Stabilization of active form of rabbit liver phosphofructokinase

(1) The active form of rabbit liver phosphofructokinase when preincubated in presence of F^? and effectors of the enzyme is stabilized against its conversion to less active form as a result of dilution. (2) The stabilized active form of enzyme has a Km value of 0.01 mM for fructose 6-phosphate, the same as measured in presence of all the positive effectors, and is lower, by 13 times, than the Km value of the non-stabilized control enzyme, and exhibits normal Michaelis-Menten kinetics, in contrast to the non-stabilized control enzyme which shows sigmoidal kinetics. (3) The stabilized active form of enzyme is neither inhibited by excess concentration of ATP nor activated by activators of phosphofructokinase. (4) The data thus support the proposition that the enzyme does indeed exist in two interconvertible forms with enormous difference in their affinities for fructose 6-phosphate and effectors.     

Inhibitory effects of active vitamin D preparations on PTH secretion in rats

To study the effects of various vitamin D preparations on PTH secretion, serum calcium and urinary excretion of cAMP were monitored in conscious perfused rats, and the influences of a bolus iv injection of the preparations on these parameters were examined. Three hours after the administration of 0.25 microgram/kg (0.6 nmol/kg) of 1 alpha, 24(R)-dihydroxycholecalciferol [1 alpha, 24(OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized (PTX) rats (50% of initial value; p less than 0.05) with no change in the concentration of serum calcium (total and ionized). In PTX rats supplemented with bovine PTH (1 U/h), the vitamin D preparation showed no significant effects either on the urinary excretion of cAMP or on serum calcium. These effects were rather specific for active vitamin D preparations, i.e. 1 alpha, 25(OH)2D3 (0.25 micrograms/kg) and 1 alpha OHD3 (1.25-6.25 micrograms/kg). However, 24,25(OH)2D3 (up to 25 micrograms/kg) had no significant effect on these parameters. These results suggest that, in rats, active vitamin D preparations specifically inhibit PTH secretion without causing a significant increase in the serum calcium concentration, reflecting a direct feedback mechanism between active vitamin D metabolite and the parathyroid glands.     

Specificity and sodium dependence of the active nucleoside transport system in choroid plexus

The transport of [3H]deoxyuridine by the active nucleoside transport system into the isolated rabbit choroid plexus was measured in vitro under various conditions. Choroid plexuses were incubated in artificial CSF containing 1 microM [3H]deoxyuridine and 1 microM nitrobenzylthioinosine for 5 min under 95% O2-5% CO2 at 37 degrees C and the accumulation of [3H]deoxyuridine measured. Nitrobenzylthioinosine was added to the artificial CSF at a concentration (1 microM) that did not inhibit the active nucleoside transport system but did inhibit the separate, saturable nucleoside efflux system. The active transport of deoxyuridine into the choroid plexus depended on Na+ in the medium, as ouabain, substitution of Li+ and choline for Na+, and poly-L-lysine all inhibited deoxyuridine transport. Thiocyanate in place of chloride and penetrating sulfhydryl reagents also inhibited the active transport of deoxyuridine into choroid plexus. The active transport of deoxyuridine into choroid plexus, which is inhibited by naturally occurring ribo- and deoxyribonucleosides (IC50 = 7-21 microM), was not inhibited (IC50 much greater than 150 microM) by nucleosides with certain alterations on the 2', 3', or 5' positions in D-ribose or 2-deoxy-D-ribose (e.g., adenine arabinoside, 3'-deoxyadenosine, xylosyladenosine); or the pyrimidine or purine rings (e.g., 6-azauridine, xanthosine, 7-methylinosine, or 8-bromoadenosine). Other analogues were effective (IC50 = 8-26 microM; e.g., 5-substituted pyrimidine nucleosides, 7-deazaadenosine, 6-mercaptoguanosine) or less effective (IC50 = 46-145 microM; e.g., 5-azacytidine, 3-deazauridine) inhibitors of deoxyuridine transport into the isolated choroid plexus.     

An active site of transforming growth factor-beta(1) for growth inhibition and stimulation.

Transforming growth factor-beta (TGF-beta) is a bifunctional growth regulator. It inhibits growth of many cell types, including epithelial cells, but stimulates growth of others (e.g. fibroblasts). The active site on the TGF-beta molecule, which mediates its growth regulatory activity, has not been defined. Here, we show that antibody to a TGF-beta(1) peptide containing the motif WSLD (52nd to 55th amino acid residues) completely blocked both (125)I-TGF-beta(1) binding to TGF-beta receptors and TGF-beta(1)-induced growth inhibition in mink lung epithelial cells. Site-directed mutagenesis analysis revealed that the replacement of Trp(52) and Asp(55) by alanine residues diminished the growth inhibitory activity of TGF-beta(1) by approximately 90%. Finally, while wild-type TGF-beta(1) was able to stimulate growth of transfected NIH 3T3 cells, the double mutant TGF-beta(1) W52A/D55A was much less active. These results support the hypothesis that the WSLD motif is an active site of TGF-beta(1), which is important for growth inhibition of epithelial cells and growth stimulation of fibroblasts.     

