Aryl sulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is some evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Incorporation of radiolabel from [1-14C]5-hydroperoxy-eicosatetraenoic acid into slow reacting substance

When synthetic [1-¹⁴C]5-hydroperoxy-eicosatetraenoic acid was incubated with rat basophilic cells, incorporation of the radiolabel into slow reacting substance (SRS) could be demonstrated as evidenced by comigration of spasmogenic activity and radioactivity after purification by high pressure liquid chromatography. This provides direct evidence that SRS is a product of the lipoxygenase pathway.     

Formation of the cysteinyl form of slow reacting substance (leukotriene E4) in human plasma

The two major species of slow reacting substance (SRS) contain either a glutathionyl or cysteinyl-glycyl side chain. Incubation of these SRS's with undiluted or diluted (usually 1:10 or 1:50) human plasma at 37°C resulted in marked losses of smooth muscle contracting activity due primarily to conversion of their oligopeptide side chains to cysteine.     

Sequential conversion of the glutathionyl side chain of slow reacting substance (SRS) to cysteinyl-glycine and cysteine in rat basophilic leukemia cells stimulated with A-23187

In rat basophilic leukemia (RBL-1) cells stimulated with A-23187, the major slow reacting substance (SRS) species contain glutathione, cysteinyl-glycine, or cysteine in their side chains, corresponding or closely related to leukotrienes LTC₄, LTD₄, and LTE₄, respectively.³ Evidence is presented that most of the SRS produced during the first few minutes of stimulation by the ionophore has a glutathionyl side chain which is sequentially converted to cysteinyl-glycine and cysteine.     

Effects of alloxan-diabetes on the sodium-potassium adenosine triphosphatase enzyme system in dog hearts

The effects of alloxan-diabetes on the partial reaction of (Na⁺+K⁺)-ATPase, K⁺-activated para-nitrophenylphosphatase, and on ouabain binding were studied in isolated adult dog heart myocytes. The Km of K⁺-activated para-nitrophenylphosphatase for K⁺ activation was increased from 2.5 to 7.7 mM with no change in Vmax. The Scatchard plots for ouabain binding between control and diabetic animals were indistinguishable. These results indicate that in acute diabetes induced by alloxan, the number of Na⁺-K⁺ pumping sites in the heart is not altered but the affinity of the system for K⁺ is decreased. It is suggested that the decrease in K⁺ affinity of the (Na⁺+K⁺)-ATPase enzyme system is at least in part responsible for the altered K⁺ homeostasis in the diabetic state.     

Localization of a site interacting with human platelet receptor on carboxy-terminal segment of human fibrinogen gamma chain

We report that the 27-residue carboxy-terminal cyanogen bromide fragment of human fibrinogen γ chain inhibits binding of [¹²⁵I]fibrinogen to human platelet receptors and blocks fibrinogen-mediated aggregation of ADP-treated human platelets. The blocking activity of the peptide was preserved after proteolysis of the isolated peptide with staphylococcal protease to generate a mixture of a dodecapeptide and a pentadecapeptide. Trypsin treatment destroyed blocking activity of the isolated peptide. These results indicate that the site responsible for the interaction of human fibrinogen with the platelet receptor resides in the 27-residue carboxy-terminal region of the γ chain.     

Metabolism of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in cultured human fetal aortic smooth muscle cells.

Cultured human fetal aortic smooth muscle cells derived from the abdominal aorta converted benzo[a]pyrene (BaP) and 7,12-dimethylbenz[a]anthracene (DMBA) $\text{via}$ cytochrome P-450-dependent monooxygenation to metabolites detectable by both a highly sensitive radiometric assay and high pressure liquid chromatography (HPLC). Cells incubated with ³H-BaP transformed this substrate primarily to phenols. ¹⁴C-DMBA was converted to metabolites that cochromatographed with 12-hydroxymethyl-7-methylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz-[a]anthracene, 7,12-dihydroxymethylbenz[a]anthracene, and trans-8,9-dihydrodiol-7,12-DMBA. Exposure of cells in culture to 13 μM 1,2-benz[a]anthracene resulted in increased oxidative metabolism of both BaP and DMBA. In the case of BaP, total phenol formation was increased, while with DMBA all metabilities detected by HPLC were increased. Support for the potential role of metabolism of polycyclic aromatic hydrocarbons by aortic smooth muscle cells in the etiology of atherosclerosis was obtained.     

