Two forms of pyruvate kinase in Escherichia coli. A comparison of chemical and molecular properties.

The two forms of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Escherichia coli have been purified from the same cultures and crystallized. A modified procedure for the purification of type I pyruvate kinase is described. Molecular weight, subunit structure, amino acid composition, NH2-terminal amino acid, maps of tryptic peptides and conditions for crystallization have been determined for the two forms. A comparison of these data shows that the two forms are different proteins, each being a tetramer of identical subunits.     

Acidic ribosomal proteins of Neurospora crassa

Neurospora crassa acidic ribosomal proteins from the high salt-ethanol extract of 80 S ribosomes have been fractionated by DEAE-cellulose chromatography. Six acidic ribosomal proteins were purified. All resemble Escherichia coli L7 and L12 in amino acid composition and molecular weight but each has a slightly different net charge at pH 3.2. Four have an apparent molecular weight of approx. 14 000, and two have a molecular weight of approx. 14 800. The amino acid compositions and circular dichroism (CD) spectra of the purified Neuropsora proteins are identical for the four 14 kDa proteins, but clearly distinguishable from the two 14.8 kDa proteins. The latter are also identical in amino acid composition and CD spectra. This suggests that there are two Neurospora acidic, or 'A', proteins, one of which exists in four microheterogeneous forms and the other exists in two forms.     

The biological significance of methionine sulfoxide stereochemistry

Methionine can be oxidized by reactive oxygen species to a mixture of two diastereomers, methionine-S-sulfoxide and methionine-R-sulfoxide. Both free amino acid and protein-based forms of methionine-S-sulfoxide are stereospecifically reduced by MsrA, whereas the reduction of methionine-R-sulfoxide requires two enzymes, MsrB and fRMsr, which act on its protein-based and free amino acid forms, respectively. However, mammals lack fRMsr and are characterized by deficiency in the reduction of free methionine-R-sulfoxide. The biological significance of such biased reduction of methionine sulfoxide has not been fully explored. MsrA and MsrB activities decrease during aging, leading to accumulation of protein-based and free amino acid forms of methionine sulfoxide. Since methionine is an indispensible amino acid in human nutrition and a key metabolite in sulfur, methylation, and transsulfuration pathways, the consequences of accumulation of its oxidized forms require further studies. Finally, in addition to methionine, methylsulfinyl groups are present in various drugs and natural compounds, and their differential reduction by Msrs may have important therapeutic implications.     

Evidence for the existence of two isocolipases in horse pancreas

The N-terminal amino acid sequences of two forms of colipase isolated from horse pancreas have been compared. Four sequence differences were found in the first 51 amino acids. This lead us to conclude that there are two distinct colipases in the horse pancreas.     

Evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical

Bovine erythrocyte green heme binding protein and bovine erythrocyte flavin reductase have been isolated in highly purified forms and subjected to amino acid analysis and N-terminal amino acid sequence analysis. The two proteins possess similar amino acid compositions and identical N-terminal amino acid sequences. Moreover, the two proteins are immunochemically cross-reactive and are indistinguishable when compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by double diffusion technique. This study provides evidence that the protein components of bovine erythrocyte green heme binding protein and flavin reductase are identical.     

Multiple molecular forms of glucagon and insulin in the kaluga sturgeon, Huso dauricus

Five molecular forms of glucagon and two molecular forms of insulin were characterized from the kaluga sturgeon. Substitutions occurred at two to thirteen internal amino acid residues among the five molecular forms of glucagons, indicating that these glucagons were encoded by five distinct genes. The amino acid sequences of two insulins from the kaluga sturgeon were identical to those of paddlefish insulin-II and Russian sturgeon insulin except that kaluga sturgeon insulin-I had an extension of five residues at the B-chain N-terminus. This is the first demonstration that more than two molecular forms of glucagon have been characterized from a single animal species.     

