Purification, N-terminal amino acid sequence and characterization of pH 2.5 optimum acid phosphatase (E.C. 3.1.3.2) from Aspergillus ficuum

An acid phosphatase from crude culture filtrate of Aspergillus ficuum was purified to homogeneity using three ion exchange chromatographic steps. SDS-PAGE of the purified enzyme gave a single stained band at approximately 68-KDa. The mobility of the native enzyme in gel filtration chromatography, however, indicated that the molecular mass to be about 130-KDa implying the active form to be a dimer. On the basis of a molecular mass of 68-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 3.4 x 10(5) M-1 cm-1. The isoelectric point of the enzyme, as judged by chromatofocusing, was about 4.0. The purified enzyme is highly stable at 0 degree C. Thermal inactivation studies have indicated that the enzyme is unstable at 70 degrees C. The enzyme, however, exhibited a broad temperature optima with a maximum catalytic activity at 63 degrees C. The Km of the enzyme for p-nitrophenylphosphate is about 270 microM with an estimated turnover number of 2550 per sec. The enzyme is a glycoprotein as evidenced by the positive PAS staining; the sugar composition suggests the presence of N-linked high mannose-oligosaccharides. A partial N-terminal amino acid sequence up to the twenty-third residue was obtained. The enzyme was inhibited competitively by inorganic orthophosphate (Ki = 185 microM) and non-competitively by phosphomycin (Ki = 600 microM).     

Aspergillus ficuum phytase: partial primary structure, substrate selectivity, and kinetic characterization

Purified Aspergillus ficuum phytase's partial primary structure and amino acid and sugar composition were elucidated. Determination of kinetic parameters of the enzyme at different pH values and temperatures indicated no significant alteration of the Km for phytate while the Kcat was affected. The enzyme was able to release more than 51% of the total available Pi from phytate in a 3.0 hr assay at 58 degrees C, but the Kcat dropped to 15% of the initial rate. Substrate selectivity studies revealed phytate to be the preferred substrate. The pH optima of phytase was 5.0, 4.0, and 3.0 for phytate, ATP, and polyphosphate, respectively. The enzyme had varied sensitivity towards cations. While Ca++ and Fe++ produced no effect on the catalytic rate of the enzyme, Cu+, Cu++, Zn++, and Fe were found to be inhibitory. Mn++ was observed to enhance enzyme activity by 33% at 50 microM. Known inhibitors of acid phosphatases e.g. L (+)-tartrate, phosphomycin, and sodium fluoride had no effect on enzyme activity.     

Primary structure of the hemoglobin α-chain of rose-ringed parakeet (Psittacula krameri)

The structure of the hernoglobin α-chain of Rose-ringed Parakeet was determined by sequence degradations of the intact subunit, the CNBr fragments, and peptides obtained by digestion with staphylococcal Glu-specific protease and trypsin. Using this analysis, the complete α-chain structure of 21 avian species is known, permitting comparisons of the protein structure and of avian relationships. The structure exhibits differences from previously established avian α-chains at a total of 61 positions, five of which have residues unique to those of the parakeet (Ser-12, Gly-65, Ser-67, Ala-121, and Leu-134). The analysis defines hemoglobin variation within an additional avian order (Psittaciformes), demonstrates distant patterns for evaluation of relationships within other avian orders, and lends support to taxonomic conclusions from molecular data.     

The use of the probit model to predict pregnancy status of buffalo based on physio-chemical properties of estrual mucus

Improper timing of artificial insemination with respect to ovulation is one of the major factors hampering the conception rate in buffalo. The present study was an attempt to relate physio-chemical changes in estrual mucus to subsequent pregnancy status in order to find their optimal values for determining the time for artificial insemination (AI). Serum estradiol, total protein and dry matter contents of estrual mucus were evaluated to predict the subsequent pregnancy in 36 buffalo during October 1988 to February 1989. Serum estradiol was determined by radioimmunoassay (RIA); spinnbarkeit, dry matter and total protein were determined by standard methods. Multivariate probit analyses were carried out to relate these variables to subsequent pregnancy status. Elasticity and protein concentration were significantly related to prediction probability of pregnancy status, and they predicted the pregnancy status 86% of the times correctly (P < 0.05). The probability of pregnant animals being correctly classified was 0.76, whereas the corresponding value for non-pregnant animals was 0.95. The present study demonstrated the possibility of using such a statistical model on mucus characteristics for determining proper AI time for better conception rates in Nili-Ravi water buffalo.     