A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation

Microsomal fractions from arterial walls of pigs and rabbits and fundus of rat stomach generate from prostaglandin endoperoxides (PGG₂ or H₂) an unstable substance, prostaglandin X (PGX) which is a potent inhibitor of platelet aggregation induced by several different substances.     

Effects of freeze-preservation on some pollen enzymes: II. Freezing and freeze-drying stresses

The isozymes of lily and corn pollen esterases and acid phosphatase were studied in relation to freeze-drying and vacuum-drying. Fresh samples of Lilium longiflorum L. and Zea mays L. pollen were frozen at rates ranging between 200 and 100 °C/min and freeze-dried at temperatures from 0 to ?70 °C for approximately 48 to 70 hr. Freeze-dried samples were rehydrated slowly (10% relative humidity) and rapidly (90% relative humidity). Vacuum-drying was performed at room temperature (22 °C).Soluble pollen enzymes were analyzed by disc electrophoresis on polyacrylamide gels stained with substrate specific reagents. The stained gels were evaluated by densitometry for migration rate, isozyme pattern, and relative activity. The numerical data generated in this manner were then statistically analyzed.The following conclusions resulted from this study: (i) freeze-drying and freeze-thawing treatments were comparable except for corn esterases; (ii) freeze-drying induced alterations in enzyme activity except for corn acid phosphatase; (iii) the freezing rate, the final freezing temperature, and exposure to various relative humidities produced few changes in freeze-dried material; (iv) freeze-drying was less detrimental than vacuum-drying to the enzyme characteristics of corn; (v) freeze-drying yielded higher viabilities than vacuum-drying; and (vi) acid phosphatase alterations appeared to be related to pollen viability in most cases.     

Teratogenic bioactivation of cyclophosphamide in vitro.

Day 10 rat embryos grown as cultured explants $\text{in vitro}$ developed characteristic defects when culture media contained cyclophosphamide (6.25 micrograms per milliliter or more), an hepatic microsomal fraction, and cofactors for a monooxygenase system. Cyclophosphamide concentrations as high as 250 micrograms per milliliter were innocuous when either the microsomal material or cofactors were omitted from the medium. These experiments represent the first direct demonstration of bioactivation of a proteratogen.     

Relative contracting adn relaxing potencies of a series of prostaglandins on isolated canine mesenteric artery strips

The contracting and relaxing potencies of anf interactions between a number of prostaglandins (PGs) were studied $\text{in vitro}$ on spiral strips of small canine mesenteric arteries (outside diameter < mm). PGF₂α and PGE₂, the most potent contracting PGs, were nearly equal in potency (EC₅₀ 4 × 10^?7M) and did not cause relaxation under our experimental conditions. PGI₂ and PGE₁ were equal and the most potent relaxing PGs (EC₅₀ 3 × 10^?9M). PGE₁ also caused contraction, but this effect was not consistent. PGI₂ did not cause contraction in concentrations up to 3 × 10^?6M. In higher concentrations, however, it caused abrupt and near maximal contraction. PGD₂ was weak in both respect, causing incomplete relaxation and contraction or biphasic effects. Interaction studies showed that PGE₁ and PGI₂ mutually excluded the relaxing effects of each other. PGE₁ also reversed the relaxing effect of isoproterenol. However, pre-exposure to PGD₂ did not attenuate the relaxing effect of PGE₁ or PGI₂ nor was the relaxing effect of PGD₂ changed by pre-exposure to PGE₁. Two different orders of potency of PGs suggest two PG receptors subserving contraction and relaxation, respectively. Further, it appears that several PGs can act upon both receptors which may explain unusual interactions between the PGs and some of their atypical effects. Finally, the data also suggest that there may be subtypes of the PG receptors subserving contraction and relaxation.     

Evidence for two classes of rat plasma fibrinogen γ chains differing by their COOH-terminal amino acid sequences

Human fibrinogen molecules contain two classes of functionally equivalent γ chains (termed γ and γ′) differing by their COOH-terminal amino acid sequences. We investigated rat plasma fibrinogen for the presence of this heterogeneity using DEAE-cellulose chromatography to separate reduced S-carboxymethylated chains. Like human γ′ chains, rat γ′ chains were more negatively charged, somewhat larger (～1000 daltons), had a different COOH-terminal acid than γ chains, and were functionally equivalent to other γ chains. The γ′ chain population from normal and turpentine-stimulated animals amounted to 28 and 30% of all γ chains, respectively, suggesting that regulation of their production is not sensitive to stimulation of fibrinogen synthesis.     

