Purification and characterization of aldolase from human erythrocytes.

A procedure has been developed for the purification of human erythrocyte aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.1.3). The process involves a specific substrate elution of the enzyme from phosphocellulose followed by a reverse ammonium sulfate fractionation. The preparation has been shown to be homogeneous by analytical ultracentrifugation, thin-layer electrophoresis, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme exhibits a specific activity of 16 I.U./mg protein, a Km of 7.1-10(-6) M for fructose 1,6-bisphosphate, and a substrate specificity (Fru-1,6-P2/Fru-1-P) of 40. The native protein in a tetramer of 158 000 molecular weight possessing identical or nearly identical subunits, an isoelectric point of 8.9, a diffusion coefficient of 4.68-10(-7) cm2/s, and a molecular radius of 4.56 nm. The study shows the enzyme to be a type A aldolase resembling other muscle forms in chemical and physical properties as well as amino acid composition.

Ascaris suum hexokinase: purification and possible function in compartmentation of glucose 6-phosphate in muscle.

Hexokinase (EC 2.7.1.1) is present in a soluble and a bound form in homogenates of Ascaris suum muscle. Cellulose acetate electrophoresis, isoelectric focusing, and ion exchange chromatography confirmed the presence of only one molecular form of hexokinase in this muscle. A procedure for purifying hexokinase from Ascaris muscle has been developed utilizing ion-exchange chromatography, ammonium sulfate fractionation and gel filtration. The enzyme is a monomer with a molecular weight of 100 000 as determined by sodium dodecyl sulfate gel filtration. The Stokes' radius, diffusion coefficient, and frictional ratio have been determined. The apparent Michaelis constants for glucose and ATP are 4.7-10(-3) M and 2.2-10(-4) M, respectively. Ascaris hexokinase also exhibits end-product inhibition by glucose 6-phosphate and ADP. It is postulated that the kinetic parameters of the enzyme are the results of its function, that of generating glucose 6-phosphate primarily for glycogen synthesis.

Isolation and characterization of yeast nicotinamide adenine dinucleotide kinase.

NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23) from yeast has been purified utilizing ion-exchange and NAD+-agarose affinity chromatography to give a 2100-fold purification. The apparent homogeneity of the enzyme preparation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a subunit molecular weight of 31,000, and a native molecular weight of 124,000, and is, thus, probably a tetramer. The single form of the enzyme has an apparent isoelectric point of 5.85. Initial velocity studies in the forward direction with both substrates gave intersecting Lineweaver-Burk plots, and this suggests a sequential mechanism in which both substrates are bound before products are released. Replots of these data were linear and gave Km values for NAD+ and ATP of 0.68 mM and 2.3 mM, respectively.

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum.

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.

Purification of malic enzyme from Ascaris suum using NAD+-agarose.

Malic enzyme has been purified from Ascaris suum by polyethylene glycol precipitation, ion-exchange chromatography, ammonium sulfate precipitation, and NAD-agarose affinity chromatography to a specific activity of 80 units/mg (V/[E]t = 350 s-1). The preparation was shown to be homogeneous by SDS polyacrylamide disc gel electrophoresis. The procedure can be accomplished in a maximum of four days with a 74% yield.

Phosphofructokinase from Dirofilaria immitis: effect of fructose 2,6-bisphosphate and AMP on the non-phosphorylated and phosphorylated forms of the enzyme.

The enzyme responsible for the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), 6-phosphofructo-2-kinase, was shown to be present in the heart worm, Dirofilaria immitis. The level of Fru-2,6-P2 was determined to be 4 +/- 0.3 nmol(g wet weight)-1 in the tissues of the filariid. Fru-2,6-P2 stimulated the activity of both the non-phosphorylated and phosphorylated forms of the D. immitis phosphofructokinase (PFK). The Kact values for Fru-2,6-P2 were 378 +/- 18 nM and 65 +/- 6 nM for the non-phosphorylated and phosphorylated forms, respectively, at 1 mM fructose 6-phosphate (Fru-6-P) and 1 mM ATP at pH 6.8. AMP also stimulated the activity of both forms of the enzyme with Kact values of 230 +/- 10 microM and 37.3 +/- 6.1 microM for the non-phosphorylated and phosphorylated forms, respectively. In the absence of any effectors, the S0.5 values for Fru-6-P were 17.4 mM and 11.0 mM for the non-phosphorylated and phosphorylated forms, respectively, of the D. immitis PFK at 1 mM ATP, pH 6.8. These S0.5 values were lowered to 0.03 mM by the combined effects of saturating levels of Fru-2,6-P2 and AMP. A physiological assay was developed based on the level of metabolites in the parasite that influence the activity of PFK. This assay contained the known effectors of the PFK at concentrations approximating those found in the parasite. Under these conditions the KFru-6-P values were 153 microM and 60 microM for the non-phosphorylated and phosphorylated forms of the PFK, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoretic analyses of myofibrillar proteins from the body wall muscles of Ascaris suum.