Synthesis of deoxy and acylamino derivatives of lactose and use of these for probing the active site of Neisseria meningitidis N-acetylglucosaminyltransferase

Derivatives of lactose with the galactose ring substituents replaced by deoxy or acylamino functions were prepared. The 2'-, 3'-, 4'- and 6'-deoxy, 3'-acetamido and 3'-benzamido derivatives of phenyl 4-O-(beta-D-galactopyranosyl)-beta-D-glucopyranoside (phenyl beta-lactoside) were synthesized from disaccharide or monosaccharide precursors. The derivatives were tested as substrates for the N-acetylglucosaminyltransferase from Neisseria meningitidis, which uses lactosyl derivatives as acceptors and UDP-GlcNAc as the donor in a beta-(1-->3) glycosylation reaction. The 6'-deoxy derivative was nearly threefold as active as phenyl beta-lactoside, whereas the 2'- and 4'-deoxy derivatives were less active. The other derivatives were inactive, as expected.     

Purification and characterization of active component and active fragment of colicin E3.

1. Two components of colicin E3, namely proteins A and B, were prepared by means of an improved method. 2. Protein A thus obtained was more than a thousand times as active as native colicin E3 when they were assayed in terms of activity for ribosome inactivation. 3. Protein A was reconstituted to colicin E3 simply by mixing with protein B. 4. Trypsin digestion of colicin E3 yielded two fragments, T1 and T2, probably by cleaving one specific bond of the A moiety of colicin E3. 5. T2 was a complex of T2A and B proteins. T2A showed an activity equivalent to that of protein A when assayed in the in vitro system, and its activity was neutralized by protein B. Thus T2A was assigned as an active fragment of protein A. 6. T2A has a characteristic amino acid composition rich in the basic amino acid, lysine. 7. The structure and function of the colicin E3 molecule is discussed based on the results obtained with its components as well as with fragments of the components.     

Specific spin-labeling at trypsin active site. Application of 'inverse substrate' to the structural analysis of the active site

Specific and reversible spin-labeling of trypsin (EC 3.4.21.4) active site was carried out by 'inverse substrate', 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolinyloxyl-p-amidinophenyl esters. The paramagnetic resonance spectra of the labeled trypsin in the form of acyl enzyme intermediate were investigated. The 2T value, separation of the outer hyperfine extrema, was observed to be sensitive to pH of the medium. These results are discussed in terms of pH-dependent conformational change at the vicinity of active site.     

Relationships between skeletal muscle characteristics and aerobic performance in sedentary and active subjects

Forty-eight sedentary and 39 quite active or well-trained men participated in this study. Muscle biopsy samples were taken from the vastus lateralis for the determination of fiber type composition (I, IIa, IIb), fiber type area, and assay of the following enzymes: malate dehydrogenase (MDH), 3-hydroxyacyl CoA dehydrogenase (HADH) and oxoglutarate dehydrogenase (OGDH). Maximal oxygen uptake (VO2max) was determined with a progressive cycle ergometer test, while endurance performance or maximal aerobic capacity (MAC) was defined as the total work output during a 90-min cycle ergometer test. Correlation analysis revealed no evidence of association between fiber type composition and VO2max kg-1 or MAC kg-1 in sedentary subjects, while active men exhibited significant correlation between % type I (r = 0.52), % type IIb (r = 0.31) and VO2max kg-1. Enzyme activities were not significantly correlated with MAC kg-1 and VO2max kg-1 in sedentary men while active men exhibited significant correlation for the three enzymes (0.37 less than or equal to r less than or equal to 0.51) with VO2max kg-1. These results show that the contribution of muscle fiber type and enzyme activities to aerobic performance may be inflated from a statistical point of view by the training status heterogeneity of subjects. They also suggest that variation in these muscle characteristics does not account for the individual differences in aerobic performance of subjects who have never trained before. Therefore, the assessment of muscle characteristics is not as useful as originally thought for the detection of individuals with a high potential for endurance performance among untrained subjects.     

Effect of prostaglandin inhibition by indomethacin on plasma active and inactive renin concentration in men.

