Secretion heterogeneity of lysosomal enzymes in Tetrahymena pyriformis

A number of lysosomal enzymes are secreted from Tetrahymena pyriformis during growth and during starvation. The secretion is energy-dependent and kinetically different among hydrolases. On the basis of the secretion kinetics under starvation conditions, Tetrahymena hydrolases can be separated into three classes. The first group containing acid phosphatase, beta-glucosidase and alpha-galactosidase, are secreted slowly. Within this group about 4% of the initial cellular activity is released per hour. The second group of enzymes, including alpha-glucosidase, alpha-mannosidase and beta-galactosidase, exhibit moderate secretion (11-15% of the initial cellular activity per hour). The third group, N-acetyl-beta-hexosaminidase, has the highest rate of secretion (22% of the initial cellular activity per hour). N-Acetyl-beta-hexosaminidase shows a continuous increase in overall activity during starvation, which is completely blocked by adding cycloheximide; its secretion is also suppressed. Such involvement of enzyme biosynthesis was not seen in the first and second groups. Furthermore, treatment with weak bases caused inhibited secretion of differing degree among acid phosphatase (group I), alpha-glucosidase (group II) and N-acetyl-beta-hexosaminidase (group III).     

High-molecular-weight cadmium-binding proteins in rat liver cytosol: isolation and partial characterization of an approximately 50-kDa protein

The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.     

Maternal contamination of some NZB sublines and genetic profiles of NZ-strains

Six sublines of NZB mice bred in Japan were collected and their mitochondrial DNA (mtDNA) was examined by restriction analysis. The phenotypes of at least three of these sublines (NZB/Nrs, NZB/Nga and NZB/KlJms) differed from a standard one (NZB/BlWehi). Since mtDNA is inherited maternally, all sublines of a single inbred strain should share the same mtDNA phenotype. Therefore, b-type of mtDNA should be observed in all NZB sublines. Nevertheless, the above-mentioned sublines showed d-type mtDNA. These results suggested a genetic contamination of these sublines. This was confirmed by the finding that six aberrant alleles were detected also in their nuclear genomes using biochemical markers. For elucidation of the cause of contamination, we characterized the genetic profiles of four standard NZ-strains, NZB/BlWehi NZO/BlWehi, NZC/BlWehi and NZX/BlWehi, and of common inbred strains with black coat color, C57BL/6J, C57BL/10Sn, C57BL/Ks, C58/J and AU/SsJ. We found that five of the six aberrant alleles most strongly corresponded with those of C57BL/Ks. These results suggest that this contamination was ascribable to cross of NZB mice with a certain C56BL strain. We also deduced that NAB/BlPt and NZB/Füll also probably were contaminated strains, suggesting that this contamination was not restricted to Japan.     

A new variant of the Es-1 locus in the rat

A new prealbumin plasma esterase was demonstrated by the use of miniaturized polyacrylamide slab gel electrophoresis. Genetic analysis indicated that the new variant is controlled by the Es-1 locus and this gene was designated Es-1c. Among 11 rat strains only one strain, WJ, possessed the gene. Two random-bred stocks, Jcl:Wistar and Jcl:SD, also maintained it in their populations. Miniaturized polyacrylamide slab gel electrophoresis showed that the ES-1 band consisted of two close bands with the cathodal one staining darker.     

Novel fatty acyl substrates for myristoyl-CoA:protein N-myristoyl-transferase

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristic acid to the NH2-terminal Gly residues of a number of viral and cellular proteins. The remarkable specificity of this enzyme for myristoyl CoA observed in vivo appears to arise in large part from a cooperativity between NMT's acylCoA and peptide binding sites: the length of the acylCoA bound to NMT influences the interactions of peptide substrates with NMT. We have previously synthesized analogs of myristic acid with single oxygen or sulfur for methylene substitutions. These heteroatom substitutions produce significant reductions in acyl chain hydrophobicity without accompanying alterations in chain length or stereochemical restrictions. In vitro studies have shown that the CoA thioesters of these analogs are substrates for S. cerevisiae NMT and that the efficiency of their transfer to octapeptide substrates is peptide sequence-dependent. In vivo studies with cultured mammalian cells have confirmed that these fatty acid analogs are selectively incorporated into a subset of cellular N-myristoylproteins, that only a subset of analog-substituted proteins undergo redistribution from membrane to cytosolic fractions, and that these analogs can inhibit the replication of human immunodeficiency virus I and Moloney murine leukemia viruses--two retroviruses that depend upon N-myristoylation of their gag polyprotein precursors for assembly. We have now extended our analysis of NMT-acylCoA interactions by synthesizing additional analogs of myristic acid and testing them in a coupled in vitro assay system. Myristic acid analogs with two oxygen or two sulfur substitutions have hydrophobicities comparable to that of hexanoic acid and decanoic acid, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new variant of the Es-4 locus in the rat

