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.     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

Effects of castration and androgen replacement on male courtship behavior in the red-sided garter snake (Thamnophis sirtalis parietalis)

Following artificial hibernation, sexually mature male garter snakes (Thamnophis sirtalis parietalis) exhibited a decline in courtship behavior irrespective of castration, sham operation, or castration with testosterone replacement therapy. Behavior declined more rapidly in castrated animals with testosterone replacement than in castrated or sham-operated animals. In sham-operated animals, the decline in courtship was accompanied by changes in testicular weight and spermatogenic state from small spermatogenically inactive testes to large spermatogenically active testes. Serum androgen levels were more than fourfold greater in sham-operated animals than in castrated animals; cell height of the androgensensitive renal sex segment was greatest in castrated animals with testosterone replacement and least in castrated animals. These findings indicate that following artificial hibernation, male courtship behavior of T.s. parietalis is independent of the presence of the testes.     

Proline synthesis and redox regulation: Differential functions of pyrroline-5-carboxylate reductase in human lymphoblastoid cell lines

Pyrroline-5-carboxylate (P5C) reductase not only catalyzes the final step in proline biosynthesis but also mediates redox regulation. We examined these dual functions in human lymphoblastoid cell lines (REH, LHN₁₃) one of which (REH) is deficient in proline synthesis. In spite of the deficiency in proline synthesis in REH, the PC-mediated redox effect was relatively intact. The dissociation of these functions was due to qualitative as well as quantitative differences in the REH enzyme relative to its utilization of NADPH and NADH.     

Microinjection studies of protein transit across the nuclear envelope of human cells

Proteins of various molecular weights were conjugated with rhodamine and microinjected into the cytoplasm or nucleus of HeLa cells. The injected proteins were then localized within the cells at various times thereafter with fluorescence microscopy. Proteins below approx. 60 kD rapidly crossed the HeLa nuclear envelope. Some larger proteins also were able to pass into or out of the nucleus, while others were unable to do so, indicating the selective permeability of the HeLa nuclear envelope to large proteins. The nuclear protein HMG17 accumulated within the nucleus shortly after cytoplasmic microinjection.     

Response of selected fetal organs to antineoplastic agents

This study was designed to quantitate the effects of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DIC) and 5-(3,3-bis(2-chlorethyl)-1-triazeno)-imidazole-4-carboxamide (BIC) on growth and selected components of rat fetal organs. Twelve-day pregnant rats were given single intraperitoneal injections of 600 mg/kg of DIC and 900 mg/kg of BIC and autopsied on day 21 of gestation. Fetal liver, brain, kidney, and placenta were removed, weighed, and assayed for total DNA, RNA, and protein. DIC significantly reduced weight, total DNA, RNA, and protein of all four fetal organs as compared to age-matched controls. The brain was most severely affected by this compound. BIC also significantly reduced weight, DNA, RNA, and protein of fetal brain, kidney, and placenta, but in fetal liver only weight and total protein were significantly depressed, while DNA and RNA remained essentially unchanged. The effect of BIC was maximal on the placenta.     

The generation of memory B-cell subpopulations capable of proliferation and expansion of the pool: effect of time and antigen

The ability of antigen to induce proliferation of memory B lymphocytes, thus perpetuating and expanding the memory cell pool, has been examined using an antibody-forming cell (AFC) assay. This method provided confirmation of previous studies in which serum antibody titer was used as a relative measure of pool size and demonstrated directly that the number of antibody-forming cells is increased. Memory cell subpopulations were prepared by lg velocity sedimentation from recently immunized donors (2 weeks) and tested for their ability to proliferate, thus expanding the memory cell pool. Both large and small immature DNP-specific memory cells displayed antigen-dependent and antigen-independent proliferation while mature cells (8 weeks postpriming) were capable only of antigen-dependent proliferation. Chicken γ-globulin (CGG) specific memory cells were also evaluated in this system and were found to differ from DNP-specific cells in several ways. (A) DNP-specific AFC were found to be concentrated in the spleen while CGG-specific AFC were found predominantly in the bone marrow early after transfer and in the spleen upon delayed challenge. (B) The rate of maturation of CGG-specific memory cells capable of antigen-driven proliferation and pool expansion was delayed in comparison to DNP-specific memory cells. The relationship of these functionally defined subsets to previously described memory cell subpopulations is discussed.     