Multiple molecular forms of glucagon and insulin in the kaluga sturgeon, Huso dauricus

Five molecular forms of glucagon and two molecular forms of insulin were characterized from the kaluga sturgeon. Substitutions occurred at two to thirteen internal amino acid residues among the five molecular forms of glucagons, indicating that these glucagons were encoded by five distinct genes. The amino acid sequences of two insulins from the kaluga sturgeon were identical to those of paddlefish insulin-II and Russian sturgeon insulin except that kaluga sturgeon insulin-I had an extension of five residues at the B-chain N-terminus. This is the first demonstration that more than two molecular forms of glucagon have been characterized from a single animal species.     

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.     

Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas

Carboxylester lipase (cholesterol esterase, EC 3.1.1.13) has been purified to homogeneity from porcine pancreas. The enzyme is isolated in two molecular mass forms, a monomer of 74 kDa and a dimer of 167 kDa. The dimer consists of two catalytically-active subunits which have molecular masses approximately 9 kDa greater than the monomers. The difference in size was not attributable to carbohydrate or lipid content. The catalytic properties of the two forms are comparable on a weight basis, the amino acid compositions are quite similar, and the N-terminal sequences are nearly identical for 24 residues. These similarities suggest a possible precursor-product relationship between the two carboxylester lipase forms.     

Isolation and sequence analysis of three cloned cDNAs for rabbit liver proteins that are related to rabbit cytochrome P-450 (form 2), the major phenobarbital-inducible form

We have isolated from rabbit liver three cDNA clones of 1400-1800 base pairs that hybridize selectively to RNA from animals treated with phenobarbital. The nucleotide sequences of the cDNAs have been determined. In the protein coding region the nucleotide sequences of two of the cDNAs are 88% homologous, and the third cDNA is about 72-74% homologous to the other two. All three are 55-60% homologous to rat liver cytochrome P-450b cDNA. The amino acid sequences derived from the cDNA sequences are about 50% homologous to those of rat liver cytochrome P-450b and rabbit liver cytochrome P-450 (form 2). The degree of homology differs substantially in different regions of the protein. The hydrophobicity profiles of these five mammalian cytochromes P-450 are very similar and contain up to eight regions of hydrophobicity that are long enough to span a membrane. These results indicate that these three cDNAs code for rabbit liver cytochromes P-450 which are different from any rabbit liver cytochrome P-450 for which amino acid sequence information is published. These cDNAs are part of a family of genes that are related to rabbit liver cytochrome P-450 (form 2) and rat liver cytochrome P-450b which are the major phenobarbital-inducible forms. The divergence of amino acid sequence between the rat and rabbit forms and the divergence of nucleotide sequences of silent sites in the two most closely related rabbit forms suggest that cytochromes P-450 have a relatively high rate of amino acid divergence compared to many other vertebrate proteins.     

Two forms of GABA transaminase in pig brain

Two forms of GABA transaminase which could be distinguished by ion-exchange chromatography have been separated and purified in pig brain. The two forms have differentK _m values for -ketoglutarate and show different degrees of inhibition by various salts. Although the two forms are separable, they have identical antigenic properties, pH optima, and NH₂ terminal amino acid composition, and they appear to be of the same molecular size. The biological significance or the relationship between multiple forms of GABA transaminase is not yet understood.     

Extracellular lipases of the fungus Rhizopus microsporus UzLT-1

Lipase biosynthesis by the fungus Rhizopus microsporus UzLT-1 was investigated. Under certain conditions the fungus could produce two forms of extracellular lipases. The paper describes the process of purification of the enzyme L-I,N- and C-terminal amino acids of the lipases have been determined. Identity of terminal amino acids of both enzymes has been demonstrated. It has been shown that both forms of lipase from Rhizopus microporus hydrolyze predominantly alpha-ester bonds.     

Structure of two human ovarian inhibins

The complete amino acid sequences of two forms of human ovarian inhibin have been determined through cloning and nucleotide sequencing of cDNAs encoding their individual subunit precursors. The alpha subunit common to both forms of human inhibin is homologous (84 percent conserved) to its equivalent porcine alpha subunit; the subunits which are different in both inhibins (beta A and beta B) are identical to their porcine equivalents in all but one of 232 sequence positions.     

