Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.     

Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes

An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.     

A method for direct incorporation of radiolabeled cholesteryl ester into human plasma low-density-lipoproteins: preparation of tracer substrate for cholesteryl ester transfer reaction between lipoproteins

[14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density greater than 1.006 at 37 degrees C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.     

Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers

A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation.     

Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C4 Plants II. Peroxisomes of Panicum miliaceum L.

Peroxisomes were isolated by sucrose density gradient centrifugationfrom mesophyll and bundle sheath protoplasts of a C₄ plant,Panicum miliaceum L. The equilibrium density in the gradientwas 1.25 for bundle sheath peroxisomes and 1.23 for mesophyllperoxisomes, the former density being similar to that of peroxisomesof wheat mesophyll protoplasts. Photorespiratory and other microbody enzymes were assayed forthe peroxisomes of P. miliaceum to detect possible differentiationat an enzyme level. The specific activities of photorespiratoryenzymes, except for hydroxypyruvate reductase, in bundle sheathperoxisomes were 40–60% of those in wheat peroxisomes,when compared on a protein basis, and only 20–30% in mesophyllperoxisomes. However, peroxisomes from both cell types containedsignificant levels of all the enzymes involved in the photorespiratoryglycolate pathway, when compared with castor bean glyoxysomes.The activity of hydroxypyruvate reductase in the peroxisomesof P. miliaceum was comparable to or higher than that in wheatperoxisomes. Two ß-oxidation enzymes and urate oxidasewere detected in the peroxisomes in a similar level to thatin wheat peroxisomes. These results suggest that the peroxisomes of mesophyll andbundle sheath cells of P. miliaceum are essentially similarto those of C₃ plants, and that they cannot be differentiatedexcept for a difference in equilibrium density in a sucrosegradient. (Received December 24, 1984; Accepted April 9, 1985)     

A simple preparation method for apoaspartate aminotransferase from Escherichia coli B, and its application for the assay of pyridoxal and pyridoxamine 5'-phosphate

A simple and rapid preparation method for apoaspartate aminotransferase from Escherichia coli B was developed. A crude extract of the bacterial cells was treated batchwise with DEAE-cellulose. The enzyme fraction obtained was then applied to a pyridoxamine-Sepharose column. Apoaspartate aminotransferase was eluted with 50 mM potassium phosphate buffer (pH 7.0), and found to be electrophoretically homogeneous. The apoenzyme preparation thus obtained showed very low holoenzyme activity (only 0.4% of the activity seen in the fully saturated condition with pyridoxal 5'-phosphate) and was successfully used for assaying pyridoxal and pyridoxamine 5'-phosphate.     

Difference in DNA strand break by gamma- and beta-irradiations: an in vitro study

Lambda DNA (125 micrograms/ml in Tris buffer, pH 7.4) was irradiated with 60Co gamma-rays and 3H beta-rays, respectively, and the number of strand breaks was determined by electrophoresis. Number of single-strand breaks increased linearly with radiation dose in both gamma- and beta-radiations and the relative effectiveness (beta/gamma) was found to be 1.82 in N2 and 1.16 in O2. Number of double-strand breaks increased with the square of the radiation dose in gamma-irradiation, but it increased linearly with radiation dose in beta-irradiation. Therefore, the relative effectiveness (beta/gamma) is higher at lower doses. O2 effects was observed by gamma-irradiation but was minimal after beta-irradiation.     

Urinary excretion of isomers of biliverdin after destruction in vivo of haemoproteins and haemin.

The amount and isomeric composition of urinary biliverdin in rabbits were analysed by h.p.l.c. Physiological values were maintained after the injection of haemin. On the other hand, when haemoglobins from several mammalian species were injected into rabbits, the excretion of biliverdin-IX alpha and biliverdin-IX beta were increased 6-18-fold and 32-66-fold respectively over physiological excretion. Injection of myoglobin resulted in a 44-fold increase in excretion of the IX alpha-isomer. Coupled oxidation with ascorbate of haemoglobin and myoglobin by oxygen produced mainly the IX alpha- and IX beta-isomers from haemoglobin and the IX alpha-isomer from myoglobin. The destruction of part of the haem from injected haemoproteins by non-enzymic chemical degradation would account for the observed respective increases in the excretion of biliverdin isomers. The excretion of biliverdin isomers after the injection of phenylhydrazine into rabbits was similar to that after the injection of haemoglobin.     

Passive immunization of mice with monoclonal antibodies to glycoprotein gB of herpes simplex virus

To investigate the protective ability of monoclonal antibodies (MCAs) to viral glycoprotein in herpes simplex virus (HSV) infection, athymic nude mice were inoculated intracutaneously with HSV type-1 (HSV-1) in the midflank. Three hours after inoculation, one group of mice was passively immunized with one of a series of MCAs to glycoprotein gB of HSV-1, and a control group of mice was given phosphate buffered saline alone. The control mice died within 16 days after infection, whereas the mice passively immunized with any of the MCA showed suppressed development of skin lesions. Three of six mice given MCA failed to develop any visible lesions and no HSV could be isolated from the lumbar dorsal root ganglia of these mice 60 days after the challenge. BALB/c mice were also protected from infection with HSV type 2 by passive immunization with MCA to HSV-1 gB.