Preparation and use of α-maltosyl fluoride as a substrate by beta amylase

The preparation of pure (amorphous) α-maltosyl fluoride is described. A modification of the procedure of Brauns was used to obtain analytically pure, crystalline hepta-O-acetyl-α-maltosyl fluoride, the structure of which was assigned by¹⁹F-and¹H-n.m.r. spectroscopy. α-Maltosyl fluoride was obtained by deacetylating the heptaacetate. It behaved as a single compound on thin-layer and paper chromatography, and was essentially completely hydrolyzed to maltose and hydrogen fluoride by 0.01M sulfuric acid in 10 min at 100°. Crystalline beta amylase, likewise, catalyzed essentially complete hydrolysis of α-maltosyl fluoride to give maltose and hydrogen fluoride. The rates of hydrolysis catalyzed by beta amylase preparations from sweet potatoes and soybeans acting on a range of concentrations of the substrate produced linear curves for the relationship, 1/v vs 1/S; reaction constants for crystalline, sweet-potato enzyme were K_m 3.6 mM and V_max ~ 2 μ mol/min/mg. The finding that α-maltosyl fluoride is hydrolyzed 30–60 times faster than maltotriose demonstrates for the first time that beta amylase is capable of effecting hydrolysis at an appreciable rate of a substrate having only two d-glucose residues.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Alterations in the developmental patterns of enzymes in chicken embryos infected with West Nile virus.

Infection of chicken embryos with West Nile (WN) virus, a group B togavirus containing structural lipids, caused a rapidly developing hypertriglyceridemia. Changes in the activity of several hepatic regulatory enzymes in glycolytic and lipogenic pathways occurred during infection. Compared to control values in embryos of the same age (16 days), an 8.8-fold increase in the specific activity of ATP-citrate lyase and a 5.6-fold increase in that of hexokinase were observed on the third day of WN virus infection. Hexose monophosphate shunt dehydrogenase specific activities were elevated twofold in virus-infected livers. Activities of malic enzyme and phosphofructokinase were also elevated in WN virus-infected livers. Malate dehydrogenase and NADP-linked isocitrate dehydrogenase levels showed little or no change during infection. The levels of pyruvate kinase and lactate dehydrogenase were decreased in virus-infected livers. Hepatic acetyl-CoA carboxylase activity was at least twofold higher in virus-infected embryos; however, following removal of low-molecular-weight compounds, the specific activities of this enzyme from infected and control embryos were virtually identical. The results of mixing experiments suggest that the low levels of carboxylase activity in control embryos may be due to the presence of enzyme inhibitor(s) which can be removed by gel filtration.The incorporation of radiolabeled precursors into cellular lipids by liver minces from virus-infected and uninfected embryos was measured. There was a twofold increase in carbohydrate incorporation in virus-infected liver as compared to uninfected liver; [¹⁴C]pyruvic acid was incorporated into lipids to the greatest extent. [1-¹⁴C]acetic acid, [U-¹⁴C]alanine, and [U-¹⁴C]leucine were incorporated very poorly in both infected and control livers. Twice as much [1-¹⁴C]oleic acid or [1-¹⁴C oleic]triolein was incorporated in WN-infected livers as in control. The relative distribution of neutral and polar lipids formed from each precursor was generally similar in infected and uninfected livers as determined by thin-layer chromatography of radiolabeled lipids. Except for a threefold increase in oxidation of [¹⁴C]glucose by virus-infected livers, the oxidations of carbohydrates and fatty acids were similar in infected and uninfected livers. The pentose phosphate pathway appears to be the major pathway utilized in glucose oxidation for both control and virus-infected livers. The results indicate that enhanced flux of metabolites into lipids reflects a virus-induced alteration in embryonic development: The enzyme patterns of infected embryos are more characteristic of older embryos or even newly hatched chicks.     

Membrane (Na+ + K+)-ATPase of canine brain,heart and kidney. Tissue-dependent differences in kinetic properties and the influence of purification procedures

Effects of commonly used purification procedures on the yield and specific activity of (Na⁺ + K⁺)-ATPase (Mg²⁺-dependent, Na⁺ + K⁺-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP-dependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na⁺ + K⁺)-ATPase and the ratio between (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na⁺ + K⁺)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na⁺ + K⁺)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzyme preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purification procedure. These results indicate that the presently used purification procedures may alter.     