Anomalous rates of evolution of pancreatic polypeptide and peptide tyrosine-tyrosine (PYY) in a tetraploid frog, Xenopus laevis (Anura:Pipidae)

The South African clawed frog Xenopus laevis is believed to have arisen as a result of a tetraploidization event occurring approximately 30 million years ago. Two molecular forms of pancreatic polypeptide (PP) have been isolated from an extract of the pancreas of this species and two molecular forms of peptide tyrosine-tyrosine (PYY) from the intestine. Despite the fact that the amino acid sequence of PP has, in general, been very poorly conserved during the evolution of tetrapods (only Pro(5), Pro(8), Gly(9), Ala(12), Tyr(27), Arg(33) and Arg(35) are invariant among species studied so far), the two Xenopus PPs differ by only a single amino acid substition (Asp(22)-->Glu). In contrast the two molecular forms of PYY differ by six amino acid substitutions (Glu(15)-->Gln, Thr(18)-->Ala, Leu(21)-->Met, Ile(22)-->Thr, Ile(28)-->Val, Val(31)-->Ile). The data imply that strong evolutionary pressure is acting to conserve the functional domain in both genes encoding PP and so suggest that PP may have an important physiological role in amphibians (although the nature of this role has yet to be determined). The more rapid mutation of the functional domain in the genes encoding PYY, a peptide whose amino acid sequence has been quite well conserved in tetrapods and whose physiological significance is well established, suggests that one of the PYY genes may be evolving towards a new function or towards becoming a pseudogene.     

Trypsin Inhibitor Polymorphism: Multigene Family Expression and Posttranslational Modification

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Based on amino acid composition, molecular mass, and N-terminal sequence, the six inhibitors are closely related to one another and belong to the Bowman–Birk family of inhibitors. To define the relations among them, molecular mass and amino acid composition of peptides obtained from digestion with trypsin were determined. The sequence and the biosynthetic mechanism of the isoform formation have been partially resolved for four major isoforms. Two isoinhibitor forms (PSTI IVa, IVb) in pea seeds are due to expression of two distinct genes; PSTI IVa has four amino acid replacements when its sequence is compared with the sequence of PSTI IVb. Two others (PSTI I, II) result from posttranslational proteolytic cleavage of nine C-terminal residues of forms PSTI IVa and IVb, respectively.     

Immunological behavior of two alloforms of ATPase fromMicrococcus lysodeikticus

Antisera were raised in rabbits to two alloforms of ATPase isolated from two substrains ofMicrococcus lysodeikticus. These alloforms show a similar amino acid composition but differ in the associated carbohydrate components. Similarities and differences in the immunological behavior of the two forms have been assessed by immunodiffusion, immunoelectrophoresis, and crossed immunoelectrophoresis. The ATPase forms show a great extent of homology as might be expected from their relatedness in amino acid composition. Differences in immunological properties are also evident. They do not seem primarily to reflect the differences in the glycan constituents, but do result from the polymorphism of the purified ATPase molecule of each form. This heterogeneity (microheterogeneity) is a consequence of the lability of the ATPase molecule and influences its behavior as antigen and immunogen.     

Multiple forms of pig somatotropin]

The multiple forms of pig somatotropin were investigated. Four monomer hormone variants were isolated and some of their physico-chemical characteristics were determined. It is shown that they differ in their molecular mass, N-terminal amino acid residues and amino acid composition. The results of work permit supposing that the identified somatotropin forms are controlled by two different genes. The age dependence of spectrum of somatotropin monomer forms is demonstrated.     

Amino terminal sequence of the bacteriophage T5-coded gene A2 protein

The first twenty-eight amino acid residues from the amino terminal of the T5 phage-coded gene A2 protein were determined. Some evidence is presented which suggests the existence of two forms of the protein; one in the cytoplasm which may have a signal sequence and another form present in the outer membrane. The amino terminal A2 protein sequence shows some sequence homology to the amino terminal region of the T4 phage-coded gene 32 protein. Finally it is important to note that residues ten through twenty-one of the A2 protein are amino acids with low ambiguity codons which should facilitate in the DNA sequencing of the A2 gene.