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S Kaufman

Publications and source records attributed to S Kaufman.

At least 181 records · Page 10Linked to original sources
PubMed1992

Role of spleen in ANF-induced reduction in plasma volume.

Atrial natriuretic factor (ANF) causes an increase in hematocrit that cannot be accounted for by urinary losses. The mechanism behind this phenomenon was studied in intact and splenectomized rats. Rat ANF 99-126 was infused i.v. for 30 min into conscious rats at rates of 0 (saline control), 0.05, or 0.1 microgram/min. Plasma volume was then determined by dilution of the dye, Evan's Blue. In one group of rats, red cell volume was determined using 51Cr-labelled erythrocytes. ANF infusion was continued uninterrupted throughout the experiments. In the intact rats, ANF (0.10 microgram/min) caused hematocrit to increase from 38.9 +/- 0.5 to 41.2 +/- 0.4% (p < 0.005). Splenectomy so attenuated this response to ANF that it failed to reach significance. Similarly, ANF (0.10 microgram/min) caused plasma volume to fall from 5.1 +/- 0.1 to 4.5 +/- 0.1 mL/100 g body wt. (p < 0.005) in the intact rats, but did not affect plasma volume in the splenectomized rats. As a result, blood volume was significantly reduced by ANF in the intact rats, but remained unchanged in the splenectomized rats. Red cell volume did not change in response to infusion of ANF, nor did ANF affect the rate of clearance of Evan's Blue out of the plasma. It is concluded that the spleen is an important site of movement of protein-poor fluid out of the vasculature, and that this exchange is influenced by ANF.

Animals↗
PubMed1992

Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.

Animals↗
PubMed1991

High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme.

A rat cDNA containing the complete coding sequence for rat tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) was isolated from a rat PC12 cDNA library and subcloned in a bacterial expression plasmid, and large amounts of functional enzyme were produced in Escherichia coli. The recombinant enzyme was purified approximately 20-fold to a final specific activity of 1.8 mumol/min per mg of protein, with a yield of 30%. As much as 1 mg of pure protein could be obtained from 1 g of wet bacterial cells. The purified hydroxylase was shown to be homogeneous by denaturing polyacrylamide electrophoresis and isoelectric focusing. Amino acid analysis of the N terminus (25 residues) revealed 100% identity with rat PC12 tyrosine hydroxylase, as deduced from its cDNA sequence. Several of the kinetic properties of the recombinant enzyme resembled those of the native PC12 hydroxylase. However, in contrast to the native enzyme, the purified recombinant hydroxylase was shown to be in an activated form. Phosphorylation with cAMP-dependent protein kinase resulted in stoichiometric incorporation of phosphate, but the kinetic profile of the recombinant enzyme was unaffected. Several clues to these differences are considered that may provide insight into the structural features important to the regulation of tyrosine hydroxylase.

Adrenal Gland Neoplasms↗
PubMed1991

Decreased CD45RA T cells in B-cell chronic lymphatic leukemia patients: correlation with disease stage.

T-cell subsets CD4, CD8 and suppressor-inducers (CD45RA) were determined in 20 patients with B-cell chronic lymphatic leukemia (B-CLL). The proportion of CD4 and CD45RA was decreased when compared with T cells from normal subjects. CD8 was markedly increased. The activity of concanavalin A-induced suppressor cells was not significantly different from that of normal controls and was negatively correlated to the percentage of CD4 of B-CLL patients. The selective loss of CD45RA cells was more prominent in patients in advanced Rai stages of the disease (III to IV) than in early stages (0 to II). Six patients of the advanced stages group suffered from autoimmune hemolytic anemia, whereas no patient in the early stages of disease showed an autoimmune phenomenon. Our results may indicate a mechanism of autoimmunity in B-CLL similar to that of patients with autoimmune diseases.

Aged↗
PubMed1991

The hydroxylation of phenylalanine and tyrosine by tyrosine hydroxylase from cultured pheochromocytoma cells.

