Guinea pig intestinal iron binding protein

An iron binding protein, isolated from guinea pig intestinal mucosa, was compared to guinea pig transferrin. Both had a molecular weight of approximately 80,000. The intestinal iron-binding protein consisted of 2 subunits of equal molecular weight; transferrin had no subunits. Transferrin showed an absorbance peak at 470 nm; the intestinal iron-binding protein had no visible absorbance but did have a peak at 336 nm. Electron spin resonance spectra of the two proteins dfffered. Significant differences on amino acid analysis were also identified.     

The Arabidopsis phenylalanine insensitive growth mutant exhibits a deregulated amino acid metabolism

Amino acids and amino acid analogs have been used in numerous genetic screens to isolate mutants deficient in amino acid biosynthetic pathways or in the regulation of amino acid metabolism. Several of these mutants exhibit relaxed feedback control of branched amino acid biosynthetic pathways and are thus resistant to accumulation of pathway end products. For example, feedback-regulated enzymes of the shikimate pathway are anthranilate synthase on the branch leading to Trp and chorismate mutase on the branch leading to Phe and Tyr. A feedback-insensitive mutant of anthranilate synthase alpha, trp5-1, is resistant to toxic Trp analogs. Mutants resistant to Phe have not previously been reported, and this article describes the isolation of the recessive Arabidopsis Phe insensitive growth mutant pig1-1 by a forward genetic screen. pig1-1 was not only tolerant to Phe, Tyr, and Trp, but also to other, not biosynthetically related amino acids. Amino acid contents in pig1-1 were significantly elevated with respect to wild-type controls but, in contrast to the wild type, dramatically decreased when plants were supplemented with 2 mm Phe. Protein contents were similar in the mutant and the wild type at all tested conditions. Phe catabolism was similar to the wild type in pig1-1 roots but was significantly increased in pig1-1 shoots. Phenylalanine uptake into the root, its root-to-shoot translocation, and Phe and phenylpropanoid contents were unaltered in pig1-1, indicating that pig1-1 is not affected in amino acid translocation or the shikimate pathway. Instead, the response of pig1-1 toward amino acid feeding indicates that amino acid metabolism is generally deregulated in pig1-1.     

Isolation and physico-chemical properties of lactoferrin from swine neutrophils

Lactoferrin, a non-heme iron-binding protein was isolated from pig neutrophils. The purification procedure included initial extraction of the protein in the presence of cetyltrimethylammonium bromide followed by chromatography on carboxymethyl-cellulose and Sephadex G-100. The thus obtained protein was found to be homogeneous on polyacrylamide gel (PAAG) electrophoresis at acidic values of pH. PAAG electrophoresis in the presence of sodium dodecyl sulfate revealed a single component with a molecular weight of 75 000-80 000. The resulting protein is capable of binding two atoms of iron molecule. The absorbance spectra for the pig neutrophil lactoferrin are identical to those for cow milk lactoferrin in the visible region and have a maximum at 465 nm. The amino acid composition of pig lactoferrin was determined. Isoelectric focusing of the protein obtained in a PAAG stabilized pH gradient revealed a component with pI of about 6.8. A single precipitin line was observed with rabbit antipig lactoferrin when examined by immunodiffusion. No immunological cross-reactions were observed between pig lactoferrin and bovine lactoferrin.     

Isolation and structural determination of rat neuromedin U

Rat neuromedin U was isolated from the small intestine using mainly immunoaffinity chromatography and radioimmunoassay for pig neuromedin U-8. The amino acid sequence of rat neuromedin U was determined by microsequence analysis to be Tyr-Lys-Val-Asn-Glu-Tyr-Gln-Gly-Pro-Val-Ala-Pro-Ser-Gly-Gly- Phe-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2, and this structure was confirmed by synthesis. Although the C-terminal heptapeptide amide structure of pig neuromedin U is completely conserved in rat neuromedin U, the remainder of the peptide reveals nine amino acid replacements and two amino acid deletions when compared to pig neuromedin U-25. Rat neuromedin U exerts two-fold potent uterus stimulant activity as compared to pig neuromedin U-25.     

Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase

Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified. A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe. The complete nucleotide sequence of the 1.5-kilobase cDNA was determined. The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase. The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides. A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified. To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]. The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met. A glutamate-requiring (citrate synthase deficient), recA- E. coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS. pT7-7PCS complemented the E. coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure comparison of the proposed low density lipoprotein (LDL) receptor binding domain of human and pig apolipoprotein B: implications for LDL-receptor interactions

Apolipoprotein B (apoB) is the predominant protein in low density lipoprotein (LDL) and is responsible for LDL binding to the LDL receptor. Although the primary amino acid sequence of human apoB has been determined, little is known about the structural domains involved in mediating apoB binding to the LDL receptor. Amino acid sequence comparisons across species lines provide a means of defining structures that are essential for function. We have sequenced a l.l kb fragment of pig apoB genomic DNA, corresponding to a 363 amino acid segment proposed to mediate human apoB binding to the LDL receptor. In human apoB this domain contains two regions enriched in positively charged amino acids flanking two disulfide-linked cysteine residues. The pig amino acid sequence shared 72% identity with the human sequence. However, there were differences that have significant structural and functional implications. Human apoB arginine-3,359 corresponds to a critical arginine (position 142) residue in the apoE LDL receptor binding domain. In the pig, this arginine residue was not conserved. Also, the two disulfide-linked cysteine residues found near the proposed apoB binding domain were not conserved in the pig. Despite these differences, pig LDL had a higher affinity than human LDL for both the pig and human LDL receptor. Thus, these features are not required for high affinity binding of pig LDL to the LDL receptor, and may not be necessary for the binding of human LDL to the LDL receptor.     

Some Effects of Protein Deficiency in Young Growing Pigs. III. Ferrokinetic Investigations

Ferrokinetic investigations have been carried out in pigs which had undergone protein starvation and rehabilitation. After long term protein deprivation haematocrit levels, blood haemoglobin levels, serum iron concentrations, serum total iron binding capacity and blood volume per kg body weight were all reduced in the deficient animals. The values for the rehabilitated animals were in the normal range. The erythrocytes of the protein starved pigs showed an increased rate of uptake of radioactive iron compared with the control pig. However, the turnover of iron as a whole was reduced greatly in the protein deprived group compared to the rehabilitated and control pigs. Accumulation of iron occurred in the livers, spleens and kidneys of the protein starved animals. The rehabilitated pigs had lower liver stores of iron than the control pig. On the basis of the data presented it has been concluded that these effects are mainly due to a shortage of amino acids for protein synthesis.     

Molecular cloning of the structural gene for porcine thyroid peroxidase

We have isolated and determined the nucleotide sequence of overlapping cDNA clones, representing the entire structural gene for pig thyroid peroxidase. The protein coding region extends from an ATG residue at base 252 to a termination codon at base 3030, coding for a 100.4-kDa apoprotein of 926 amino acids. The derived amino acid composition agrees well with the experimentally determined amino acid composition of purified pig thyroid peroxidase. Five potential glycosylation sites are present in the protein. Potential membrane spanning regions are present at the amino-terminal end (1-23) and near the carboxyl-terminal end (845-870) of the protein. These data indicate that pig thyroid peroxidase is synthesized as a single polypeptide that is membrane-bound.     

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares approximately 80-90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76-80% identity with the nucleotide and amino acid sequences of these other species. [(3)H]nicotinic acid binding affinity at guinea pig and hamster receptors is similar to that in human (dissociation constant = 121 nM for guinea pig, 72 nM for hamster, and 74 nM for human), as are potencies of nicotinic acid analogs in competition binding studies. Inhibition of forskolin-stimulated cAMP production by nicotinic acid and related analogs is also similar to the activity in the human receptor. Analysis of mRNA tissue distribution for the hamster and guinea pig nicotinic acid receptors shows expression across a number of tissues, with higher expression in adipose, lung, skeletal muscle, spleen, testis, and ovary.     

Structure-activity studies of heptapeptide derivatives related to substance P, neurokinin A, B and other tachykinins on smooth muscles

Diverse C-terminal heptapeptide derivatives related to substance P, kassinin, physalaemin, neurokinin A and B were synthesized and the contracting activities on the guinea pig ileum as well as rat duodenum were compared. In the partial sequence of C-terminal of tachykinin peptides, -I-II-Phe-III-Gly-Leu-Met-NH2, the combination of amino acid residues at positions I and III have significant roles in contraction of smooth muscle. For the activation of rat duodenal muscle (SP-E), Asp(I) and aliphatic amino acid(III), and for guinea pig ileal muscle(SP-P), Gln(I) and aromatic amino acid(III) are essential. Moreover, guinea pig ileum is sensitive to a full sequence of neurokinin peptides.     

