The low-molecular-weight acid phosphatase from bovine liver: Isolation,amino acid composition,and chemical modification studies

The smallest of the three molecular weight forms of acid phosphatase from bovine liver was purified to a specific activity of 100 μmol min^?1 mg^?1 (measured at pH 5.5 and 37 °C with p-nitrophenyl phosphate). Using several chromatographie and electrophoretic methods, no evidence of heterogeneity was detected. The enzyme was characterized with respect to its stability as a function of pH, molecular weight, amino acid composition, steady-state kinetic parameters in the pH range 4–7 and inhibition by common acid phosphatase inhibitors at pH 5.5. The amino acid composition differed somewhat from a previous literature report. The enzyme was stoichiometrically inactivated upon incubation with Hg²⁺, Ag⁺, and iodoacetate. Inactivation also occurred upon photoinactivation in the presence of Rose Bengal but no inactivation occurred with diethyl pyrocarbonate. The alkylation of one of five cysteine residues by iodoacetate was shown to cause complete inactivation of the enzyme. This alkylation was prevented by the presence of phosphate ion. A tryptic dipeptide containing this essential cysteine was isolated following inactivation with iodo[2-¹⁴C]acetate.     

Resonance Raman spectroscopy of chemically modified retinals: Assigning the carbon-methyl vibrations in the resonance Raman spectrum of rhodopsin

To assign the observed vibrationsl modes in the resonance Raman spectrum of the retinylidene chromophore of rhodopsin, we have studied chemically modified retinals. The series of analogs investigated are the n-butyl retinals substituted at C₉ and C₁₃. The results obtained for the 11-cis isomer have clearly assigned the CCH₃ vibrational frequencies observed in the spectrum of the retinylidene chromophore. The data show that the C₍₉₎CH₃ stretching vibration can be assigned to the vibrational mode observed in the 1017 cm^?1 region, and the vibration detected at 997 cm^?1 can be assigned to the C₍₁₃CH₃ vibration. The C₍₅₎CH₃ stretching mode does not contribute to the vibrations observed in this region. The splitting in the C_(n)CH₃ (n = 9, 13) vibration is characteristic of the 11-cis conformation. The results on the modified retinals do not support the hypothesis that the splitting arises from equilibrium mixtures of 11-cis, 12-s-cis and 11-cis, 12-s-trans in solution. Thus, this splitting cannot be used to determine whether the chromophore in rhodopsin is in a 12-s-cis or 12-s-trans conformation. However, our results demonstrate that there are other vibrational modes in the spectra which are sensitive to this conformational equilibrium and we use the presence of a strong ~ 1271 cm^?1 mode in bovine and squid rhodopsin spectra as an indication that the chromophore in these pigments is 11-cis, 12-s-trans.     

Isolation and characterization of the cytosolic and mitochondrial superoxide dismutases of maize

The cytosolic and mitochondrial forms of Superoxide dismutase have been purified to homogeneity from an inbred line of maize. The cytosolic isozymes SOD-2 and SOD-4 are dimers with a molecular weight of 31,000–33,000, composed of apparently equal subunits, and are remarkably similar with respect to their ultraviolet absorption spectra, antigenic specificity, and sensitivity to cyanide, azide, hydrogen peroxide, and diethyldithiocarbamate. These and other data suggest that both isozymes belong to the family of copper and zinc-containing Superoxide dismutases. The mitochondrial isozyme, SOD-3, is unlike the cytosolic isozymes in every parameter studied and appears to be similar to the mitochondrial manganese-containing Superoxide dismutases purified from other eukaryotic organisms. It is a tetramer with a molecular weight of approximately 90,000, composed of apparently equal subunits, and is insensitive to both 1 mm cyanide and hydrogen peroxide.     

Isolation of the outer acrosomal membrane from bull sperm

A procedure is described for subcellular fractionation of bull sperm which allows the isolation of outer acrosomal membrane without the use of detergent. After washing to remove seminal plasma contaminants, the acrosomal membrane is removed by homogenization and separated on a two-step sucrose gradient. The isolated membranes have been characterized by light and electron microscopy and enzyme analysis. While the acrosomal enzymes hyaluronidase and acrosin are bound to the isolated membranes, they represent only a small percentage of the total activity and therefore do not provide reliable marker enzymes for this fraction.Subcellular fractionation of sperm also yields information on the solubility of acrosomal enzymes. Two types of acrosomal enzymes have been identified on the basis of their distribution in gradient fractions. Both α-fucosidase and β-N-acetyl glucosaminidase are concentrated in the soluble fraction of the gradient. In contrast, over 70% of the acrosin and hyaluronidase activity remains associated with the sperm pellet. These differences in solubility of these enzymes may reflect differences in their function in fertilization.     

