Amino acid sequence surrounding the lipoic acid cofactor of bovine kidney 2-oxoglutarate dehydrogenase complex

The 2-oxoglutarate dehydrogenase complex was succinylated using 2-oxo[5-14C]glutarate in the presence of N-ethylmaleimide to label the lipoic acid cofactor of the transuccinylase (E2) component. Following peptic digestion, 14C-lipoate-containing peptides were purified and subjected to automated Edman degradation and amino acid analysis. The amino acid sequence surrounding the lipoyllysine residue is reported.     

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).     

Characterization of a heterogeneous camel milk whey non-casein protein

A milk protein, occurring in the whey fraction, has been characterized from camel milk. Determination of the primary structure reveals the existence of two related types of chain with residue differences in at least the N-terminal region. A fragment representing an N-terminal part of the protein was also recovered (heterogeneous at the same positions). The absence of cysteine residues in the protein shows that no disulphide bridges are present. The pattern of fragments and a parent protein resembles that for casein and its fragments, showing that fragments and a multiplicity of forms may be typical for different milk proteins.     

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     

Induction of NF-kappa B DNA-binding activity during the G0-to-G1 transition in mouse fibroblasts.

A DNA-binding factor with properties of NF-kappa B and another similar activity are rapidly induced when growth-arrested BALB/c 3T3 cells are stimulated with serum growth factors. Induction of these DNA-binding activities is not inhibited by pretreatment of quiescent cells with the protein synthesis inhibitor cycloheximide. Interestingly, the major NF-kappa B-like activity is not detected in nuclear extracts of proliferating cells, and thus its expression appears to be limited to the G0-to-G1 transition in 3T3 cells. These DNA-binding activities bind many of the expected NF-kappa B target sequences, including elements in the class I major histocompatibility complex and human immunodeficiency virus enhancers, as well as a recently identified NF-kappa B binding site upstream of the c-myc gene. Furthermore, both the class I major histocompatibility complex and c-myc NF-kappa B binding sites confer inducibility on a minimal promoter in 3T3 cells stimulated with serum growth factors. The results demonstrate that NF-kappa B-like activities are immediate-early response proteins in 3T3 cells and suggest a role for these factors in the G0-to-G1 transition.     

Calcium channels that are required for secretion from intact nerve terminals of vertebrates are sensitive to omega-conotoxin and relatively insensitive to dihydropyridines. Optical studies with and without voltage-sensitive dyes

Extrinsic absorption changes exhibited by potentiometric dyes have established the ionic basis of the action potential in synchronously activated populations of nerve terminals in the intact neurohypophyses of amphibia and mammals (Salzberg et al., 1983; Obaid et al., 1983, 1985b). Also, large and rapid changes in light scattering, measured as transparency, have been shown to follow membrane depolarization and to be intimately associated with the release of neuropeptides from the nerve terminals of the mouse neurohypophysis (Salzberg et al., 1985; Gainer et al., 1986). We report some experiments that help to define the pharmacological profile of the calcium channels present in intact neurosecretory terminals of vertebrates. For these, we used the peptide toxin omega-conotoxin GVIA (1-5 microM) and the dihydropyridine compounds Bay-K 8644 and nifedipine (2-5 microM), together with the after-hyperpolarization of the nerve terminal action potential. This undershoot depends upon the activation of a calcium-mediated potassium channel, as suggested by its sensitivity to [Ca++]o and charybdotoxin. omega-conotoxin GVIA substantially reduced the after-hyperpolarization in neurosecretory terminals of Xenopus, while neither of the dihydropyridine compounds had any effect under conditions that mimic natural stimulation. The effects of these calcium channel modifiers on the action potential recorded optically from the terminals of the Xenopus neurohypophysis were faithfully reflected in the behavior of the light-scattering changes observed in the neurohypophysis of the CD-1 mouse. omega-conotoxin GVIA (5 microM) reduced the size of the intrinsic optical signal associated with secretion by 50%, while the dihydropyridines had little effect. These observations suggest that the type of calcium channel that dominates the secretory behavior of intact vertebrate nerve terminals is at least partially blocked by omega-conotoxin GVIA and is insensitive, under normal conditions, to dihydropyridines.