scholarly journals Mobilization of granulose in Clostridium pasteurianum. Purification and properties of granulose phosphorylase

1974 ◽  
Vol 144 (3) ◽  
pp. 513-517 ◽  
Author(s):  
R L Robson ◽  
J G Morris
Keyword(s):  
Energy Charge ◽  
Clostridium Pasteurianum ◽  
Adenylate Energy Charge ◽  
Ph Optimum ◽  
Exogenous Glucose ◽  
Bivalent Cations ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Nucleotide Sugars

1. The granulose of Clostridium pasteurianum ATCC 6013 is degraded when the organism is incubated in a medium containing no utilizable source of carbon and energy. 2. Mobilization of the polyglucan does not occur in the presence of exogenous glucose. 3. Breakdown of granulose is effected by a constitutively synthesized α-1,4-polyglucan phosphorylase. 4. Partial (530-fold) purification of this granulose phosphorylase was facilitated by its being loosely bound to the native granules of its substrate polyglucan. 5. The enzyme (pH optimum 6.4) was assayed both (a) in the degradative direction, Km for Pi=2.2mm, and (b) in the synthetic direction, Km for glucose 1-phosphate=0.05mm. No requirement for bivalent cations was evidenced. 6. Granulose phosphorylase was inhibited by various nucleotide sugars; GDP-glucose, ADP-glucose (Ki=20μm) and UDP-glucose (Ki=60μm) were particularly potent competitive inhibitors. ATP, NADP+and NADPH (at 1mm) were less effective inhibitors, whereas AMP was slightly stimulatory. 7. It would appear that granulose mobilization is favoured under conditions of low adenylate energy charge, but is prevented under conditions of ‘glucose excess’ chiefly by ADP-glucose-mediated inhibition of granulose phosphorylase.

scholarly journals Purification and properties of β-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller

1978 ◽  
Vol 175 (2) ◽  
pp. 743-750 ◽  
Author(s):  
P Calvo ◽  
A Reglero ◽  
J A Cabezas
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mixed Substrates ◽  
Ph Optimum ◽  
Terrestrial Mollusc ◽  
Buffer Solutions ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Ph Range

1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8–4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.

Changes in nucleotide pools during conidial germination in Neurospora crassa

1981 ◽  
Vol 59 (11-12) ◽  
pp. 899-905 ◽  
Author(s):  
Richard L. Sabina ◽  
Paulette Dalke ◽  
Alan R. Hanks ◽  
Jane M. Magill ◽  
Clint W. Magill
Keyword(s):  
Neurospora Crassa ◽  
Adenine Nucleotide ◽  
Energy Charge ◽  
Fold Increase ◽  
Conidial Germination ◽  
Adenylate Energy Charge ◽  
Wild Type ◽  
Nucleotide Pools ◽  
Nucleotide Sugars ◽  
Purine Pathway

The acid-soluble nucleotide pools of wild type and several adenine auxotrophs of Neurospora crassa were studied immediately prior to and during conidial germination in the presence of adenine. A two- to four-fold increase in most nucleotide pools was observed after 6 h of germination at 33 °C indicating a general increase in nucleotide pools during this developmental period. The largest components of the acid-soluble nucleotide pools were uracil-containing nucleotide–sugars, which are precursors of chitin and glucan, the major constituents of cell walls. On removal of adenine, the UDP–sugar pools decreased significantly, in adenine auxotrophs, while the pools of UTP increased significantly. ATP levels increased approximately twofold in the first 6 h of germination. After 1 h without exogenous adenine, ATP dropped twofold or more in adenine auxotrophs but not in wild type. There was a net decrease in all adenine nucleotide pools during adenine starvation and a much greater decrease was seen in adenine auxotrophs than in wild type. The adenylate energy charge remained stable despite major changes in the adenylate pools.Accumulation of intermediates was observed in germinated conidia from purine auxotrophs blocked at various steps in the purine pathway. IMP accumulated in ungerminated and in starved conidia of the adenine-8 (ad-8) strain. Ungerminated conidia of the ad-5 strain contained a large pool of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) while the ad-1 strain had smaller amounts of AICAR, but significantly more than any other strain tested. The AICAR pools disappeared from ad-5 and ad-1 in the presence of 50 mg histidine/100 mL. Similarly the IMP pools in ad-8 decreased markedly in the presence of histidine, indicating that the contribution from the histidine biosynthetic pathway to purine nucleotide formation is significant.