Comparison of the transposon-like structures encoding clindamycin resistance in Bacteroides R-plasmids

The R-plasmids pBF4, pBFTM10, and pBI136 encode transmissible clindamycin resistance (Ccr) in Bacteroides spp. These plasmids are distinct replicons but the regions implicated in Ccr share some homology and appear to have a transposon-like structure. To better understand the mechanism of dissemination and to locate the Ccr determinant(s), the genetic and structural properties of the Ccr regions of each plasmid were compared and contrasted. For this work a single EcoRI restriction fragment containing the Ccr region from each plasmid was cloned into pBR322 in Escherichia coli. Results of restriction mapping and heteroduplex experiments showed that the pBF4 EcoRI-D and pBFTM10 EcoRI-B fragments shared more than 90% base sequence homology but that the EcoRI-C fragment of pBI136 had diverged significantly. The pBI136 fragment also did not confer tetracycline resistance in E. coli as shown for the pBF4 EcoRI-D fragment (D.G. Guiney, P. Hasegawa, and C. E. Davis, 1984, Plasmid 11, 248-252). Heteroduplex experiments showed that the pBI136 EcoRI-C and pBF4 EcoRI-D fragments shared a 1.2-kb region of homology attributed to a directly repeated sequence which bounds the Ccr region. Southern hybridization studies indicated that an additional 0.85 kb of the pBI136 EcoRI-C fragment was homologous to the EcoRI-D fragment of pBF4. This region was characterized by its sequential restriction endonuclease sites for HindIII, AvaII, and DdeI, and it is proposed that the Ccr gene(s) resides in this area.     

Size and sequence homology of masked maternal and embryonic histone messenger RNAs.

The messenger RNAs for five classes of histone proteins are shown by competitive RNA-DNA hybridization to be stored in the unfertilized egg of the sea urchin, Lytechinus pictus. The masked mRNAs for f2b, f2a2, f3 and f2al histones migrate in polyacrylamide slab gels with the same mobility as the histone mRNAs that are synthesized after fertilization and are found engaged in protein synthesis on polysomes. The masked maternal and embryonic mRNAs for histone f2a1 are identical in mobility when analyzed in a gel system capable of resolving differences estimated as small as 4–5 nucleotides in length. We conclude that these histone mRNAs synthesized during oogenesis and inactive prior to fertilization are not activated during embryogeny by alteration in their molecular size.     

A family of phosphohydrolases from bovine intestinal mucosa: 5'-nucleotidase

Bovine intestinal 5'-nucleotidase has been partially purified and characterized for comparison with two other phosphohydrolases from the same tissue, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which are closely related structurally and mechanistically. Kinetic studies with a variety of nucleotides and phosphonate analogs show that, although 5'-nucleotidase is a monoesterase like alkaline phosphatase, it more closely resembles 5'-nucleotide phosphodiesterase in its high affinity and specificity for nucleotide binding. 5'-Nucleotidase is bound very strongly by an affinity column containing a bound phosphonate analog of ADP but is not bound by an affinity column containing a non nucleotide phosphonate which selectively binds alkaline phosphatase. 5'-Nucleotidase is strongly bound by immobilized antibodies prepared against 5'-nucleotide phosphodiesterase, and is less strongly bound by immobilized antibodies prepared against alkaline phosphatase. We conclude that 5'-nucleotidase is structurally more similar to 5'-nucleotide phosphodiesterase than to another monoesterase, alkaline phosphatase.     

Mitochondrial matrix granules in soft tissues. II. Isolation and initial characterization of a calcium-precipitable, soluble lipoprotein subfraction from brown fat and liver mitochondria

