Preparation and characteristics of monoclonal antibodies against GTP-binding protein from the bovine retina

Five monoclonal antibodies against the native GTP-binding protein (transducin) from bovine retina have been prepared. By immunoblotting and immunoenzymatic analysis of the isolated alpha- and gamma-subunits of transducin and the beta gamma-subunit complex it was determined that two monoclonal antibodies A3G7 and A3C10 recognize linear antigenic determinants on the alpha-subunit, two other, A3E4 and 3B3, bound specifically to the gamma-subunit, and monoclonal antibodies 1C3 interact only with native transducin. Both antibodies against the alpha-subunit inhibited transducin GTPase activity, whereas antibodies A3E4, 3B3 and 1C3 did not affect it.     

Primary structure of bovine cerebellum GTP-binding protein G39 and its effect on the adenylate cyclase system

The primary structure of bovine cerebellum GTP-binding protein alpha-subunit, protein G39, was determined by parallel analysis of the protein amino acid sequence and the corresponding cDNA nucleotide sequence. The protein consists of 354 amino acid residues and has a molecular mass of 40064 Da. High homology between G39 and other G-proteins, especially rat brain G0, was shown. An assumption is made that certain brain adenylate cyclase system properties are determined by the presence of G39.     

The complete primary structure of the cellular retinaldehyde-binding protein from bovine retina

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol and 11-cis-retinaldehyde as endogenous ligands and may be a functional component of the visual cycle. The complete amino acid sequence of CRALBP from bovine retina has been determined by direct microanalysis of the protein. Bovine CRALBP contains 316 residues in a single amino-terminal-blocked chain corresponding to a molecular weight of 36,421, inclusive of the blocking group. Overlapping peptides were generated by cleavage of lysyl, arginyl, methionyl, glutamyl, and one tryptophanyl bond and sequenced by gas-phase Edman degradation. Analysis of amino-terminal arginyl and methionyl peptides by fast atom bombardment mass spectrometry identified the N alpha-blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 9 residues. Comparison of CRALBP with other known protein sequences reveals no significant structural relatedness. The present results provide a basis for relating CRALBP domains with physiological function and for the future development of a more detailed three-dimensional model of the interaction of 11-cis-retinaldehyde with protein.     

Primary culture of microvascular endothelial cells from bovine retina

Summary To provide an in vitro system for studying retinal capillary function we have developed methods for isolation and culture of microvascular endothelial cells from retina. Retinal microvessels were isolated by homogenization of the retina and collection of the microvessels onto nylon mesh. Treatment of the isolated microvessels with collagenase and dispase followed by Percoll gradient centrifugation yielded endothelial cells that were largely free of pericytes. A homogeneous population of endothelial cells that were capable of at least six population doublings was obtained by plating onto a fibronectin coated substrate in plasma derived serum. The endothelial origin of these cells was confirmed by the presence of Factor VIII antigen, angiotensin converting enzyme activity, numerous tight junctions, and a cell surface that did not bind platelets. A second cell type, which did not exhibit these cell markers and which is presumably the intramural pericyte, was obtained when the isolated microvessels were plated on tissue culture grade plastic in fetal bovine serum. Supported by Research Grants 5R01-EY03772 and 5R01-ES02380 from the U.S. Public Health Service (G. W. G.) and Established Investigator Award 31-107 from the American Heart Association (A. L. B.).     

The gamma-subunit of ATP synthase from spinach chloroplasts. Primary structure deduced from the cloned cDNA sequence

cDNA clones encoding the gamma-subunit of chloroplast ATP synthase were isolated from a spinach library using synthetic oligonucleotide probes. The predicted amino acid sequence indicated that the mature chloroplast gamma-subunit consists of 323 amino acid residues and is highly homologous (55% identical residues) with the sequence of the cyanobacterial subunit. The positions of the four cysteine residues were identified. The carboxyl-terminal region of the chloroplast gamma-subunit is highly homologous with those of the gamma-subunits from six other sources (bacteria and mitochondria) sequenced thus far.     