Enzymatic Properties of 5′-AMP Deaminase in Platelet Lysates

1977 ◽  
Vol 37 (03) ◽  
pp. 380-395 ◽  
Author(s):  
H Holmsen ◽  
A.-C Østvold ◽  
M. A Pimentel
Keyword(s):  
Heavy Metals ◽  
Resting State ◽  
Human Platelet ◽  
Energy Charge ◽  
Maximal Activity ◽  
Monovalent Cation ◽  
Adenylate Energy Charge ◽  
Amp Deaminase ◽  
Optimal Activity ◽  
Competitive Inhibitors

SummaryMetabolic ATP is converted to hypoxanthine during platelet secretion, metabolic shock and slowly in the resting state. This conversion involves deamination of 5′-AMP to 5′-IMP which has been studied by incubating 5′-AMP (3–40 μM) with human platelet lysates and quantifying the metabolites formed. Deamination occurred only when EDTA was present or endogenous ATP absent, showing that 5’-AMP deaminase (EC 3.5.4.6) was the only enzyme attacking AMP. EDTA stimulated AMP deamination, probably by removal of endogenous heavy metals which were powerful inhibitors of deamination. The experiments were therefore performed with EDTA. 5′-AMP deaminase was soluble, and had optimal activity at pH 6.5; however, the rate of AMP deamination was highly dependent on the type of buffer used. Km was 0.92 × 10–3M and Vmax was 0.26 μmoles/min x mg protein. The deamination required presence of monovalent cation with Li+ = Na+ >K+ >NH4 +, a sequence distinctly different from that seen with erythrocyte and muscle deaminase. 50 mM Na+ gave maximal activity with [math]. Tris+ and K+ were competitive inhibitors with respect to Na+, a feature not reported for the enzyme from other tissues. Above 60 mM K+ there was no effect by Na+ (0–100 mM). PPi, GTP, ITP, CTP and UTP gave greater inhibition than Pi and phospho-esters. Unlike AMP deaminase from other tissues, the platelet enzyme was insensitive to fluoride. ATP counteracted Pi and GTP inhibition and activated with or without Na+; ATP-and Na+-activation were additive. The activity increased as the adenylate energy charge was lowered, but was linearly related to the AMP concentration, thus in sharp contrast to deaminase from the liver. It is suggested that in the intact platelet AMP deamination is regulated chiefly through variations in the AMP level and not likely through variations in the ATP/Pi ratio.

scholarly journals The purification and properties of a β-N-acetylhexosaminidase from Trichomonas foetus

1975 ◽  
Vol 151 (1) ◽  
pp. 145-148 ◽  
Author(s):  
R G Edwards ◽  
P Thomas ◽  
J H Westwood
Keyword(s):  
Affinity Chromatography ◽  
Active Site ◽  
Substrate Binding ◽  
Hydroxyl Group ◽  
Ph Optimum ◽  
Dual Specificity ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Catalytic Rate

A β-N-acetylhexosaminidase was purified 800-fold from extracts of Trichomonas foetus by affinity chromatography on a column of N-(epsilon-aminohexanoyl)-2-acetamido-2-deoxy-β-D-glucopyranosylamine bound to CNBr-activated Sepharose. The enzyme has a dual specificity for the p-nitrophenyl β-D-glycosides of N-acetylglucosamine and N-acetyl-galactosamine. The parent sugars are both competitive inhibitors. The enzyme has a mol. wt. approx. 150000 and a pH optimum of 6.2. It is suggested that the same active site catalyses both activities and that no part is played by the 4-hydroxyl group in substrate binding, but it is involved in determining the catalytic rate.

scholarly journals Partial purification and properties of guinea-pig liver polynucleotide phosphorylase

1970 ◽  
Vol 119 (3) ◽  
pp. 517-524 ◽  
Author(s):  
Yew Phew See ◽  
P. S. Fitt
Keyword(s):  
Guinea Pig ◽  
Small Extent ◽  
Partial Purification ◽  
Polynucleotide Phosphorylase ◽  
Ph Optimum ◽  
Pig Liver ◽  
Bivalent Cations ◽  
Purification And Properties ◽  
Liver Nuclei ◽  
Guinea Pig Liver

1. Polynucleotide phosphorylase has been isolated and partially purified from crude preparations of guinea-pig liver nuclei. 2. The enzyme is particulate and associated with RNA and lipids characteristic of membranes. 3. It has phosphorolysis and exchange activities, but the latter may be due to a contaminating enzyme. 4. The phosphorolysis activity is dependent on bivalent cations, preferably Mg2+, has a pH optimum between 8.6 and 9.2 and is inhibited by potassium chloride and sodium chloride. 5. The enzyme catalyses phosphorolysis of poly A, poly C, poly U, rRNA and tRNA. Poly G is only phosphorolysed to a very small extent and DNA is not a substrate. 6. The enzyme appears to lack nucleoside diphosphate polymerization activity.

scholarly journals Purification and properties of threonine aldolase from Clostridium pasterianum