Degradation of ¹²⁵I-labelled HDL ([¹²⁵I]HDL) was measured in isolated rat hepatocytes that had been preincubated with [¹²⁵I]HDL and then reincubated in fresh medium without [¹²⁵I]HDL. About 5 % of the [¹²⁵I]HDL associated with the cells in advance were degraded per hour at 37 °C. This in vitro degradation was inhibited about 50% by lysosomal inhibitors such as chloroquine, ammonia and leupeptin. Depolymerization of microtubuli by colchicine inhibited the degradation of [¹²⁵I]HDL to about 65–75 % of the control cells. Cytochalasin B (CB), a destabilizer of microfilaments, had a less marked effect on the degradation in vitro. Degradation of [¹²⁵I]HDL associated with cells in vivo after intravenous injection was also studied in isolated cells. About 8.5% of the [¹²⁵I]HDL associated with the cells in vivo were degraded per hour in the isolated cells. The effects of ammonia, chloroquine, leupeptin and colchicine on HDL degradation were similar for [¹²⁵I]HDL taken up in vivo and in vitro. Subcellular fractionation by centrifugation in sucrose gradients indicated that [¹²⁵I]HDL associated with hepatocytes in vivo are primarily accumulated in lysosomes. [¹²⁵I]HDL associated with the cells in vitro are located in organelles whose distribution coincides with that of 5′-nucleotidase. These organelles may be endocytic vesicles. It is concluded that the internalization of [¹²⁵I]HDL in rat hepatocytes is relatively slow. The intracellular degradation of the apoproteins of HDL is at least partly lysosomal.     

Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina

Messenger RNA has been isolated from day-old chick lens. Size characterization and heterologous cell-free translation demonstrate that the predominant species of mRNA present code for α-, β- and δ-crystallins. Total polysomal RNA and polysomal RNA which did not bind to oligo (dT)-cellulose translate in the cell-free system to give a crystallin profile qualitatively similar to that of poly(A)⁺ mRNA. RNA from postribosomal supernatant which binds to oligo(dT)-cellulose also translates to give crystallins, but the products are enriched for β-crystallins. Messenger RNAs isolated from 15-day embryo lens fiber and lens epithelium cells give products on translation which reflect the different protein compositions of these two cell types, as do mRNAs isolated from chick lenses at various developmental stages. Messenger RNAs were isolated from freshly excised 8-day embryo neural retina and from this tissue undergoing transdifferentiation into lens cells in cell culture. Cell-free translation demonstrates no detectable crystallin mRNAs in the freshly excised material, but by 42 days in cell culture, crystallin mRNAs are the most prominent species.     

Isolation of phospholipase A2 from sheep erythrocyte membranes in the presence of detergents

Isolation of phospholipase A₂ (EC 3.1.1.4) from sheep erythrocyte membranes was carried out by a combination of (1) extraction of membranes at low ionic strength, (2) solubilization of extracted membranes with sodium dodecyl sulfate, (3) replacement of dodecyl sulfate with cholate by means of gel exclusion chromatography and (4) affinity chromatography on dialkyl-phosphatidylcholine-Sepharose in the presence of cholate. The phospholipase was prepared with good yield and purified to near homogeneity, as judged by sodium dodecyl sulfate gel electrophoresis. The protein is a minor component of the sheep erythrocyte membrane and has an apparent molecular weight of 18 500.     

The physical association between rat liver mitochondria and rough endoplasmic reticulum : I. Isolation, electron microscopic examination and sedimentation equilibrium centrifugation analyses of rough endoplasmic reticulum-mitochondrial complexes

Rough endoplasmic reticulum-mitochondrial (RER-MT) complexes have been isolated from rat liver homogenates by rate zonal Centrifugation using a reorienting zonal rotor. Electron microscopic examination of the isolated complexes reveals a close association between rough endoplasmic reticulum (RER) and mitochondria. The associated RER appears as bilamellar sheets as it does in intact liver tissue, not as microsomal vesicles. When the complexes are subjected to sedimentation equilibrium Centrifugation, the marker enzymes for mitochondria and RER coband at an equilibrium density of 1.190. Electron microscopic analysis of the complexes after sedimentation equilibrium Centrifugation again reveals a close association between RER and mitochondria. Treatment of the complexes with 500 mM KCl or 500 mM KCl plus 20 mM EDTA resulted in a shift in the equilibrium density of the complexes to 1.180 and 1.176, respectively. Concomitant with the density shift was a release of A₂₆₀ units to the top of the gradient. After incubating KCl-EDTA stripped complexes with cytoplasmic ribosomes and ribosomal subunits, the complexes band at the same equilibrium density, 1,190, as do untreated complexes. In order to completely remove the associated RER it is necessary to treat the complexes with digitonin at a concentration of 0.13 mg digitonin/mg protein. Our data suggest that a fraction of the total cellular RER is physically associated with rat liver mitochondria.     

Isolation and characterization of acetylornithine delta-transaminase of wild-type Escherichia coli W. Comparison with arginine-inducible acetylornithine delta-transaminase.