Crystal structure of the GTP-binding protein Obg from Thermus thermophilus HB8

Obg comprises a unique family of high-molecular mass GTPases conserved from bacteria to eukaryotes. Bacterial Obg is essential for cellular growth, sporulation, and differentiation. Here, we report the crystal structure of the full-length form of Obg from Thermus thermophilus HB8 at 2.07 A resolution, in the nucleotide-free state. It reveals a three-domain arrangement, composed of the N-terminal domain, the guanine nucleotide-binding domain (G domain), and the C-terminal domain. The N-terminal and G domains have the Obg fold and the Ras-like fold, respectively. These global folds are similar to those of the recently published structure of the C-terminal domain-truncated form of Obg from Bacillus subtilis. On the other hand, the C-terminal domain of Obg was found to have a novel fold (the OCT fold). A comparison of the T.thermophilus and B.subtilis nucleotide-free Obg structures revealed significant conformational changes in the switch-I and switch-II regions of the G domain. Notably, the N-terminal domain is rotated drastically, by almost 180 degrees, around the G domain axis. In the T.thermophilus Obg crystal, the nucleotide-binding site of the G domain interacts with the C-terminal domain of the adjacent molecule. These data suggest a possible domain rearrangement of Obg, and a potential role of the C-terminal domain in the regulation of the nucleotide-binding state.     

Photochemistry and stereoselectivity of cellular retinaldehyde-binding protein from bovine retina

11-cis-Retinaldehyde bound to cellular retinaldehyde-binding protein (CRALBP) is unaffected in bovine eyecup preparations by illumination that bleaches approximately 70% of the rhodopsin. Illumination of retinal homogenates to which CRALBP X [3H]11-cis-retinaldehyde had been added did not result in a reduction of the specific activity of recovered 11-cis-retinaldehyde, ruling out a bleaching regeneration cycle. The quantum efficiency of photoisomerization for CRALBP X 11-cis-retinaldehyde was determined by comparing the rate of photoisomerization of 11-cis-retinaldehyde bound to purified CRALBP and opsin. The low value obtained (0.07), coupled with a low molar extinction coefficient (15,400 M-1 cm-1), results in a photosensitivity only about 4% that of rhodopsin. CRALBP binds 9-cis- and 11-cis-retinaldehyde, producing complexes with absorption maxima at 405 and 425 nm, respectively. No complexes were detected with 13-cis- and all-trans-retinaldehyde. Following incubation of CRALBP X 11-cis-retinol with an equimolar mixture of 9-, 11-, 13-cis-, and all-trans-retinaldehydes, only 11-cis-retinaldehyde and residual 11-cis-retinol are present on the protein following separation from excess retinoids. A similar result is obtained following incubation of CRALBP X 11-cis-retinol with mixtures of 9- and 11-cis-retinaldehyde ranging in composition from 9:1 to 1:9 (9-cis-:11-cis-,mol/mol). The results indicate that CRALBP X 11-cis-retinol is sufficiently stereoselective in its binding properties to warrant consideration as a component of the mechanism for the generation of 11-cis-retinaldehyde in the dark.     

Primary structure of hepatoredoxin from bovine liver mitochondria

The primary structure of hepatoredoxin from bovine liver mitochondria was established. It consists of 117 amino acid residues. The identity of the amino acid sequences of bovine hepatoredoxin and adrenodoxin from bovine adrenal cortex mitochondria was shown. It is assumed that the role of a ferredoxin component in mitochondrial steroid-hydroxylating systems from different organs is played by the same [2Fe-2S]-protein.     

Primary structure of a ribonuclease from bovine brain

The primary structure of a pyrimidine base-specific ribonuclease from bovine brain was determined. The sequence determined is (sequence; see text). Although the sequence homology of this RNase with bovine pancreatic RNase A is 78.2%, it consists of 140 amino acid residues, and it is 16 amino acid residues longer than RNase A at the carboxyl-terminal. In addition to an N-glycosylated long carbohydrate chain, the bovine brain RNase has two short O-glycosylated carbohydrate chains at the 129th and the 133rd serine residues. The additional C-terminal tail of the bovine brain RNase has a unique composition: 6 proline, 5 hydrophobic amino acids, and two basic amino acids, arginine and histidine.     

Primary structure of ferredoxin from bovine kidney mitochondria

Kidney mitochondrial ferredoxin (renodoxin) is a component of the cytochrome P-450-dependent enzymatic system whose main function is the hydroxylation of vitamin D₃ in the 1a- and 24-positions. The complete amino acid sequence of renodoxin was determined by protein chemistry and mass spectrometry. The mature renodoxin has 128 amino acid residues. The N- and C-terminal regions of renodoxin are subject to proteolytic modification, this being the origin of heterogeneous molecular mass (from 14,200 to 12,400 kD) of purified protein preparations. The antigenic structure of renodoxin was studied using antibodies to peptide fragments of a homologous protein, adrenodoxin.     