A new variant of kidney esterase in the DK/Nac rat strain is reported. The new esterase was tentatively named ES-4C determined by a third allele of the Es-4 locus of Linkage Group V (LGV). Strain distribution was surveyed using 17 inbred strains, but no strain except for the DK/Nac strain possessed the ES-4C type. Although we surveyed outbred stocks (Jcl: Wistar and Jcl: SD) we could not find rats carrying the ES-4C type. Genetic analysis of the ES-4C type was carried out using mating experiments between DK/Nac and BUF/Nac (ES-4B). The results indicated that the new variant was controlled by the Es-4 locus and it was named the Es-4c allele.     

Quantitative Determination of Changes in the Number and Size of Chloroplasts in Naturally Senescing Leaves of Rice Seedlings

Changes in the number and size of chloroplasts in senescingleaves of rice seedlings were determined. The method employedinvolves electron microscopic examination of large numbers ofcells and chloroplasts in the mesophyll of leaves at differentstages of senescence with the aid of a microcomputer. Analysisshowed that, once leaves had been fully expanded, the numberand size of the mesophyll cells remained unaltered throughoutthe course of senescence. By contrast, the quantity of chloroplastspresent in leaves decreased with advancing senescence. Whencompared with the newly expanded 6th leaves, the chloroplastnumber per unit area of mesophyll section was reduced by 40%and the mean cross section area of chloroplasts by 23% in theoldest leaves (3rd leaves) of seedlings. Chloroplasts occupied33% of the mesophyll section area in the 6th leaves and thepercentage decreased slightly in the 5th leaves and markedlyin lower leaves to reach 17% in the 3rd leaves. The rate ofoxygen evolution decreased approximately in parallel to thedecline in the chloroplast content. Thus, sequential decreasein the amount of chloroplasts is a main cause of loss of photosynthesisduring foliar senescence of rice seedlings. (Received May 31, 1989; Accepted October 17, 1989)     

Supramolecular Assembly of Fucoxanthin-Chlorophyll-Protein Complexes Isolated from a Brown Alga, Petalonia fascia. Electron Microscopic Studies

Using a mild detergent, octyl sucrose, light-harvesting fucoxanthin-Chla/c-protein complexes of a brown alga, Petalonia fascia, wereisolated in the form of supramolecular assemblies. Negativelystained images of these assemblies (FCPAs) were extremely uniformin size and shape. Each was discoidal in shape, being 11.2 nmin diameter and 10.2 nm in height, with a small pit at the centerof disc. From the sedimentation rate (S_{20, w} = 21.6) and theobserved dimensions, the molecular mass (Mr) of FCPA was calculatedas about 697?10³, and each FCPA was deduced to contain 128 moleculesof Chl a 27 of Chl c, 69 of fucoxanthin and 8 of violaxanthin.Fresh FCPA showed highly efficient transfer of excitation energyfrom fucoxanthin to Chl a but the energetic coupling was disruptedon storage with accompanying distortion of fine structures.Given the occurrence of similar supramolecular assemblies offucoxanthin-chlorophyll a/c-protein complexes in another brownalga, Dictyota dichotoma [Katoh et al. (1989) Biochim. Biophys.Acta 976: 233], the molecular assemblies of fucoxanthin-Chla/c-protein complexes is assumed to be common to the light harvestingsystems in all brown algae. (Received December 28, 1989; Accepted March 5, 1990)     

Scale-up of hydrophobic interaction chromatography for purification of antitumor antibiotic SN-07

Scale-up of hydrophobic interaction chromatography for purification of SN-07 was studied. The height equivalent to a theoretical plate of the adsorbent gel (Sepabeads FP-PH 12) was kept constant for various superficial liquid velocities and column diameters. The efficiency of purification was also constant for the Sepabeads FP-PH 12 columns ranging from 16 mm to 113 mm in inner diameter. Effects of the ligand concentration on the adsorption capacity were also studied, and the adsorption capacity increased with an increase in the ligand concentration. These effects resulted in decreasing the required amount of salt to purify SN-07.