Positive identification of a lambda gt11 clone containing a region of fungal phytase gene by immunoprobe and sequence verification

Summary As the initial step in a project to provide a more cost-effective source of the phytase enzyme, this paper reports on the use of a polyclonal antibody raised to phytase purified from an isolate of Aspergillus niger (A. ficuum) to screen an A. niger lambda gt11 expression library and the use of amino acid sequencing to identify a clone containing part of the fungal phytase gene. The described use of amino acid sequence fragments to verify the cloning of a gene has potential applications in other cloning projects.Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable Offprint requests to: E. Mullaney     

Coexistence of cytoplasmic and nuclear genes for male sterility in Petunia

Summary Cytoplasmic male sterility (cms) and nuclear male sterility (nms) in Petunia were described respectively as possible autonomous and integrated states of the same genetic element by Frankel (1971). In the present study we describe genetic analysis of the interaction between the cms, the nuclear gene for male sterility (e) and the fertility restorer allele (Rf). The main findings in this study are: (1) The nuclear sterility allele can coexist in one or two dosages with the cytoplasmic male sterility elements (ste) in somatic cells or female gametes; (2) the presence of the fertility restorer allele Rf is not required for the coexistence of ste and e and (3) Rf does not interact epistatically with e, e.g., the expression of e is independent of Rf—the genotypes (S) RfRfee and (S) Rfrfee are male sterile.Contribution from the Agricultural Research Organization. The Volcani Center, Bet Dagan, Israel. 1983 series No. 846 E     

Quantitative generation of singlet (1Δg) oxygen from acidified aqueous peroxynitrite produced by the reaction of nitric oxide and superoxide anion

Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet (¹Δ_g) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO^? generated in the diffusion - controlled reaction of cellular NO^? and O. The experiments consist of (i) chemical generation of ONOO^? from NO^? gas and KO₂ powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO^? in the near-UV with maximum at 302 nm; (iii) spectroscopic identification of ¹O₂ by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of ¹O₂ generated in ONOO^?/H⁺ reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H₂O₂/OCl^? reaction that generates ¹O₂ with unit efficiency at alkaline pH. ¹O₂ was generated with unit efficiency with respect to ONOO^? concentration by the ONOO^?/H⁺ reaction.     

Involvement of two differenturf-s related mitochondrial sequences in the molecular evolution of the CMS-specificS-Pcf locus in petunia

In petunia, a mitochondrial (mt) locus,S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). TheS-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF,Pcf, contains parts of theatp9 andcoxII genes and an unidentified reading frame,urf-s. The second and third ORFs contain NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) sequences, respectively. Thenad3 andrps12 sequences included in theS-Pcf locus are identical to the corresponding sequences on the mt genome of fertile petunia. In both CMS and fertile petunia, only a single copy ofnad3 andrps12 has been detected on the physical map of the main mt genome. The origin of theurf-s sequence and the molecular events leading to the formation of the chimericS-Pcf locus are not known. This paper presents evidence indicating that two different mt sequences, related tourf-s and found in fertile petunia lines (orf-h and Rf-1), might have been involved in the molecular evolution of theS-Pcf locus. Southern analysis of mtDNA derived from both fertile and sterile petunia plants suggests that one of theseurf-s related sequences (showing 100% homology tourf-s and termedorf-h) is located on a sublimon. An additional, low-homologyurf-s related sequence (Rf-1) is shown to be located on the main mt genome 5′ to thenad3 gene. It is, thus, suggested that the sequence of events leading to the generation of theS-Pcf locus might have involved introduction of theorf-h sequence, via homologous recombination, into the main mt genome 5′ tonad3 at the region where the Rf-1 sequence is located.