Interaction of a new fluorescent reagent with sulfhydryl groups of the (Na+ + K+)-stimulate ATPase.

The (Na+ + K+)-ATPase enzyme of rat brain microsomes can be reversibly inhibited by a new fluorescent sulfhydryl (SH) probe, dimethylaminoaphthalenecysteine-Hg+ (Dn-cys-Hg+). This reagent is more reactive than N-ethylamaleimide (MalNEt) toward membrane sulfhydryl groups. A procedure using the two SH reagents sequentially seems to permit a more selective labelling of the SH groups involved in the (Na+ + K+)-ATPase than is possible by using MalNEt alone. Brain microsomes treated by this method incorporate the fluorescent label within or near the active site of the enzyme. When the probe was bound a blue shift of its fluorescence emission maximum (from 540 to 495 nm) and a 20-fold increase in relative fluorescence occurred. This indicates that the Dn moiety is within a very non-polar region of the membrane.     

Purification and characterization of rat liver microsomal beta-glucuronidase.

Beta-Glucuronidase (EC 3.2.1.31) has been isolated from rat-liver microsomes by a novel chromatographic method employing antibody to rat preputial gland beta-glucuronidase coupled to Sepharose. The purified enzyme, homogeneous by several methods, was purified some 1700-fold. The microsomal beta-glucuronidase has been characterized with respect to catalysis, stability, and molecular weight. The purified enzyme is a tetramer of 290 000 daltons. Comparative studies with lysosomal beta-glucuronidase indicate that while these two enzymes are electrophoretically distinct, they are catalytically and immunologically identical and have indistinguishable molecular dimensions. The results suggest that microsomal and lysosomal beta-glucuronidase are charge isomers.     

Histochemical localization of potassium-stimulated P-nitrophenylphosphatase activity in the somatosensory cortex of the rat.

Potassium-stimulated p-nitrophenylphosphatase (K+-pNPPase) activity was investigated in rat somatosensory cortex where 64-88% of enzymatic activity survived 5-10 min of fixation with 3% formaldehyde in 0.1 M cacodylate buffer, pH 7.4. Potassium-stimulated activity was inhibited by 1-10 mM ouabain. Levamisole (1.7 mM) inhibited brain alkaline phosphatase activity, facilitating the detection of K+-pNPPase activity. Strontium (10-20 mM) inhibited enzymatic activity by 38-75%. In parallel histochemical studies reaction product was found in strata, with cortical layers 2, 3, 4 and the outer portion of 5 containing the heaviest deposits. Highly reactive, vertically oriented, large diameter fibers were seen as groups between the outer portion of layer 5 and the pail surface. These fibers apparently arborize in the superficial layers. Smaller fibers were also positive and were oriented in various planes. The highest density of smaller, positive fibers occurred in layers 2 through 5. All positive fibers appeared to be axons or dendrites. Reaction product was not heavily concentrated in neuron perikarya or in glial elements. Sections did not contain reaction product when incubated in media lacking K+ or containing ouabain. The convergence of data from parallel histochemical and biochemical approaches supports the conclusion that the reactivity localized in the cerebral cortex represented the site of K+-pNPPase, a known component of the Na+,K+-adenosine triphosphatase complex. Neuronal processes demonstrated the highest enzymatic activity and may be most important in the active transport of Na+ and K+ in somatosensory cortex.     

Innervation of the synovial membrane of the cats joint capsule

Summary Results of investigations on the occurrence of nerve fibres and endings in the synovial membrane of the knee and elbow joint in the cat are reported. The stratum synoviale contains only autonomic fibres, running in the adventitia of arteries.Free nerve endings are lacking in the stratum synoviale. Simple Pacinian corpuscles with an inner core are occasionally observed in the border zone between the stratum synoviale and fibrosum. The ultrastructure of these endorgans resembles that of Pacinian corpuscles in the hairless and hairy skin of the cat.     

Chromosomal constitution of nucleolus-associated chromatin in man

Summary Use of specific stains permits analysis of the frequency of nucleolus-associated heterochromatin in chromosomes 1 and 9 from human fibroblasts. In 81% of interphase nuclei the heterochromatic segment of both No. 1 chromosomes is associated with the nucleolus, while in 19% only one heterochromatic segment shows such an association with the other occupying a random position in the nucleoplasm. The nucleolar association of chromosome 9 heterochromatin is less constant: in 42.3% of the nuclei both segments are associated with the nucleolus, in 39% of the nuclei only one heterochromatic segment presents such an association, and in 18.7% neither of the two heterochromatic segments is in nucleolar association. In 6% of the cells, one or two chromosome 9 heterochromatic segments are in contact with the nuclear membrane.In situ hybridization using tritium-labeled 28S and 18S RNA shows that in the interphase nucleus the acrocentric short arms, carriers of ribosomal cistrons, are associated with the nucleolus.These observations demonstrate the complexity of the nucleolus-associated chromatin which, in addition to segments of chromosomes 1, 9, 13, 14, 15, 21 and 22, may include the Y chromosome. They also confirm that the nucleolus constitutes one of the orientation points determining the relative localization of chromosomes in the interphase nucleus.     

