A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

Specificity of the dopamine sensitive adenylate cyclase for antipsychotic antagonists

Chlorpromazine, haloperidol and clozapine are approximately equipotent in antagonizing dopamine sensitive adenylate cyclase activity in homogenates of rat brain striatum, in contrast to the differences in clinical antipsychotic potencies reported by others. The antagonism appeared to occur at a structurally specific dopamine site, as inhibition by a series of chlorpromazine analogues of similar hydrophobicity exhibited a structural specificity similar to that found for their neuroleptic and cataleptic activities. Sulpiride, a dopamine antagonist with antipsychotic activity, and metoclopramide, a structurally related central dopamine antagonist, failed to inhibit the dopamine sensitive adenylate cyclase. Pre-treatment of rats with haloperidol (3 mg/kg per day) for 6 or 28 days did not induce a supersensitive response of the adenylate cyclase to stimulation by dopamine or apomorphine or inhibition by clozapine. It was concluded that the dopamine sensitive adenylate cyclase may not be the site of action of all anti-psychotic agents.     

Diurnal rhythmicity in plasma 24,25-dihydroxyvitamin D3: correlation with intestinal calcium transport and plasma minerals

Plasma 24,25-(OH)₂D₃ exhibits diurnal rhythmic variations in the adult rat. The decrease in 24,25-(OH)₂D₃ occurring at the onset of feeding during light-dark transition coincides with a decrease in plasma calcium but is inversely related to plasma phosphate and to increased intestinal calcium transport activity.     

Platelet plasma membrane lectin activity

The lectin activity of human platelet and erythrocyte membranes was evaluated using trypsinized, formalinized erythrocytes from eight species. Platelet membranes had the greatest lectin activity against cow erythrocytes, but also had significant activity against human, sheep, electric eel, and rabbit erythrocytes. In contrast, erythrocyte membranes only had low lectin activity against electric eel erythrocytes with no activity against the other types of erythrocytes tested. The platelet membrane lectin activity was found to reside in protein molecules on the external surface of the platelet plasma membrane. The lectin activity of platelet membranes was inhibited by amino sugars and some basic amino acids: N-acetylated amino sugars and other neutral sugars were without effect. These results demonstrate that the external surface of the platelet plasma membrane has a specific lectin activity.     

Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) substrate.

This report describes the preparation of a sodium (4-methylumbelliferyl-α-d-N-acetylneuraminate) substrate and its use in a sensitive fluorometric assay of neuraminidase (EC 3.2.1.18) from Vibrio cholerae, cultured fibroblasts, and human leucocytes. V. cholerae neuraminidase showed maximum activity at pH 4.6 and an apparent K_m of 1.5 mm and was activated by CaCl₂ and inhibited by ethylenediaminetetraacetate, NaCl, and N-acetylneuraminic acid. The inhibition by N-acetylneuraminic acid was competitive (K_i = 6.1 mm). Cultured fibroblast and leucocyte neuraminidases showed maximum activity between pH 4.2 and 4.4 and apparent K_m values of 0.13 and 0.22 mm, respectively. Neuraminidase activity was considerably reduced in cultured fibroblasts of patients with mucolipidosis types I, II, and III.     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

The utility of the microsomal 4-chlorobiphenyl hydroxylase enzyme assay in distinguishing between phenobarbitone- and 3-methylcholanthrene-induced microsomal monooxygenases