The enzyme that catalyzes the reversible conversion of N-acetylglutamic γ-semialdehyde and l-glutamate to α-N-acetyl-l-ornithine and α-ketoglutarate, acetylornithine δ-transaminase, has been isolated in homogeneous form and crystallized from both the wild-type and the arginine-inducible strains of Escherichia coli W. The molecular weight of the wild-type transaminase is 119,000 while the molecular weight of the arginine-inducible enzyme is 61,000. However, the arginine-inducible acetylornithine δ-transaminase is not a breakdown product of the wild-type, arginine-repressible transaminase. Analysis of crude extracts of the wild-type and arginine-inducible strains by varying the acrylamide concentration in polyacrylamide disc gel electrophoresis showed that arginine-inducible and wild-type transaminases differed in ionic charge. Immunochemical analysis of the two transaminases showed that neither enzyme would cross-react with antibodies prepared against its counterpart. Treatment of the two enzymes with sodium dodecyl sulfate, followed by disc gel electrophoresis revealed that both transaminases were composed of 31,000-dalton subunits. Tryptic digestion of the two transaminases showed that nearly identical peptides were present. The overall data suggest that the wild-type and inducible transaminases were products of two different structural genes. The two transaminases have different molecular weights, ionic charges, and antigenic determinants, but both are composed of similar molecular weight subunits and show a high degree of similarity in amino acid content and peptide composition.     

Effects of gonadotropins and steroids on the subsequent fertilizability of extrafollicular bovine oocytes cultured in vitro

Effects of gonadotropins (2 i.u./ml follicle stimulating hormone, FSH and 10 μg/ml luteinizing hormone, LH) and steroids (1 μg/ml oestradiol, E and progesterone, P) on the fertilizability of extrafollicular bovine oocytes cultured in vitro and transferred in either the rabbit oviduct (Experiment I) or glass test tubes (Experiment II) were investigated. Bovine oocytes collected from follicles of 2–5 mm in diameter were cultured in vitro for 27 h in a medium containing Ham's F-12, 20% (v/v) bovine fetal serum and antibiotics. The combination of the hormones added to the medium was as follows; (1) none (control), (2) E, (3) LH, (4) LH + E, (5) FSH + LH + E, and (6) FSH + LH + E + P. All oocytes were recovered 24 h after insemination and examined for the presence of the pronuclei and a sperm tail with the midpiece in the oocyte cytoplasm, and the extrusion of the second polar body.In Experiment I, 630 of 704 transferred oocytes (85.7%) were recovered from the rabbit oviduct. The maturation rates of these oocytes (overall 61.1%) were not significantly affected by gonadotropins and steroids. Of the 741 of 920 oocytes recovered from test tubes in Experiment II, the maturation rates of them (overall 64.2%) were significantly increased (P < 0.05) by addition of LH (72.9%) and FSH + LH + E + P (74.1%) as compared with controls (55.4%). Fertilization rates were increased (P < 0.05) by the addition of FSH + LH + E compared with the controls in both Experiments I (31.1% and 14.0%) and II (36.2% and 20.8%). Furthermore, the proportion of fertilized eggs in hormone treated groups was the highest in each experiment. The present study indicates that the addition of FSH, LH and E to a medium has improved the fertilizability of extrafollicular bovine oocytes cultured in vitro.     

Mutation leading to gene fusion in the histidine operon of Salmonella typhimurium

A spontaneous polar mutation located in the region of an intercistronic border in the hiatidine operon of Salmonella was isolated in our laboratory. The mutant, R81, tests as a frameshift in reversion experiments but is prototrophic, capable of growth without histidine supplements despite lowered levels of certain histidine enzymes. The mutation affects the operator distal end of the D gene, causing production of an active histidinol dehydrogenase enzyme with an altered C-terminus. The mutation severely affects expression of the immediately succeeding gene in the translation sequence, hisC, suggesting either that the D–C border and possibly hisC are physically altered or that their normal function in translation is seriously impaired. We have previously described the fortuitous production from R81 of a non-polar derivative with fused D and C genes. This strain produces a bifunctional enzyme with normally separate dehydrogenase and aminotransferase activities present on dimers or multimers of a single fused polypeptide chain. We have now investigated in greater detail the R81 mutation by amino acid sequencing of the C-terminus of altered histidinol dehydrogenase. We find that the R81 mutation causes the addition of a “tail” of four amino acid residues to an otherwise normal dehydrogenase polypeptide chain. The results support our previous suggestion that the R81 mutation profoundly effects the D–C gene border and that this effect is prerequisite to gene fusion.     