A myofibrillar protein extract has been isolated from the muscle of Ascaris suum. Two-dimensional electrophoresis of this extract revealed that the myosin light chain 1 (ALC1) migrates as 3 components with approximate isoelectric points in the range of 5.3-5.6. The most acidic component of ALC1 appeared to be phosphorylated when the myofibrillar extract was incubated for 10 s with catalytic subunit of cAMP dependent protein kinase and [gamma-32P] ATP. The myosin light chain 2 (ALC2) migrated as a single component in isoelectric focusing with an approximate isoelectric point of 5.5 Actin was resolved into 2 components with identical molecular weight but isoelectric points differing by approximatley 0.2 pH units. A protein was tentatively identified in the myofibrillar extract as tropomyosin. It migrated as a single band with an approximate isoelectric point of 5.0 and a molecular weight of 39 000. None of the troponin components could be identified in the myofibrillar extract. It is postulated that muscle contraction in A. suum muscle could be controlled by phosphorylation of myosin.

Trypsin modification of phosphofructokinase from Ascaris suum.

Phosphofructokinase from Ascaris suum is a tetramer with subunits of 90 kDa. Treatment of the native enzyme with trypsin (10%, w/w) followed by SDS-gel electrophoresis was shown to immediately generate a 40-kDa fragment followed by a gradual formation of two other fragments of 37 and 32 kDa. The loss of catalytic activity during the digestion was less than 50%. Gel filtration of the digested enzyme under non-denaturing conditions showed a Mr almost that of the native enzyme. Digestion of the phosphorylated enzyme resulted in an 80% release of the phosphorylated peptide over the period of 1 h. The digested enzyme was inhibited less by ATP than the native enzyme, but it was still positively affected by the effectors, fructose 2,6-bisphosphate and AMP. The results are interpreted to suggest that the structure of the ascarid phosphofructokinase is similar to that of the mammalian enzyme.

Purification and characterization of phosphorylase B from Ascaris suum.

Glycogen phosphorylase b (EC 2.4.1.1) has been purified from the muscle of the roundworm, Ascaris suum. The 223-fold purified enzyme was shown to be homogenous by high performance liquid chromatography (HPLC), gel filtration column chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. The apparent native molecular weight of the enzyme determined by size exclusion chromatography by HPLC and gel filtration corresponded to 200 000 and 199 000, respectively. The subunit molecular weight of the enzyme was determined to be 100 000 by electrophoresis in the presence of SDS. Therefore, the enzyme appears to be a dimer with identical or near identical subunits. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol subunit and exhibited an absorbance index E1% 280 of 13.8. The apparent isoelectric point of the enzyme is 5.53. The enzyme, inactive in the absence of AMP, can be converted to the active form by rabbit muscle phosphorylase kinase and MgATP. The molecular weight of the activated form of the enzyme is 200 000. Kinetic studies showed apparent Km values of 0.17% for glycogen, 36 mM for Pi and 52 mM for glucose-1-P. The apparent Ka for AMP was 0.22 mM.

Structure and toxicity of alkaloids and amino acids of Sophora secundiflora.

Seeds from Sophora secundiflora were extracted into three major fractions: lipids, alkaloids, and amino acids. Most of the lipid material was composed of steroid esters. These esters were hydrolyzed, and the fatty acid compositions were determined. The major alkaloid component was cytisine, with N-methylcytisine, anagyrine, and the fatty acid compositions were determined. The major alkaloid component was cytisine, with N-methylcytisine, anagyrine, and termopsine in lower concentration. The major free ninhydrin-positive compound was gamma-glutamyltyrosine. In addition to several common amino acids, pipecolic acid and 4-hydroxypipecolic acid were identified. Both the amino acid fraction and the alkaloid fractions caused minor pharmacological disorder when injected into rats. However, when both fractions were simultaneously administered, they were lethal.

Activity of enzymes regulating glycogen metabolism in perfused muscle-cuticle sections of Ascaris suum (Nematoda).