4-Chlorobiphenyl was used as a substrate for the in vitro determination of rat hepatic microsomal, cytochrome P-450-dependent monooxygenase activity. The 4-chlorobiphenyl hydroxylase assay was tested for its ability to distinguish between a variety of phenobarbitone- and 3-methylcholanthrene-type inducers. Two radiometric procedures were employed to investigate the metabolism of 4-chlorobiphenyl. First, the metabolite profile of 4-chlorobiphenyl was analyzed by radio-thin-layer chromatography. This procedure permitted an assessment of the effects of microsomal enzyme inducers on both the qualitative and quantitative aspects of 4-chlorobiphenyl metabolism. Second, the rate of 4-chlorobiphenyl metabolism was determined by a differential extraction procedure which separated unreacted starting material (hexane phase) from metabolites (base phase). This procedure provided a rapid measurement of the overall activity of 4-chlorobiphenyl hydroxylase. Irrespective of the animal pretreatment, the metabolite profile of 4-chlorobiphenyl was dominated by 4′-chloro-4-biphenyl. Unlike the qualitative aspects, the quantitative aspects of 4-chlorobiphenyl metabolism were markedly influenced by animal pretreatment. Specifically, 3-methylcholanthrene-type inducers (3-methylcholanthrene and 3,3′,4,4′-tetrachlorobiphenyl) enhanced the activity of 4-chlorobiphenyl hydroxylase at least 10 times more than phenobarbitone-type inducers (phenobarbitone, 2,2′,4,4′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl) enabling these two classes of inducers to be clearly distinguished. It is concluded that 4-chlorobiphenyl is preferentially metabolized by the 3-methylcholanthrene-inducible form(s) of cytochrome P-450 and that this class of microsomal enzyme inducers can be readily distinguished from phenobarbitone-type inducers by means of the 4-chlorobiphenyl hydroxylase assay.     

A radiochemical assay for N5-formyltetrahydrofolic acid (citrovorum factor) in serum and urine.

N⁵-Methyltetrahydrofolate, but not N⁵-formyltetrahydrofolate, can be measured in biological fluids by ligand-binding radioassay. Therefore, in order to measure N⁵-formyltetrahydrofolate by radioassay, it is chemically converted to N⁵-methyltetrahydrofolate by acidification followed by reduction with borohydride. By this method, 70–113% of N⁵-formyltetrahydrofolate added to serum and urine was recovered. The plasma clearance of the mixture of diastereoisomer of N⁵-formyltetrahydrofolate (Leucovorin) following intravenous administration to two normal subjects was rapid for the first 30 min, but then plateaued and cleared very slowly over the next 90 min, most probably because of the accumulation of the inactive isomer which was slowly excreted in the urine during this time period.     

A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts.

A radioisotope assay for the measurement of glutamine synthetase activity has been developed in which tandemly arranged ion-exchange columns of Dowex 1-acetate and Amberlite CG-50 (H⁺) are used to separate the product, [¹⁴C]glutamine, from unreacted [U-¹⁴C]glutamate and other labeled compounds, particularly γ-aminobutyrate, that are formed by competing reactions. The technique is sensitive, reproducible, and suitable for multiple determinations. The assay has been used successfully to measure glutamine synthetase activity in neural and nonneural tissues which contain appreciable amounts of glutamate decarboxylase activity.     

A rapid radioenzymatic assay for dopamine and N-acetyldopamine.

Dopamine (DA) was measured in various tissue extracts as [³H]methoxy-N-acetyldopamine after incubation with two partially purified enzymes, catechol-O-methyl transferase (EC 2.1.1.1) and N-acetyltransferase (EC 2.3.1.5), in the presence of [³H]adenosylmethionine and acetyl-CoA. This product can be separated quantitatively from labeled products of norepinephrine and epinephrine by solvent extraction. N-Acetyl-DA can be assayed by omitting the acetylating system from the incubation mixture. The procedure is rapid, convenient for processing large numbers of samples, and has a sensitivity of approximately 0.1 pmol. It has been used to measure DA in ganglia and in individual neurons from gastropod mollusks.     

Effect of histamine and acetylcholine on hypophysial stalk plasma dopamine and peripheral plasma prolactin levels.

To further define the role of dopamine in the regulation of prolactin secretion, we studied the effect on prolactin and hypothalamic dopamine secretion of histamine and acetylcholine (ACh) injected into the lateral ventricle of urethane anesthetized diestrus-1 rats. Histamine (10 μg) caused a 592% increase in plasma prolactin levels and a 26% decrease in stalk plasma dopamine levels. ACh (50 μg) caused a 2090% increase in plasma prolactin levels but no significant change in stalk plasma dopamine concentration.To determine if the 26% fall in stalk plasma dopamine following histamine administration could account for the 6-fold increase in plasma prolactin, we measured the effect on prolactin secretion of a similar decrease in administered dopamine. During an infusion of physiologic levels of dopamine, a 25% decrease in arterial plasma dopamine concentration resulted in only a 2-fold increase in prolactin secretion.The results of these experiments suggest that the effect of histamine on prolactin secretion may be mediated in part by decreased hypothalamic secretion of dopamine but that an additional hypothalamic hormone is probably involved. The stimulatory effect of ACh on prolactin secretion is not mediated by dopamine. These data are consistent with the growing evidence for the participation of multiple hypothalamic factors in the regulation of prolactin secretion.     