Pheochromocytoma tyrosine hydroxylase was reported to have unusual catalytic properties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S. J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the apparent inability to hydroxylate phenylalanine and an unprecedented reactivity with hydrogen peroxide were investigated further in the present study. Tyrosine hydroxylase was purified to apparent homogeneity from cultured pheochromocytoma PC12 cells. The purified tumor enzyme was entirely dependent on tetrahydrobiopterin (BH4) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hydrogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (S0.5 Tyr = 15 microM) and BH4 (apparent Km BH4 = 210 microM). In addition, the enzyme catalyzed the hydroxylation of substantial amounts of phenylalanine to tyrosine and 3,4-dihydroxyphenylalanine (apparent Km Phe = 100 microM). Phenylalanine did not inhibit the enzyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations greater than or equal to 50 microM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or exceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hydroxylase from other tissues.

Animals↗
PubMed1991

Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine.

Studies with purified nitric oxide synthase from rat cerebellum have confirmed previous reports that product formation is enhanced by tetrahydrobiopterin [H4B; 6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin]. The effect of the natural isomer, (6R)-H4B, is observed at extremely low (less than 0.1 microM) concentrations and is remarkably selective. At these concentrations, only the diastereoisomer (6S)-H4B, the structural isomer 7-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin, and 7,8-dihydrobiopterin showed detectable effects. Our observations are inconsistent with a stoichiometric role for H4B in the oxygenation of arginine [e.g., Stuehr, D. J., Kwon, N. S., Nathan, C. F., Griffith, O. W., Feldman, P. L. & Wiseman, J. (1991) J. Biol. Chem. 266, 6259-6263]. Activity is initially independent of added H4B; enhanced product formation with H4B is observed only as incubation progresses. The effect of H4B is catalytic, with each mole of added H4B supporting the formation of greater than 15 mol of product. Recycling of H4B was excluded by direct measurement during nitric oxide synthesis and by the demonstration that nitric oxide synthase is not inhibited by methotrexate. These combined results exclude H4B as a stoichiometric reactant and suggest that H4B enhances product formation by protecting enzyme activity against progressive loss. Preliminary studies indicate that the decreased activity in the absence of added H4B does not depend on catalytic turnover of the enzyme. The role of H4B may be allosteric or it may function to maintain some group(s) on the enzyme in a reduced state required for activity.

Amino Acid Oxidoreductases↗
PubMed1991

7-Tetrahydrobiopterin is an uncoupled cofactor for rat hepatic phenylalanine hydroxylase.

Rat hepatic phenylalanine hydroxylase requires both a tetrahydropterin cofactor and molecular oxygen to convert phenylalanine to tyrosine. During the physiological hydroxylation, a single mol of the natural cofactor, tetrahydrobiopterin, is oxidized for each mol of phenylalanine converted to tyrosine. Artificial conditions have been devised in which the oxidation of the tetrahydropterin is uncoupled from the hydroxylation of the aromatic amino acid substrate. Recently, an isomer of tetrahydrobiopterin, 7-tetrahydrobiopterin, has been isolated from the urine of certain mildly hyperphenylalaninemic children. We report in this communication that 7-tetrahydrobiopterin may be an inefficient cofactor for phenylalanine hydroxylase because, in vitro, the phenylalanine-dependent oxidation of 7-tetrahydrobiopterin is accompanied by the hydroxylation of the aromatic amino acid substrate only about 15% of the time, i.e. the enzymatic oxidation of 7-tetrahydrobiopterin is about 85% uncoupled from the hydroxylation of the amino acid substrate.

Animals↗
PubMed1991

A staphylococcal plasmid that replicates and expresses ampicillin, gentamicin and amikacin resistance in Escherichia coli.

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.

Amikacin↗
PubMed1991

Leukocyte alkaline phosphatase and carcinoembryonic antigen in metastatic colorectal cancer patients.

Peripheral blood leukocyte alkaline phosphatase scores and plasma carcinoembryonic antigen levels in 26 patients with metastatic colorectal cancer were compared to those in 30 healthy controls. Patients had metastases to the liver and abdomen. The mean leukocyte alkaline phosphatase score in the metastatic colorectal cancer patients was significantly higher than in the control group (246 +/- 65 vs, 52 +/- 26, p less than 0.001); and the mean carcinoembryonic antigen level in the patients was also significantly higher than in the controls (110 +/- 100 vs, 4.9 +/- 3 ng/ml, p less than 0.001). One hundred percent of the metastatic cancer patients had elevated LAP scores and 73% of these patients had elevated CEA levels. There was a difference between the mean CEA levels in the patients with liver metastases and those with abdominal metastases (162 +/- 135 vs, 39 +/- 53 ng/ml, p less than 0.04). The results suggest that although both markers were elevated in metastatic colorectal cancer, the LAP score seems to be more useful in detecting metastatic disease, since we found 11% false negatives with the CEA level and 0% false negatives with the LAP score.