The effect of inhibition of prostaglandin synthesis by indomethacin on active renin and on acid-activable inactive renin was studied in nine healthy, sodium-replete men, both at rest and exercise. These volunteers were investigated after pretreatment with placebo or indomethacin, 150 mg daily for 3 days. Indomethacin induced a decrease in active (p = 0.004), total (p less than 0.001), and inactive (p = 0.02) renin at rest recumbent on average by 42, 19, and 8%, respectively, and at rest sitting on average by 45, 15, and 3%, respectively. Inhibition of prostaglandins with indomethacin reduced (p less than 0.001) active and total renin at each level of work load but not (p = 0.32) inactive renin. However, the exercise-induced stimulation (p less than 0.05) of active and total renin still occur during indomethacin. Indomethacin reduced (p less than 0.001) at rest sitting and at maximal exercise the plasma concentrations of immunoreactive prostaglandins E2 by 50 and 54%, respectively, prostaglandin F2 alpha by 36 and 39%, respectively, and 13,14-dihydro-15-keto-prostaglandin F alpha by 38 and 60%, respectively. The urinary excretion of immunoreactive prostaglandin E2 and F2 alpha was also reduced.     

The volume and structure of the chymotrypsin active site

It was found that the chymotrypsin active site is located in the largest cleft on the enzyme surface approximated by a sphere with a radius of 20 angstroms. The active site cleft volume is about 2 nm3, as computed by the Monte-Carlo method. The size and shape of the active site cleft-- the intersection of two unequal spheres--are sufficient for large (about 1 nm3) fragments of substrate molecules to enter the active site. The active site bottom and the adjacent narrow section are about 600 angstroms3 in volume and may serve as a combustion chamber of a water-substrate mixture during the operation of the enzyme machinery. Intrinsic water molecules inside the combustion chamber can take part in heat exchange during different steps of the enzymatic process.     

Sodium fluxes through the active transport pathway in toad bladder.

To assess the active components of sodium flux across toad bladder as a function of transepithelial potential, unidirectional sodium fluxes between identical media were measured before and after adding sufficient ouabain (1.89 X 10(-3)M) to eliminate active transport, while clamping transepithelial potential to 0, 100 or 150 mV. Evidence was adduced that ouabain does not alter passive fluxes, and that fluxes remain constant if ouabain is not added. Hence, the ouabain-inhibitable fluxes represent fluxes through the active path. Results were analyzed by a set of equations, previously shown to describe adequately passive fluxes under electrical gradients in this tissue, here modified by the insertion of E, the potential at which bidirectional sodium fluxes (beta E, and theta E) through the active pathway are equal. According to these equations, beta E and theta E are the logarithmic mean of bidirectional fluxes through the active path at any potential, and the flux ratio in this path is modified by a constant factor Qia, which represents the ratio of the bulk diffusion coefficient to the tracer diffusion coefficient in this pathway. The data are shown to conform closely to these equations. Qia averages 2.54. Hence, serosal-to-mucosal flux vanishes rapidly as potential falls below E. Mean E in these experiments was 158 +/- 1 mV. Thus, linear dependence of net flux in both active and passive pathways on potential is present, even though the sodium fluxes in both paths fail to conform to the Ussing flux ratio equation. Qip less than 1 in the passive path (qualitatively similar to exchange diffusion) and Qia greater than 1 in the active path (as in single file pore diffusion). Both of these features tend to reduce the change in serosal-to-mucosal sodium flux induced by depolarization from spontaneous potential to zero potential ("short-circuiting").     

Molecular modeling of sialyloligosaccharide fragments into the active site of influenza virus N9 neuraminidase

Molecular modeling studies have been carried out to investigate the interactions between substrate sialyloligosaccharide (SOS) fragments bearing different glycosidic linkages and influenza virus N9 neuraminidase, a surface glycoprotein of influenza virus subtype N9. The studies revealed that the allowed orientation for sialic acid (SA) is less than 1% in the Eulerian space at the active site. The active site of this enzyme has enough space to accommodate various SOS fragments, NeuNAcalpha(2-3)Gal, NeuNAcalpha(2-6)Gal, NeuNAcalpha(2-8)NeuNAc and NeuNAcalpha(2-9)NeuNAc, but on specific conformations. In the bound conformation, among these substrates there exists a conformational similarity leading to a structural similarity, which may be an essential requirement for the cleavage activity of the neuraminidases irrespective of the type of glycosidic linkage.     

The structure of active serpin 1K from Manduca sexta.