Regulation of the amphibian oocyte plasma membrane ion permeability by cytoplasmic factors during the first meiotic division.

The denuded Rana pipiens oocyte depolarizes from 80–90 mV inside negative to 3–5 mV? inside positive during progesterone-induced meiotic maturation, apparently due to decreased K⁺ permeability. Depolarization is dependent upon protein synthesis and coincides with breakdown of the oocyte nucleus, but occurs even in the absence of the nucleus, suggesting cytoplasmic regulation of cation selectivity of the oocyte plasma membrane.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Skin banking methodology: An evaluation of package format,cooling and warming rates,and storage efficiency

The two major skin packaging formats for transplantable human skin, flat — folded and rolled — cylindrical, were evaluated with respect to the control of cooling rate, warming rate, and storage efficiency. Experiments were performed with six amounts of skin ranging from 7.6 × 20 cm (0.17 ft²) up to 7.6 × 120 cm (1.00 ft²).Contrary to previously published statements, when skin packaged in either of the two formats is cooled at an uncontrolled rate in a low temperature (?70 °C) mechanical refrigerator or dry-ice chest, the smaller skin dimensions cool too rapidly (up to ?24 °C min^?1), while the packets containing larger skin dimensions exhibit prolonged exothermic temperature plateaus (8–44 min), allowing the possibility of significant crystallization damage to the cells. On the other hand, controlled-rate cooling of ?1 °C min^?1 can be obtained using a temperature-feedback controlled-rate freezer along with a flat skin packet geometry. Much less control is obtained if a cylindrical skin packet geometry is used with a controlled-rate freezer.Skin processed in the flat format is capable of being warmed by water immersion about 10 times more quickly than equivalent amounts of skin processed in the rolled format. The longer warming times associated with the cylindrical package format (3.5–25 min, depending upon the amount of skin per packet) result from extended endothermic temperature plateaus in the subzero region, which have been shown to damage skin cells and reduce their subsequent viability. The short warming times (0.25–3.5 min) associated with the flat skin package format are devoid of such complications, since they are within the needed warming rate of 50 °C–70 °C min^?1.Package geometry affects the storage requirements of transplantable skin. The flat format possesses a two- to threefold advantage in storage efficiency. Capital equipment and liquid nitrogen usage for storage is drastically decreased if a flat package format is chosen.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

Regulation of energy metabolism in Saccharomyces cerevisiae. Relationships between catabolite repression, trehalose synthesis, and mitochondrial development.

Changes in trehalose accumulation and in cytochromes during diauxic growth in glucose medium were examined in a normal Saccharomyces cerevisiae strain. While no appreciable disaccharide accumulation occurred during most of the logarithmic phase, a rapid synthesis took place during the final stages. The intrinsic capacity of cells to accumulate trehalose was also determined under nonproliferating conditions, in glucose medium lacking a nitrogen source. Cells harvested at an early growth stage had a much lower trehalose accumulation capacity than cells taken after glucose was exhausted from the culture medium. A high trehalose accumulation capacity could also be obtained at any growth stage by using maltose or galactose as carbon source. Since cells grown under various conditions exhibit a correlated change in cytochrome development and in trehalose accumulation capacity, it was concluded that the level of glucose repression determines the concentration and/or state of activation of the trehalose synthetase-trehalase complex. Independent control of trehalose accumulation capacity and mitochondrial biogenesis by the level of glucose repression was shown in two ways: by demonstrating derepression of trehalose accumulation without development of cytochromes a and c in microaerobic cells, and by showing repression-dependent changes in a cytoplasmic respiration-deficient (ρ^?) mutant, which lacked functional mitochondria. Therefore, the capacity of a cell to accumulate trehalose is not regulated solely by the supply of ATP generated by oxidative phosphorylation.     