Abdominal Neoplasms↗
PubMed1991

Hydroxylation of 4-methylphenylalanine by rat liver phenylalanine hydroxylase.

Rat liver phenylalanine hydroxylase that has been activated with lysolecithin catalyzes the hydroxylation of 4-methylphenylalanine in the presence of a pterin cofactor. Two products, 4-hydroxymethylphenylalanine and 3-methyltyrosine, can be detected. The total amount of amino acids hydroxylated is equal to the amount of tetrahydropterin oxidized. Isotopic labeling studies with 18O2 and H2(18)O show that the hydroxyl groups of both products are derived from molecular oxygen and not from water. Results obtained with 2H-labeled substrates support the conclusion that these products are formed via different mechanistic pathways. Our previous investigations on substrate analogs, as well as the present results, indicate that a highly reactive oxygen-containing intermediate, such as an enzyme-bound iron-oxo compound, must be the hydroxylating species. Our present results could stimulate further discussion of the possibility that the reaction mechanism for the "NIH-shift" of the methyl group may not involve the spontaneous opening of an epoxide intermediate.

Animals↗
PubMed1991

Conversion of 6-substituted tetrahydropterins to 7-isomers via phenylalanine hydroxylase-generated intermediates.

A new variant form of hyperphenylalaninemia has recently been discovered in which the patients characteristically excrete 7-biopterin in their urines in addition to the natural 6-biopterin (Curtius, H. Ch., Kuster, T., Matasovic, A., Blau, N. & Dhondt, J.-L. (1988) Biochem. Biophys. Res. Commun. 153, 715-721). This isomer had not been found previously in humans, and although its origin was not established, preliminary evidence suggested that it might be produced from 6-biopterin. We have now found that 7-biopterin can be formed in vitro from (6R)-tetrahydrobiopterin during the hydroxylation of phenylalanine catalyzed by phenylalanine hydroxylase [L-phenylalanine, tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1]. The resulting 7-biopterin was unequivocally identified by the following criteria: preparative isolation and conversion to 7-hydroxymethylpterin following periodate oxidation and borohydride reduction, quantitative conversion to pterin-7-carboxylic acid after oxidation with permanganate, and liquid chromatography/thermospray mass spectrometry. Addition of 4a-carbinolamine dehydratase, an enzyme involved in the regeneration of tetrahydrobiopterin from the pterin carbinolamine intermediate (also called 4a-hydroxytetrahydrobiopterin) formed in the phenylalanine hydroxylase reaction, greatly decreased the amount of the 7-biopterin formed. This result implies that the in vitro formation of 7-biopterin occurs via the nonenzymatic rearrangement of the unstable substrate of the dehydratase, 4a-hydroxytetrahydrobiopterin, and suggests that this new variant of hyperphenylalaninemia may be caused by a lack of 4a-carbinolamine dehydratase activity. A mechanism for the rearrangement is proposed that predicts that other 6-substituted tetrahydropterin substrates of the aromatic amino acid hydroxylases could also give rise to rearranged products from an opening of the pyrazine ring of the corresponding 4a-hydroxytetrahydropterin intermediate.

Animals↗
PubMed1991

Leukocyte alkaline phosphatase score in patients with cerebrovascular disease and in patients with primary and metastatic brain tumors.

Leukocyte alkaline phosphatase (LAP) activity in peripheral blood was determined in 54 patients with cerebrovascular disease (CVD), 11 patients with primary brain tumor, and 23 patients with metastatic brain tumor. The LAP score of the primary brain tumor patients (155 +/- 40) was significantly higher (P less than 0.03) than the LAP score for cerebrovascular disease patients (96 +/- 87). The LAP score levels of the metastatic brain tumor patients (251 +/- 55) was significantly higher as compared with both the cerebrovascular disease (P less than 0.001) and primary brain tumor groups (P less than 0.001). The LAP score could serve as an additional, although non-specific, marker to assist in the differentiation between cerebrovascular disease and brain tumors.

Adult↗