1970 ◽  
Vol 117 (3) ◽  
pp. 585-592 ◽  
Author(s):  
R. H. Dainty
Keyword(s):  
Pyridoxal Phosphate ◽  
Anaerobic Bacteria ◽  
Clostridium Pasteurianum ◽  
Strictly Anaerobic ◽  
Prosthetic Group ◽  
Ph Optimum ◽  
Threonine Aldolase ◽  
Purification And Properties ◽  
Km Value ◽  
The Relationship

1. Threonine aldolase was purified about 200-fold in 10% yield from Clostridium pasteurianum and its properties were examined. The final preparation gave three bands after ionophoresis on polyacrylamide gel. 2. The purified enzyme was shown to produce glycine and acetaldehyde in stoicheiometric amounts from threonine. The reverse reaction was demonstrated qualitatively. 3. The enzyme has a broad pH optimum at 6.5–7.0. 4. The enzyme is highly specific for l-threonine. 5. The enzyme is completely inhibited by 1mm concentrations of hydroxylamine and semicarbazide. Activity is decreased to 20% of the original by treatment with cysteine plus mercaptoethanol; most of the loss is regained on incubation with pyridoxal phosphate. It is concluded that pyridoxal phosphate is a prosthetic group. 6. The relationship between velocity and substrate concentration is atypical but indicates a Km value of 0.42mm. 7. The enzyme was demonstrated in several other strictly anaerobic bacteria.

scholarly journals Purification and properties of 3′-nucleotidase of Leishmania donovani

1992 ◽  
Vol 285 (1) ◽  
pp. 41-46 ◽  
Author(s):  
G O Gbenle ◽  
D M Dwyer
Keyword(s):  
Leishmania Donovani ◽  
Intact Cell ◽  
Surface Membrane ◽  
Low Latency ◽  
Kinetic Properties ◽  
Ph Optimum ◽  
Ph Values ◽  
Purification And Properties ◽  
Sds Page ◽  
Competitive Inhibitors

A surface membrane 3′-nucleotidase from Leishmania donovani promastigotes has been purified to SDS/PAGE homogeneity. The enzyme has apparent subunit molecular mass of 38 kDa, pI 5.8 and a broad pH optimum, 5.5-7.5. EDTA partially inhibited the enzyme activity, which was fully restored by Co2+; Mg2+, Ca2+ or Mn2+ had no effect on the activity. ZnCl2 or dithiothreitol at 1 mM was inhibitory at pH 7.5, but was without effect at pH 5.5, whereas at both pH values 5 mM of either compound inhibited the enzyme. The substrate-specificity of the purified enzyme is restricted to ribonucleoside 3′-phosphates. 3′-AMP and 3′-IMP are the best substrates, whereas ADP, ATP, 2′-deoxyadenosine 3′-phosphate and 5′-AMP are competitive inhibitors of the enzyme. The enzyme showed low latency in intact-cell preparations. The kinetic properties and the surface membrane localization of the enzyme suggest its implication in the formation of nucleosides from 3′-nucleotides of the parasite's host.

scholarly journals Sugars promote graft union development in the heterograft of cucumber onto pumpkin

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Li Miao ◽  
Qing Li ◽  
Tian-shu Sun ◽  
Sen Chai ◽  
Changlin Wang ◽  
...  
Keyword(s):  
Sugar Content ◽  
Healing Process ◽  
Energy Charge ◽  
Sugar Metabolism ◽  
Union Formation ◽  
Graft Union ◽  
Positive Role ◽  
Exogenous Glucose ◽  
Expression Of Genes ◽  
Graft Interface

AbstractThe use of heterografts is widely applied for the production of several important commercial crops, but the molecular mechanism of graft union formation remains poorly understood. Here, cucumber grafted onto pumpkin was used to study graft union development, and genome-wide tempo-spatial gene expression at the graft interface was comprehensively investigated. Histological analysis suggested that resumption of the rootstock growth occurred after both phloem and xylem reconnection, and the scion showed evident callus production compared with the rootstock 3 days after grafting. Consistently, transcriptome data revealed specific responses between the scion and rootstock in the expression of genes related to cambium development, the cell cycle, and sugar metabolism during both vascular reconnection and healing, indicating distinct mechanisms. Additionally, lower levels of sugars and significantly changed sugar enzyme activities at the graft junction were observed during vascular reconnection. Next, we found that the healing process of grafted etiolated seedlings was significantly delayed, and graft success, xylem reconnection, and the growth of grafted plants were enhanced by exogenous glucose. This demonstrates that graft union formation requires the correct sugar content. Furthermore, we also found that graft union formation was delayed with a lower energy charge by the target of rapamycin (TOR) inhibitor AZD-8055, and xylem reconnection and the growth of grafted plants were enhanced under AZD-8055 with exogenous glucose treatment. Taken together, our results reveal that sugars play a positive role in graft union formation by promoting the growth of cucumber/pumpkin and provide useful information for understanding graft union healing and the application of heterografting in the future.

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