A new perfusion system has been developed in which muscle-cuticle sections of Ascaris suum were perfused, enabling study of enzymes in vitro. Using this technique the activity of the regulatory enzymes glycogen synthase and glycogen phosphorylase was determined, and the level of glycogen in the muscle was assessed. During starvation, 98% of glycogen synthase was in the inactive D-form, and 80% of the glycogen phosphorylase activity was in the active a-form. When the ascarid muscle section was perfused with 27 mM glucose, 13.1% of the glycogen synthase was in the active I-form, whereas phosphorylase a-levels dropped to 46% and glycogen was synthesized at a linear rate of 12 mg/g/hr or 1.23 mumoles/min/g muscle-cuticle. ATP levels (3.71 +/- 0.32 mM) remained unchanged over a 4-hr perfusion period with an adenylate energy charge of 0.82. Fructose supported glycogen synthesis, though not as well as glucose. Galactose, mannose, and trehalose did not support glycogen synthesis. The new perfusion system should be useful in future, similar studies on Ascaris.

Glycogen metabolizing enzymes during starvation and feeding of Ascaris suum maintained in a perfusion chamber.

The glycogen content of muscle was correlated with the activity of glycogen synthase and glycogen phosphorylase from the parasitic roundworm Ascaris suum maintained in vitro. Adult female worms were maintained in the laboratory in a perfusion system during periods of starvation and feeding. During starvation, the levels of glucogen decreased at a rate of 0.1 to 0.2 mumoles/min/g wet weight of muscle-cuticle. During this time, 95% of the glycogen synthase (E.C. 2.4.1.11) was in the active D-form, and 48% of the phosphorylase (E.C. 2.4.1.1) was in the active a-form. Upon feeding, the rate of incorporation of glycosyl residues into glycogen proceeded at a rate of 0.75 to 1.0 mumoles/min/g muscle-cuticle. Glycogen synthase was 22% in the active I-form and phosphorylase a-levels remained virtually unchanged at 41% as compared with the starved worm. Total levels of both enzymes remained constant over the starvation-feeding period with 3.9 units/g phosphorylase and 0.4 units/g glycogen synthase. The apparent Km value for the substrate UDPG for glycogen synthase was 0.22 +/- 0.02 mM. For glycogen phosphorylase the Km value for G-1-P was 1.76 +/- 0.38 mM.

Expression of Ascaris suum malic enzyme in a mutant Escherichia coli allows production of succinic acid from glucose.

The malic enzyme gene of Ascaris suum, was cloned into the vector pTRC99a in two forms encoding alternative amino-termini. The resulting plasmids, pMEA1 and pMEA2, were introduced into Escherichia coli NZN111, a strain that is unable to grow fermentatively because of inactivation of the genes encoding pyruvate dissimilation. Induction of pMEA1, which encodes the native animoterminus, gave better overexpression of malic enzyme, approx 12-fold compared to uninduced cells. Under the appropriate culture conditions, expression of malic enzyme allowed the fermentative dissimilation of glucose by NZN111. The major fermentation product formed in induced cultures was succinic acid.

Methods and outcomes of breastfeeding instruction for nursing students.

Support from nurses can influence breastfeeding rates, but many nurses are not well-informed about breastfeeding topics. Surveys were used to assess the breastfeeding instruction provided in five nursing programs. Most students attended breastfeeding lectures, but only one-fourth received breastfeeding information during clinical activities. After completing their maternity rotation, less than 25 percent had as many as three clinical opportunities to teach breastfeeding techniques or counsel about lactation problems. Completion of maternity rotation did not improve student's knowledge of breastfeeding health benefits or clinical advice. Previous personal breastfeeding experience was associated with more accurate clinical advice and rating breastfeeding instruction as inadequate. We conclude that nursing education may not prepare students for effective breastfeeding promotion, and we suggest solutions for lactation consultants.

Expecting in prison: preparing for birth under conditions of stress.

When a pregnant woman is separated from her customary support system and placed in a high-stress environment, she experiences concurrent developmental and situational crises. A unique example of this is the incarcerated pregnant woman. A prenatal program developed to decrease the stress of being pregnant in prison is presented. The needs and concerns of the women are described. Nursing strategies are presented, with emphasis on practical interventions. Problems in implementation and ways to alleviate those problems are discussed.

Determination of metabolite and regulatory enzyme levels in Dirofilaria immitis and Ascaris suum: a comparative study.

Studies on metabolite levels in Dirofilaria immitis revealed similarities in several metabolites with those of Ascaris suum. The glycogen level in the filariid was however 3-4 times lower than that in A. suum. Levels of three regulatory enzymes were also determined in D. immitis and compared with those in A. suum. The activities of Hexokinase and Phosphofructokinase were similar. However, the levels of Glycogen phosphorylase b appeared to be much lower in the filariid than in A. suum. The subtle but important differences observed may reflect modifications of the parasite enzymes suggesting salient differences in the regulation of energy production from carbohydrates in the worms. The differences may also represent specialization required for the unique life style of the worms in their different locations in their hosts.