The fluorescein-mediated interaction of bovine serum albumin with fluorescent derivatives of prolactin and other polypeptides in polarization of fluorescence based assays

During the course of studies relating to the interaction of bovine prolactin with its receptor, it was observed that the fluorescence polarization of prolactin labeled with fluorescein isothiocyanate (fluorescein prolactin) increased from 0.10 to 0.15 upon the addition of bovine serum albumin. Dilution titration measurements show an apparent K_dissociation for the BSA-fluorescein-prolactin complex of 1.1 × 10^?7 M. The stoichiometry of the complex was shown to be approximately 2 mol of fluorescein-prolactin per mole of BSA. The fluorescence emission spectra of the fluorescein moiety in the fluorescein-prolactin is slightly red shifted and increased in intensity in the presence of BSA. The interaction between prolactin and BSA is dependent on the fluorescein attached to the prolactin since [¹²⁵I]prolactin does not form a complex with BSA under identical conditions. The fluorescence polarization of fluorescein-labeled growth hormone and α-lactalbumin also increased in the presence of BSA, suggesting that BSA may interact generally with fluorescein-labeled proteins to form complexes bridged through the fluorescein moiety.     

Isolation of iron-containing superoxide dismutase from Bacteroides fragilis: reconstitution as a Mn-containing enzyme

Superoxide dismutase from the anaerobe Bacteroides fragilis has been purified to apparent homogeneity. The protein, Mr 42,000, is a dimer of equally sized subunits joined by noncovalent interactions. Metal analysis of the native enzyme revealed 1.8-1.9 g-atoms Fe, 0.2 g-atoms Zn, and less than 0.05 g-atoms Mn per mole dimer in a preparation whose specific activity was 1200 U/mg. Exposure of the enzyme to guanidinium chloride plus 8-hydroxyquinoline (T. Kirby, J. Blum, I. Kahane, and I. Fridovich, 1980, Arch. Biochem. Biophys. 201, 551-555) resulted in complete loss of enzymatic activity. Activity could be restored by dialysis of the denatured apoprotein against Tris buffer containing either ferrous ammonium sulfate or manganous chloride. The Fe-reconstituted enzyme was inhibited by 1 mM azide and inactivated by H2O2 in a manner similar to the native enzyme. Mn-reconstituted enzyme was inhibited by azide but resisted inactivation by H2O2 comparable to other purified manganese-containing superoxide dismutases. The manganese reconstituted protein contained approximately 1 gm-atom Mn/mol dimer. Zn ion potently inhibited reconstitution of the denatured apoprotein by either Mn or Fe and bound to the protein with a stoichiometry of 2-3 g-atoms/mol dimer.     

Selective recognition in erythrophagocytosis by mouse peritoneal macrophages. Isolation and purification of a possible self-marker from mouse erythrocyte membranes

The percental participation of exogenous cytidine in liver RNA synthesis was determined after application of ³H-cytidine to rats. The amount of exogenous cytidine was varied by a factor of 5 × 10⁵, between 0.000 02 and 10.0 μg/g rat. With the ³H-cytidine doses and specific activities most frequently reported in the literature, the percental participation of the exogenous precursor is only about 0.1%, with 99.9% of the cytidylic acid incorporated into RNA under these conditions being of endogenous origin.The results show that the upper limit of the tracer dose of exogenous cytidine is about 1.0 μg/g rat. Within this tracer region 1.8% of ³H-activity—and therefore 1.8% of the amount of exogenous cytidine—is incorporated into liver RNA. The dependence of the percental participation on the duration of the experiments is examined.It is shown that autoradiographic grain density and specific activity of RNA can only be regarded as direct measures for the rate of RNA synthesis in different cells and animals if the percental participation of exogenous cytidine in RNA synthesis is generally of equal value.Comparable situations exist in the incorporation of ³H-thymidine into DNA as shown by earlier experimental work.     

Isolation of a chondroitin sulfate--dermatan sulfate proteoglycan from bovine aorta.

Material containing proteoglycans was extracted from bovine aorta by the dissociative solvent 3.0 m MgCl². The proteoglycan that remained in solution at low ionic strength was purified by isopycnic CsCl centrifugation (?, 1.75 – 1.89 g/ml). From the lower third of the gradient a proteoglycan was isolated which behaved as a homogeneous material when analyzed by the ultracentrifuge and by electrophoresis on cellulose acetate. The proteoglycan contained 12% protein, 21% uronic acid, and 28% hexosamine. Analyses by hyaluronidase digestion and gas-liquid chromatography of the polysaccharide moieties of the proteoglycan showed a composition of 56% chondroitin 6-sulfate, 20% chondroitin 4-sulfate, and 7% dermatan sulfate. A copolymeric structure for the polysaccharide of the proteoglycan is proposed.