Studies on the asymmetric arrangement of membrane-lipid-enveloped virions as a model system.

Lipids of BHK 21 cells (baby hamster kidney) grown in tissue culture were labelled with radioactive fatty acids. The enveloped vesicular stomatitis virus was propagated in this host cell type. The virions were purified by density gradient centrifugation. Neuraminidase treatment of the intact virions led to a complete transformation of hematoside [N-acetylneuraminosyl(alpha2-3)lactosyl(beta1-1)ceramide] into lactosylceramide, with identical labelling of the ceramide portion in hematoside of the untreated virions and the lactosylceramide of the neuraminidase-treated particles. The morphology of the virions appeared unchanged in electron micrographs, but the neuraminic-acid-free virions had a strong tendency to aggregate. The results of these studies are evidence that gangliosides are integrated exclusively into the outer lamella of the lipid bilayer in the viral envelope. It is also evident that the viral envelope is a suitable model for studies on membrane asymmetry.     

Multiple forms of β-glucuronidase in rat liver lysosomes and microsomes

Multiple forms of β-glucuronidase have been demonstrated using sucrose gradient and polyacrylamide gel isoelectric focusing techniques in 6 m urea. Microsomal β-glucuronidase, a membrane-bound enzyme, was solubilized from lysosome-free, Ca²⁺-precipitated microsomes by detergents and isolated by chromatography on columns of rabbit anti-rat preputial gland β-glucuronidase antibody bound to Sepharose. The enzyme has a pI of 6.7. Polyacrylamide gel isoelectric focusing resolves the microsomal enzyme into three components, each of which is protease sensitive. The protease-modified microsomal enzyme is very similar to several forms of β-glucuronidase in lysosomes. The lysosomal β-glucuronidase, isolated from osmotically shocked lysosomes, is very heterogeneous after isoelectric focusing over the range pI 5.4–6.0. The lysosomal enzyme can be resolved into 10–12 bands by polyacrylamide gel isoelectric focusing. The more acid forms of the lysosomal enzyme are neuraminidase sensitive, suggesting they may be sialoglycoproteins.     

Structure and synthesis of malformin A1

The structure of malformin A₁, a metabolic product of Aspergillus niger, was reexamined and the sequence of its amino acid constituents established as

The cyclopentapeptide-disulfide corresponding to this structure was prepared through stepwise synthesis of the protected pentapeptide derivative, benzyloxy-carbonyl-l-isoleucyl-S-benzyl-d-cysteinyl-S-benzyl-d-cysteinyl-l-valyl-d-leucine methylester, which in turn was converted to the hydrazide, partially deprotected, and cyclized via the azide. On removal of the S-benzyl groups and oxidation to the disulfide, a synthetic material was obtained that was indistinguishable from natural malformin A₁ and was as equally potent in causing curvatures on corn roots.     

Double-strand breaks can initiate meiotic recombination in S. cerevisiae

We investigated the effects of double-strand breaks on meiotic recombination in yeast. A double-strand break was introduced at the MATa locus by sporulation of a MAT alpha inc/MATa diploid under inducing conditions for the HO-encoded endonuclease; 14% of the resulting tetrads had undergone 4 alpha:0a conversion. Conversion at MAT was associated with co-conversion of a closely linked marker and an increased recombination frequency for flanking markers. We also studied the sporulation products of a diploid heterozygous at the HIS4 locus for an insertion of a 100 bp fragment of MATa containing the HO endonuclease cut site. Under inducing conditions, a significant number of tetrads were formed that had undergone gene conversions in favor of the HIS4+ allele. Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.     

An asparagine-rich protein from blood stages of Plasmodium falciparum shares determinants with sporozoites.

We describe a cDNA clone derived from mRNA of asexual blood-stages of the malaria parasite Plasmodium falciparum. This clone, designated Ag319, expresses a P.falciparum antigen fused to beta-galactosidase in Escherichia coli. Human antibodies from Papua New Guinea were affinity-purified by adsorption to extracts of Ag319 immobilized on CNBr-Sepharose. The antibodies reacted predominantly with P. falciparum polypeptides of Mr 220,000 and 160,000, and a number of ill-defined lower molecular weight species. Antibodies reacted in indirect immunofluorescence with all asexual blood-stages although the antigen appeared to be most abundance in the schizont. Surprizingly the antibodies also reacted with sporozoites. The amino acid sequence predicted from the complete nucleotide sequence of this clone is remarkable because 40% of the residues are Asn, and so the antigen has been termed the Asparagine-Rich Protein (ARP). Like other P. falciparum antigens, ARP contains tandemly repetitive sequences, based on the tetrapeptide Asn-Asn-Asn-Met and we have confirmed that these represent natural epitopes by reaction of the corresponding synthetic peptides with human antibodies. Surprisingly, ARP is also rich in Asn outside the tandem repeats.