypsin

The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (Lys-C) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of 18O atom into the alpha-carboxyl group of N(alpha)-acetyl-L-lysine from H2(18)O solvent. The optimum pHs of the carboxyl oxygen exchange reaction catalyzed by Lys-C and trypsin were found to be pH 5.0 and 6.0, respectively, which were significantly shifted toward acidic pHs compared to the most favorable pHs of their amidase activities for N(alpha)-acetyl-L-lysine amide in the pHs examined. Steady-state kinetics parameters were also determined for both enzymes at two different pHs, one at the pH optimum for their carboxyl oxygen exchange activity (pH 5-6) and the other at the favorable pH for their amidase activity (pH 8-9). Significantly lower Km (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher kcat values (1.5-fold higher for Lys-C, 5-fold higher for trypsin) were obtained at the acidic pHs compared to the alkaline pHs, suggesting that Lys-C and trypsin have higher substrate binding affinities and higher catalytic rates at the acidic pHs than at the alkaline pHs. The higher carboxyl oxygen exchange activities at the acidic pHs were also confirmed with peptide substrates derived from apomyoglobin. These findings are significant toward the goal of improving the efficiency of the Lys-C and trypsin catalyzed 18O labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing 18O labeling.     

Enantioselectivity in degradation and transformation of quizalofop-ethyl in soils

The enantioselective degradation of quizalofop-ethyl and its metabolite quizalofop-acid in two soils, a Wuhan acidic soil and a Baoding alkaline soil, was investigated. The dissipation of quizalofop-ethyl consisted of two phases, a rapidly deceasing first phase that lasted 1 day and a slowly decreasing second phase that extended till the end of the incubation. It is shown that S-quizalofop-ethyl degraded slightly faster than R-quizalofop-ethyl in the two soils. Further incubation of enantiopure enaniomers showed that quizalofop-ethyl was configurationally stable in soil. Quizalofop-acid was produced quickly, and its amount reached a maximum at 1-6 days time and then decreased slowly with half-lives ranging from 11 to 21 days. The results also showed that quizalofop-acid degraded faster in the acidic Wuhan soil than in the alkaline Baoding soil. At last, significant enantiomerization from S-quizalofop-acid to R-quizalofop-acid was observed, and the enantiomerization was fast, resulting in residues enriched with R-quizalofop-acid whatever racemic quizalofop-ethyl or pure enantiomers were initially applied in the soils.     

Carbon and nitrogen metabolism during Dendrobium huoshanense protocorm-like body development in suspension culture

Abstract

The aim of this work was to study carbon and nitrogen metabolism during suspension culture of protocorm-like bodies (PLBs) from Dendrobium huoshanense. No significant lag phase of PLB growth was found, and a maximum biomass of 288.6 g l^?1 was obtained at day 30 of culture. Sucrose concentration was halved as PLB growth proceeded, while no change in glucose and fructose levels in the medium was found in the first 3 days, followed by a gradual increase until day 9 of culture. Conversely, sucrose in PLBs accumulated dramatically in the first 6 days of culture, followed by a rapid decrease. At the same time, glucose and fructose content of PLBs increased, then declined after 9 days of culture. Soluble acidic invertase (soluble acidic IT) and alkaline invertase (alkaline IT) were activated after inoculation, and reached the highest value on day 6 and day 18, respectively whereas cell wall-bound invertase (cell wall-bound IT) seemed to be repressed throughout culture. The maximum value of sucrose synthase (SuSy) activity was observed on day 18, while sucrose phosphate synthase (SPS) stayed low and constant from inoculation to the end of culture. Ammonium concentration in the medium decreased rapidly, and was hardly detectable after 12 days, when the rapid utilization of nitrate began. Conversely, ammonium in PLBs showed a sharp increase in the first 3 days of culture, followed a rapid decrease until day 12, corresponding to nitrate depletion. Peaks in glutamine synthase (GS) and glutamate synthase (GOGAT) activity were observed on day 12 and 15, respectively. Nitrate reductase (NR) was repressed in the early culture stage, and activated from day 9 to 15 of culture. These results suggest that soluble acidic IT, alkaline IT, SuSy, GS, GOGAT and NR control carbon and nitrogen metabolism at different PLB growth stages.     