BACKGROUND: The reactive center loops (RCL) of serpins undergo large conformational changes triggered by the interaction with their target protease. Available crystallographic data suggest that the serpin RCL is polymorphic, but the relevance of the observed conformations to the competent active structure and the conformational changes that occur on binding target protease has remained obscure. New high-resolution data on an active serpin, serpin 1K from the moth hornworm Manduca sexta, provide insights into how active serpins are stabilized and how conformational changes are induced by protease binding. RESULTS: The 2.1 A structure shows that the RCL of serpin 1K, like that of active alpha1-antitrypsin, is canonical, complimentary and ready to bind to the target protease between P3 and P3 (where P refers to standard protease nomenclature),. In the hinge region (P17-P13), however, the RCL of serpin 1K, like ovalbumin and alpha1-antichymotrypsin, forms tight interactions that stabilize the five-stranded closed form of betasheet A. These interactions are not present in, and are not compatible with, the observed structure of active alpha1-antitrypsin. CONCLUSIONS: Serpin 1K may represent the best resting conformation for serpins - canonical near P1, but stabilized in the closed conformation of betasheet A. By comparison with other active serpins, especially alpha1-antitrypsin, a model is proposed in which interaction with the target protease near P1 leads to conformational changes in betasheet A of the serpin.     

Influence of an active pre-stretch on fatigue of skeletal muscle

In activities such as running, many muscles of the lower extremities appear to be actively stretched before they are allowed to shorten. In this study we investigated the effect of an active pre-stretch on the fatigability of muscles. Thus muscle contractions were compared in which shortening was preceded by an active isometric phase or by an active stretch. Rat medial gastrocnemius muscle-tendon complexes (with arrested blood flow) performed a series of ten repeated contractions (1.s-1) with either an active stretch or an isometric phase preceding the shortening. Contraction duration (0.45 s), and shortening duration (0.3 s), distance (6 mm) and velocity (20 mm.s-1) were the same in both types of contraction. Work output during the ten shortening phases was approximately 40% higher in the contractions with an active pre-stretch; in contrast, high-energy phosphate utilization was similar. Over the ten repeated contractions reduction of work output during the shortening phases of both types of contraction was similar in absolute terms (approx. 9.5 mJ). It is suggested that all the extra work performed during the shortening phases after a pre-stretch originated from sources other than cross-bridge cycling, which are hardly affected by fatigue. However, reduction of work output in relative terms, which is how the reduction is often expressed in voluntary exercise, was less after a pre-stretch (26% vs 32%), giving the impression of protection against fatigue by an active pre-stretch.     

Proteins from nuclear fractions with different contents of active genes

The protein content in four nuclear fractions was compared. The nuclear fraction of rat liver deficient in active genes was characterized by a very low content of non-histone proteins whose mobility is less than that of histone H1.. The predominant protein of this fraction is an acid-soluble protein (Mr = 41 +/- 1 kD) designated as 41K. This protein was detected in acid nuclear extracts of rat lungs, kidney and spleen but was absent (or practically absent) in four murine and rat hepatomas under study. The decreased content of protein 41K was correlated with the diminution of the content of histone H1(0) fraction. It was shown that proteins HMG 14 and 17 are readily washed off during fractionation of nuclei and they bind to DNA fragments passing into solution irrespective of whether they contain active or inactive genes. The nuclear matrix fraction rich in active genes was heterogeneous according to its protein composition. Differences in the intensity of staining and in electrophoretic mobility of some polypeptides of this nuclear fraction in normal and hepatoma cells were revealed.     

Platelet-activating factor. Evidence for 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine as the active component (a new class of lipid chemical mediators).

A glyceryl ether containing phosphoglyceride, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (Ac-GEPC), has been shown to have a biological activity indistinguishable from that of naturally generated (rabbit) platelet activating factor (PAF). Its biochemical and biological properties so closely parallel those of naturally occurring PAF that we propose they are one and the same compound. Both PAF and AcGEPC could be converted to an inactive form through base-catalyzed methanolysis and restored to 100% functional activity by reaction with acetic anhydride. The synthetic lipid, AcGEPC, elicited 50% secretion of serotonin from rabbit platelets at a level of 10(-10) M (based on phosphorus). A propionyl derivative had somewhat comparable activity towards platelets, whereas the butyryl homologue was some 7-fold less active and the stearoyl derivative was inactive. These short chain acylglyceryl ether phosphoglycerides represent an entirely new, potent and unique class of lipid chemical mediators. 1-Acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AcLL) also exhibited activity towards platelets but was some 200-fold less active than AcGepc. the propionyl lysolecithin behaved quite similarly to AcLL, but butyryl and stearoyl lysolecithins showed no activity.     

Small conformationally restricted piperidine N-arylsulfonamides as orally active gamma-secretase inhibitors

The design and development of a new class of small 2,6-disubstituted piperidine N-arylsulfonamide gamma-secretase inhibitors is reported. Lowering molecular weight including the use of conformational constraint led to compounds with less CYP 3A4 liability compared to early leads. Compounds active orally in lowering Abeta levels in Tg CRND8 mice were identified as potential treatments for Alzheimer's disease.