Detachment variants of Chinese hamster cells : Hyaluronic acid as a modulator of cell detachment

Variants of the Chinese hamster cell line CHO have been isolated and characterized with respect to attachment and trypsin- or EGTA-mediated detachment kinetics, cell morphologies, and the complex carbohydrates (labeled with [³H]glucosamine) of the cell surface. The variant which was more readily detached from the substratum exhibited a more rounded cell shape and had three times more label as hyaluronic acid on the cell surface than the parental cell. The slowly detaching variant had a morphology similar to the parental cell but only half the radioactivity ascribable to hyaluronic acid. Endogenous levels of cAMP were unaltered in the variants. Exogenous dbcAMP caused the cells to elongate and flatten but did not alter the characteristic detachment kinetics. The role of hyaluronic acid as a modulator of the cell substratum interface is discussed.     

Do mammalian collagenases and DNA restriction endonucleases share a similar mechanism for cleavage site recognition?

Despite the apparent uniformity of the collagen molecule, vertebrate and invertebrate collagenases cleave it in one region only. We suggest that the enzyme recognises the cleavage site by the arrangement of the imino acids proline and hydroxyproline on either side of a region where the helical conformation of the collagen molecule is less stable. This less stable region could fold out of the rigid collagen molecule allowing the two recognition sites to be simultaneously attached to identical subunits in the same collagenase molecule. Class II DNA restriction endonucleases are confronted by a similar recognition problem in cleaving the DNA molecule at a specific site and it is generally accepted that here recognition is achieved by a sequence of bases with two-fold symmetry. We postulate that collagenase may, like the DNA restriction enzyme, be active in the dimeric form and that it recognises its substrate site by a similar two-fold symmetric arrangement of imino acid residues.     

Transport of succinate by Pseudomonas putida

Induced succinate uptake and transport (defined as transport of a compound followed by its metabolism and transport in the absence of subsequent metabolism) by Pseudomonas putida are active processes resulting in intracellular succinate concentrations 10-fold that of the initial extracellular concentration. Uptake was studied with the wild-type strain P. putida P2 and transport with a mutant deficient in succinate dehydrogenase activity. Addition of succinate, fumarate, or malate to the growth medium induces both processes above a basal level. Induction is dependent on protein synthesis and subject to catabolite repression. When extracts of induced and noninduced wild-type cells were assayed for succinate dehydrogenase, fumarase, and malate dehydrogenase only malate dehydrogenase increased in specific activity. Transport is inhibited by iodoacetamide, KCN, NaN₃, and 2,4-dinitrophenol and shows pH and temperature optima of 6.2 and 30 °C. Kinetic parameters are: basal uptake (cells grown on glutamate) K_m 11.6 μm, v 0.32 nmoles per min per mg dry cell mass; induced uptake (cells grown on succinate plus NH₄Cl) K_m 12.5 μm, v 5.78 nmoles per min per mg dry cell mass; induced transport (cells grown on nutrient broth plus succinate) K_m 10 μm, V 0.98 nmoles per min per mg dry cell mass. It was not possible to determine the kinetic parameters of basal transport. Malate and fumarate were the only compounds exhibiting competitive inhibition of uptake and transport suggesting common transport system for all three compounds. The K_i values for competitive inhibition and the K_m for succinate indicate the order of affinity for both uptake and transport are succinate > malate > fumarate. Data from kinetic parameters of uptake and transport and studies on succinate metabolism provide evidence consistent with concurrent increases in transport and metabolism to account for induced succinate uptake by P. putida.     

Properties of xanthosine 5'-monophosphate-amidotransferase from Escherichia coli.