猪脾和马脾铁蛋白理化特性的比较

Ｈ＾＋,ＯＨ＾－均能参与猪脾和马脾铁蛋白铁核组成,迫使它们分别释放铁核中对酸碱不稳定的铁组份。在可见光谱中,猪脾和马脾铁蛋释放铁的动力学过程可分为一级快速反应和零级慢速反应,但猪脾铁蛋白释放铁一级反应速度明显大于马脾铁蛋白释放铁的一级反应的速率,推测这些现象均与各自蛋白的蛋白壳自身调节能力有着密切联系。     

NADH-ferric reductase activity associated with dihydropteridine reductase

In mammals dietary ferric iron is reduced to ferrous iron for more efficient absorption by the intestine. Analysis of a pig duodenal membrane fraction revealed two NADH-dependent ferric reductase activities, one associated with a b-type cytochrome and the other not. Purification and characterization of the non-cytochrome ferric reductase identified a 31 kDa protein. MALDI-MS analysis and amino acid sequencing identified the ferric reductase as being related to the 26 kDa liver NADH-dependent quinoid dihydropteridine reductase (DHPR). The NADH-dependent DHPR ferric reductase activity was found to be pteridine-independent since exhaustive dialysis did not reduce activity and heat-inactivation destroyed activity. In intestinal Caco-2 cells, DHPR mRNA levels were found to be regulated by iron. Thus, DHPR appears to be a dual function enzyme, a NADH-dependent dihydopteridine reductase and an iron-regulated, NADH-dependent, pteridine-independent ferric reductase.     

小鼠海帕西啶1和2的氨基酸对比分析

海帕西啶是一种含有25个氨基酸的多肽类激素,在铁代谢过程中处于重要的地位。通过比对小鼠海帕西啶1和2发现,8位的异亮氨酸和25位的苏氨酸在哺乳动物中高度保守,因此认为这两种氨基酸可能对于海帕西啶的铁代谢功能发挥着重要的作用。     

Primary structure of the alpha-subunit of Na+,K+-ATPase from the swine kidney. III. Complete nucleotide sequence corresponding to the structural region of the gene

The nucleotide sequence of the cDNA, containing coding region of the alpha-subunit of the pig kidney Na+, K+-ATPase, was determined. The region contains 3063 b.p. coding for 1021 amino acid residues. In the course of processing, five amino acid residues are cleaved to yield the mature Na+, K+-ATPase alpha-subunit containing 1016 amino acid residues.     

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.     

Amino acid sequence of normal (microheterogeneous) porcine immunoglobulin lambda chains.

The partial amino acid sequence of pooled, microheterogeneous pig immunoglobulin lambda chains was determined previously (Fran?k, F. (1970), FEBS Lett. 8, 269; Novotny, J., and Fran?k, F. (1975), FEBS Lett. 58, 24). In the present study, citraconylated pig lambda chains were digested by trypsin under conditions in which some of the epsilon-amino groups of lysine residues unmask. The resulting fragments were purified by gel filtration and ion-exchange chromatography at pH 3.0 in buffers containing urea; some of the fragments were found to be of intermediate size (i.e., larger than normal tryptic peptides but smaller than "citraconyl" peptides), thus permitting overlap information and amino acid sequences of all the 14 tryptic peptides to be deduced from amino acid compositions and partial amino acid sequences of selected fragments. In addition to completing the major amino acid sequence of pig immunoglobulin lambda chains, the present study demonstrates that it is possible to sequence microheterogeneous proteins with a suitable fragmentation strategy.     

Free Radicals Enhance Basal Release of D-[3H]Aspartate from Cerebral Cortical Synaptosomes

Abstract: Excessive generation of free radicals has been implicated in several pathological conditions. We demonstrated previously that peroxide-generated free radicals decrease calcium-dependent high K⁺-evoked l -[³H]-glutamate release from synaptosomes while increasing calcium-independent basal release. The present study evaluates the nonyesicular release of excitatory amino acid neurotransmitters, using d -[³H]aspartate as an exogenous label of the cytoplasmic pool of l -glutamate and l -aspartate. Isolated presynaptic nerve terminals from the guinea pig cerebral cortex were used to examine the actions and interactions of peroxide, iron, and desferrioxamine. Pretreatment with peroxide, iron alone, or peroxide with iron significantly increased the calcium-independent basal release of d -[³H]aspartate. Pretreatment with desferrioxamine had little effect on its own but significantly limited the enhancement by peroxide. High K⁺-evoked release in the presence of Ca²⁺ was enhanced by peroxide but not by iron. These data suggest that peroxide increases nonvesicular basal release of excitatory amino acids through Fenton-generated hydroxyl radicals. This release could cause accumulation of extracellular excitatory amino acids and contribute to the excitotoxicity associated with some pathologies.     

Characterization of two novel forms of cholecystokinin isolated from bovine upper intestine

The primary structures of two novel forms of cholecystokinin, isolated from bovine upper intestine are reported. The two peptides are composed of 33 and 39 amino acid residues, respectively, the larger being an N-terminally extended form of the shorter peptide. The primary structure of the 39 amino acid peptide is: (Formula: see text) This amino acid sequence differs from the porcine hormone at positions 13 and 15, which are Val and Met, respectively, in pig, the same amino acid substitutions have previously been found to occur also in dog.