Reversible acidic-alkaline transition of the carbon monoxide complex of cytochrome c peroxidase

The Soret absorption band of the ferrous carbon monoxide (CO) complex of cytochrome c peroxidase exhibited a blue shift from 423.7 to 420 nm upon an increase in pH from 6.5 to 8.5. The spectral change was reversible with an isosbestic point at 422 nm. The pH dependence of this spectral change gave a sigmoidal curve fitted well to a theoretical curve of a cooperative release of two protons with a pK value of 7.5, indicating the existence of the acidic and alkaline forms of the ferrous CO enzyme. Upon irradiation of light flash (100 J of power and 30-microseconds), the heme-bound CO was readily dissociated in both acidic and alkaline forms with a quantum yield of approximately unity. On the other hand, the rate of recombination of the dissociated CO with the heme iron was significantly different between these two forms; the recombination rate constants were 1.1 X 10(3) and 3.0 X 10(4) M-1 S-1 at 25 degrees C for the acidic and alkaline forms, respectively. At intermediate pH values, kinetics of recombination were biphasic, consisting of the slow and fast processes with the appropriate rate constants mentioned above. When the fraction of the fast process was plotted against pH, the pH profile coincided with the spectrophotometric pH titration curve described above. Thus, it was concluded that the acidic and alkaline forms of the enzyme were responsible for the slow and fast processes, respectively. In infrared spectroscopy, the acidic form showed a narrow CO stretching band at 1922 cm-1 with a half-band width of 12.5 cm-1, while the alkaline form exhibited a broad CO-stretching band at 1948 cm-1 with a half-band width of 33 cm-1. Significance of these results are discussed in relation to the structure of the heme vicinity on the CO complex of cytochrome c peroxidase.     

An in vitro study of LH release, synthesis and heterogeneity in pituitaries from proestrous and short-term ovariectomized rats

It is known that acute ovariectomy (OVX) greatly attenuates the pituitary luteinizing hormone (LH) response to gonadotropin-releasing hormone (GnRH) in vitro. The present study evaluated possible quantitative and/or qualitative differences in the biosynthesis and secretion of LH in pituitaries from proestrous and acutely (72 h) OVX rats. Paired anterior pituitary glands were incubated for 4 h in a medium containing +/- 10 nM GnRH. Pituitary and secreted LH were measured by radioimmunoassay with differences in total LH (tissue plus medium) +/- GnRH being indicative of GnRH-stimulated LH synthesis. Qualitative changes in LH were evaluated by isoelectrofocusing (IEF). The results show that the major form of LH stored in and released from the pituitaries consisted of LH molecules with an isoelectric point (pI) in the alkaline pH range (alkaline LH), and a lesser amount (approximately 30%) of LH molecules in the acidic pH range (acidic LH). The ratio of alkaline/acidic LH observed in the pituitary and medium was similar in the proestrous and OVX groups, although the amount of alkaline and acidic LH release in response to GnRH was 2-3 times greater in the proestrous group. In both groups, the alkaline/acidic LH ratio of secreted LH was higher in the presence of GnRH than in its absence. Alkaline LH synthesis was increased by GnRH in both groups, with the response being greater in the proestrous than in the OVX group; GnRH-stimulated acidic LH synthesis was observed only in the proestrous group. In both groups, the amount of LH synthesized was about 60% of the amount released, which suggests that LH synthesis does not fully account for differences in GnRH-stimulated LH release. Treatment of pituitary extracts with neuraminidase decreased acidic LH, and proportionately increased alkaline LH. These results suggest that the quality of LH stored in and secreted from pituitaries of proestrous and OVX rats is similar, and that there is a preferential release of the major alkaline LH isoform in response to GnRH. The ovarian steroid environment, presumably estradiol, proportionately increases the amount of alkaline and acidic LH released, and differentially affects the amounts of the various isoforms synthesized in response to GnRH. The charge heterogeneity of alkaline and acidic LH may be related to the sialic acid content of the LH molecule.     

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.     

Influence of initial pH on hydrogen production from cheese whey

Batch experiments were conducted to investigate the effect of initial pH, between 5 and 10, on fermentative hydrogen production from crude cheese whey (87.5% (v/v) by Clostridium saccharoperbutylacetonicum). Hydrogen was produced over the range of pH studied. The hydrogen production rate and yield peaked at an initial pH 6 and then steadily decreased as the pH increased. The highest rate and yield were 28.3 ml h⁻¹ and 7.89 mmol g⁻¹ lactose, respectively. Sugar consumption was unaffected between pH 5 and 9 and remained at 97%. All final pHs were acidic and increased alongside the initial pH. There was no correlation between the initial pH and the fermentation time; the times were shorter (50–52 h) between pH 6 and 8, and longer (62–82 h) outside this range. A modified Gompertz equation adequately described fermentative hydrogen production from cheese whey. The respective maximum hydrogen production rate and hydrogen potential at an optimal pH of 6 were 47.07 ml h⁻¹ and 1432 ml. Lag phase times were much longer at acidic pHs than at alkaline pHs.