Biochemical,immunological, and structural studies on a sphingolipid activator protein (SAP-1)

Sphingolipid activator protein-1 (SAP-1) is a glycoprotein found in human tissue extracts that stimulates the enzymatic hydrolysis of at least two glycosphingolipids, including GM1 ganglioside and sulfatide. The ability of purified SAP-1 to stimulate GM1 ganglioside hydrolysis by extracts of cultured fibroblasts from patients with β-galactosidase deficiency was examined, and all patients had a pronounced deficiency (under 10% of control). Using monospecific antibodies against SAP-1, the concentration was determined in cultured fibroblasts by rocket immunoelectrophoresis. Extracts from 15 control cell lines were found to have 0.72 ± 0.24 μg cross-reactive material/mg protein, while cell extracts from 8 patients with GM1 gangliosidosis involving mental retardation were found to have 1.08 ± 0.17, which is significantly elevated. When the fibroblast extracts were subjected to sodium dodecyl sulfate-polyacramide gel electrophoresis followed by electroblotting, multiple bands were observed. Controls were found to have two major bands with estimated molecular weights of 9000 and 9500, and a minor band at 7800. Extracts from patients with GM1 gangliosidosis were found to have multiple bands ranging upward to 13,000. Extracts from patients with the most severe clinical types of GM1 gangliosidosis had almost exclusively high-molecular-weight forms (molecular weights above 10,000). Treatment of SAP-1 from control liver with endoglycosidase D caused a decrease in the M_r 9500 band and increased in the M_r 7800 band. When SAP-1 from GM1 gangliosidosis liver was treated sequentially with neuraminidase, β-galactosidase, and endoglycosidase D, almost all of it was converted to the forms found in control human liver.     

Rat hepatic uroporphyrinogen III cosynthase: Purification,properties, and inhibition by metal ions

Rat hepatic uroporphyrinogen III cosynthase has been isolated and purified 50-fold with a 36% yield by ammonium sulfate fractionation and sequential chromatography on DEAE-Sephacel and Sephadex G-100SF. Inhibition of uroporphyrinogen III formation with increasing porphobilinogen concentration was observed. Cosynthase was shown to be thermolabile, and a time-dependent loss of enzyme activity during reaction with uroporphyrinogen I synthase and porphobilinogen was observed. The pH optimum for the complete system (synthase and cosynthase) was pH 7.8 in 50 mm Tris-HCl or 50 mm sodium phosphate buffer. Various metals (KCl, NaCl, MgCl₂, CaCl₂) increased formation of Uroporphyrinogen III. Heavy metals including ZnCl₂, CdCl₂, and CuCl₂ were shown to selectively inhibit cosynthase activity, whereas other metals (HgCl₂, PbCl₂) were less selective and inhibited both synthase and cosynthase at similar concentrations.     

Antigen-bearing Langerhans cells in skin,dermal lymphatics and in lymph nodes

Ferritin-challenged skin sites and draining lymph nodes were studied in normal guinea pigs and in guinea pigs which had been passively sensitized to ferritin or peroxidase by lymphoid cell transfer to ascertain whether Langerhans cells can bind antigen in skin and carry it to lymph nodes. After intradermal challenge with amounts of ferritin as small at 5 μg, ferritin-containing Langerhans cells were seen by electron microscopy in the marginal sinus and cortex of draining lymph nodes in ferritinscnsitized animals and, to an apparently lesser degree, in control animals. Lymph nodes from unchallenged normal guinea pigs contained rare Langerhans cells, none of which had ferritin. The findings indicate that Langerhans cells may pick up antigen in skin and from there circulate to draining lymph nodes, thus carrying out a function analogous to macrophages. In this way they may exhibit antigen to lymphocytes both in skin and in lymph nodes.     

Studies on photosystem I. I. Relationship of plastocyanin, cytochrome f and P700

DEAE-Sephadex column chromatography now has been used for the final step in purification of d-amino acid oxidase apoenzyme. A specific enzymatic activity of 35–37 units/mg has been obtained for the pure holoenzyme. The purity has been established by disc and SDS gel electrophoreses and by sedimentation equilibrium. The molecular weight per enzyme monomer has been found to be 38,000 ± 1000. Each enzyme monomer binds one FAD and one benzoate with dissociation constants at 23 °C and pH 8.5 of 5.35 × 10^?7m and 1.96 × 10^?6m, respectively. The holoenzyme is more negatively charged than the apoenzyme at alkaline pH. The amino acid composition and some other physicochemical properties of the oxidase are reported.