Inhibition of a nuclease contaminant in the commercial preparations of Escherichia coli alkaline phosphatase.

Escherichia coli alkaline phosphatase is a valuable reagent for removal of terminal phosphate from both ribo-and deoxyribo-oligonucleotides or from restriction enzyme fragments of DNA. Some commercial preparations of this enzyme were found to be contaminated with nucleases which could degrade both DNA and RNA. These contaminating nucleases can be completely eliminated by carrying out the enzymic reaction in the presence of 0.1-1% sodium dodecyl sulfate without any loss of phosphatase activity. This report has immediate application in the sequence analysis of DNA or RNA.     

Biosynthesis of 2-methylalkanes in the crickets Nemobius fasciatus and Gryllus pennsylvanicus.

The biosynthesis of 2-methylalkanes was studied in the crickets $\text{Nemobius}\text{fasciatus}$ and $\text{Gryllus}\text{pennsylvanicus}$. Labelled acetate, valine, and isobutyric acid were incorporated into the cuticular hydrocarbon of $\text{N.}\text{fasciatus}$ at levels of 6.0 ± 1, 6.5 ± 2, and 1.5 ± 0.7 percent respectively. The hydrocarbons of this insect are 20 percent 2-methylalkanes, primarily of even numbered carbon chain lengths, and 80% n-alkanes. Of the label incorporated into the hydrocarbon fraction, 28 ± 2 percent of sodium [1-¹⁴C] acetate, 98 ± 1 percent of L-[G-³H] valine, and 75 ± 10 percent of [1-¹⁴C] isobutyric acid were incorporated into the 2-methylalkanes. This suggests that valine is converted to isobutyric acid and is incorporated into the even numbered carbon chain length 2-methylalkanes during the initial stages of chain elongation. Similar data obtained in $\text{G.}\text{pennsylvanicus}$ suggests that leucine is converted to isovaleric acid which is then incorporated into the odd numbered carbon chain length 2-methylalkanes.     

Protein intake regulation in the weanling rat: Effects of additions of lysine,arginine and ammonia on the selection of gluten and energy

The effects of adding lysine, arginine and ammonia to gluten on the self-selection of protein and energy by the weanling rat simultaneously offered a choice of two diets differing only in gluten concentration (15 and 55%) were tested. Previous studies have shown that while lysine (6 g/100 g) additions to gluten decreased the amount of gluten selected by the rat from 40 to 20 g per 100 g of food eaten, selection was not related to the nutritional quality of the gluten. When graded levels of arginine (1.8, 3.6 or 7.2 g/100 g) were added to the gluten with or without lysine (0 or 6 g/100 g) the dietary protein selection was unaffected. The addition of ammonia (1.4 g/100 g as NH₄Cl) to gluten had initially the same effect as lysine (6 g/100 g) but with time protein intake returned to control levels. This effect of ammonia was unaltered by arginine additions. It is concluded that the mechanisms which lead to decreases in gluten selection caused by lysine or ammonia are not similar, and that the effects of lysine on gluten selection are not caused by an increased arginine requirement for urea cycle activity.     

Fluorescent thin-layer peptide mapping for protein identification and comparison in the subnanomole range

Conditions and simple precautions are presented for carrying out highly reproducible and sensitive peptide mapping by thin-layer chromatography and subsequent electrophoresis of subnanomole amounts of tryptic digest on silica gel G or GHL plates. The fluorogenic reagent “fluorescamine” is employed for visualization under long-wavelength ultraviolet illumination. Permanent photorecording of high-contrast images, using readily available filters, is substituted for subjective hand scoring of plates. Contrast reversal is used to produce peptide maps suitable for half-tone reproduction.     

The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism

Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.     

Bone marrow as a major lymphoid organ in Rana.

The bone marrow of adult frogs (Rana pipiens and R. catesbeiana) contains leukocytes equivalent in number to those present in the spleen. As in the spleen, a high percentage of these cells are morphologically identified as lymphocytes, and the bulk of these can be categorized as small or medium lymphocytes. In young frogs (R. catesbeiana), the ratio of marrow to spleen leukocytes is considerably greater than in adults, and in limited comparisons a high percentage of lymphocytes are found. A comparison of adult marrow and spleen anti-TNP (trinitrophenyl) plaque-forming cell responses at various days early in the response indicates that in the majority of animals marrow normally produces a larger total primary response.