Xanthosine 5′-phosphate (XMP)-amidotransferase catalyzes the formation of guanosine 5′-phosphate (GMP) by aminating XMP with either the amide group of glutamine (amidotransferase) or ammonia (aminase). The glutamine-supported activity of the purified enzyme from Escherichia coli has been studied, and its properties have been compared with those of other amidotransferases. The following results have been obtained. (i) The glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), irreversibly inhibits the amidotransferase activity. A maximal rate of inhibition by DON is achieved in the presence of XMP, ATP, and Mg²⁺ with a pseudo-first-order rate constant of 0.276 min^?1. (ii) The total number of sulfhydryl groups is approximately 22 per dimer (126,000 M_r). In the absence of substrates, about 8 sulfhydryl groups per dimer are titratable with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), and in the presence of XMP, ATP, and Mg²⁺ an additional 6 cysteine residues per dimer become exposed. When the amidotransferase activity is inactivated by DON, only 8 sulfhydryl groups are titratable. DTNB, p-chloromercuribenzoate, and bromopyruvate all selectively inactivate the amidotransferase activity. These results are consistent with the hypothesis that cysteine residues which are exposed by the substrates are involved in the amidotransferase activity. (iii) The purified XMP amidotransferase contains a glutaminase activity which can be measured in the absence of GMP formation. The glutaminase activity requires XMP, Mg²⁺, and either psicofuranine, an analog of adenosine, or inorganic pyrophosphate (PP_i) and is inhibited by p-chloromercuribenzoate and DON. Maximal stimulation is observed with 100 μm psicofuranine or PP_i, and there is no further stimulation in the presence of both effectors. The apparent K_m is 31 μm with PP_i and 13 μm with psicofuranine; the V for glutamine hydrolysis is about 60% of the rate of the amidotransferase activity. The cooperative interactions between the binding of PP_i and psicofuranine have been confirmed. In the presence of 2.5 μm psicofuranine the K_m for PP_i is reduced 20-fold, but the maximal velocity is unchanged. Similarly, the apparent K_m for psicofuranine is reduced by low concentrations (10 μm) of PP_i. The “uncoupling” of the hydrolysis of glutamine from the amination of XMP is the basis for the reported inhibitory effects of psicofuranine and PP_i on the amidotransferase activity. (iv) Tris buffer selectively inhibits the XMP-amidotransferase activity by inhibiting the glutaminase activity. This inhibition is time dependent and reversible and may explain the previous reports on the inability of this enzyme to use glutamine as a substrate.     

Melatonin-induced suppression of gonadotropin titers in male golden hamsters: Effect on gonadal feedback mechanisms

Daily subcutaneous injections of melatonin cause testicular regression and a decline in circulating gonadotropin levels in male hamsters maintained on long photoperiods. We examine here if a reduction in gonadotropin levels also occurs in castrates administered melatonin and if melatonin-regressed hamsters respond to castration with an increased release of pituitary gonadotropins — a typical “castration response.” Groups of intact and castrated male hamsters maintained on a photoperiod of LD 14:10 received subcutaneous injections of 15 ug melatonin/day. Controls received vehicle only. After 7 weeks of injections the intact males were castrated. All animals were sacrificed a few days later and serum was assayed for gonadotropin titers. Melatonin injections caused a marked decline in serum gonadotropins in castrates and in intact males also caused testicular regression. In the latter, no “castration response” was observed upon removal of the testes. Thus, daily injections of melatonin depress serum gonadotropins in castrate and intact males and block the castration-associated rise in circulating gonadotropins in the latter.     

Rapid preparation of covalently closed circular DNA by acridine yellow affinity chromatography

Covalently closed circular DNA can be isolated rapidly from cell lysates in a two-step process. Hydroxylapatite chromatography to prepurify the plasmid DNA from contaminating protein and RNA is followed by a step gradient elution of covalently closed circular (CCC) plasmid DNA from an acridine yellow affinity column. This procedure results in CCC DNA of a purity comparable to that obtained from ethidium bromide-CsCl gradients without lengthy centrifugation and free of contaimination by intercalating dye. Up to 250 μg of CCC pBR 322 can be isolated from 500 ml of bacterial culture in 4–6 h.     

l-Phenylalanine stereospecifically inhibits the renaturation of muscle pyruvate kinase

Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine HCl. Subsequent dilution of the enzyme into buffer containing β-mercaptoethanol or dithiothreitol results in recovery of enzymatic activity with an average half-time of 17 min at 16 °C. The addition of 1 mm l-phenylalanine increases the average half-time for recovery of enzymatic activity to 26 min, while 8 mm l-phenylalanine further increases this value to 46 min. Tyrosine and tryptophan also inhibit the reactivation but to a lesser extent than phenylalanine. Neither l-alanine, l-valine, d-phenylalanine, phosphoenolpyruvate, nor fructose 1,6-bisphosphate have any appreciable effect on activity recovery rates, either in the presence or absence of l-phenylalanine. Phenylpyruvate is a very potent inhibitor of reactivation. The addition of 5 mm phenylpyruvate increases the half-time to 57 min. The evidence presented in this paper supports the hypothesis that an l-phenylalanine-binding site which probably is distinct from the catalytic site is formed early in the renaturation process. l-Phenylalanine binds to this site and inhibits two first-order relaxations that are rate limiting for the reactivation and that have the following rate constants: 8.76 × 10^?2 and 1.24 × 10^?2 min^?1, respectively, in the absence of phenylalanine and 3.04 × 10^?2 and 7.63 × 10^?3min^?1, respectively, in the presence of 8.0 mm phenylalanine. We presume these first-order processes to be transconformational steps in the reactivation process.