Primary structure of tRNA1ser from bovine liver

The primary structure of tRNA(1Ser) from the bovine liver has been studied. pG- A-C-G-A-G-G-U-G-G-C-ac4C-G-A-G-D-Gm-G-D-D-A-A-G-G- C-m2(2)-G-A-psi-G-G-A-m3C-U-G-C-U-A*-A-psi-C-C-A-U-Um-G-psi- G-C-U-m3C-U-G-C-A-C-G-m5C-G-U-G-G-G-T-psi-C-G-m1A-A- U-C-C-C-A-U-C-C-U-C-G-U-C-G-C-C-AOH. A comparison of the nucleotide sequence of tRNA(1Ser) from the bovine liver with already known sequences of serine tRNA revealed a number of common nucleotides, some of them, probably, participated in specific interaction with seryl-tRNA synthetase.     

Primary structure of cAMP-gated channel from bovine olfactory epithelium

The complete amino-acid sequence of the bovine olfactory epithelium adenosine 3',5'cyclic monophosphate (cAMP)-gated channel has been determined by cloning and sequencing its cDNA. It exhibits a high degree of sequence homology with the cGMP-gated channel of rod photoreceptors, suggesting that cyclic nucleotide-gated channels fall into a new family of genetically related proteins.     

Primary structure of an alkaline ribonuclease from bovine liver

A pyrimidine base specific and most basic alkaline RNase named RNase BL4 was isolated from bovine liver as a protein showing a single band on slab gel-electrophoresis. The enzyme is most active at pH 7.5. The enzyme was immunologically distinguishable from the known bovine RNases such as pancreatic RNase (RNase A), seminal RNase, kidney non-secretory RNase (RNase K2), and brain RNase (RNase BRb). The primary structure of this pyrimidine base-specific RNase was determined to be less than EDRMYQRFLRQHVDPDETG- GNDSYCNLMMQRRKMTSHQCKRFNTFIHEDLWNIRSICSTTNIQCKNGQMNCHEGVVRV- TDCRETGSSRAPNCRYRAKASTRRVVIACEGNPEVPVHFDK. It consists of 119 amino acid residues, and is 5 amino acid residues shorter than RNase A. The sequence homology of RNase BL4 with RNase A is 46.2%, and optimal alignment of RNase A and RNase BL4 requires five deletions, one at the 24th position, two at the 75th and 76th positions, and two at the C-terminus in RNase BL4. The RNase BL4 was highly homologous with a porcine liver RNase (RNase PL3, 94.1% homology) studied by Hofsteenge et al. (personal communication from Hofsteenge, J., Matthies, R., and Stones, S.R.).     

Primary structure of a non-secretory ribonuclease from bovine kidney

The primary structure of a non-secretory ribonuclease from bovine kidney (RNase K2) was determined. The sequence determined was VPKGLTKARWFEIQHIQPRLLQCNKAMSGV NNYTQHCKPENTFLHNVFQDVTAVCDMPNIICKNGRHNCHQSPKPVNLTQCNFIAGRYPDC RYHDDAQYKFFIVACDPPQKTDPPYHLVPVHLDKYF. The sequence homology with human non-secretory RNase, bovine pancreatic RNase, and human secretory RNase are 46, 34.6, and 32.3%, respectively. The bovine kidney RNase has two inserted sequences, a tripeptide at the N-terminus and a heptapeptide between the 113th and 114th position of bovine pancreatic RNase; on the other hand, it is deleted of the hexapeptide consisting of the 17th to the 22nd amino acid residue of RNase A. The amino acid residues assumed to be the constituents of the bovine pancreatic RNase active site are all conserved except F120 (L in RNase K2).     

NAD glucohydrolase from bovine retina

The NAD glycohydrolase activity in the retina is very low compared with the one found in the brain. Therefore the retina extracts ahve a sufficiently high NAD level so that they are able to form "in vitro" lactate from glucose in anaerobiosis in presence of only ATP, Mg++ and glucose. The NAD glycohydrolase has been found in the retina in a great extent in the microsomes.     

Primary structure of the activation peptide from bovine pancreatic procarboxypeptidase A

The complete sequence of the 94 residues composing the activation peptide of bovine procarboxypeptidase A has been determined by automated analysis of the intact activation segment and of three peptides resulting from enzymatic cleavages of the isolated peptide. The sequencing of a CNBr peptide isolated from procarboxypeptidase A allowed to connect the activation peptide with alpha-carboxypeptidase A (peptidylprolyl-L-amino-acid hydrolase, EC 3.4.17.1). The activation segment has a high content of acidic residues and a proline-rich region. Conformational prediction studies show that the bovine peptide, as the porcine and rat peptides, contains a high proportion of secondary structure and that the structural disposition of the regions in secondary structure is similar in the three peptides. The comparison of the sequence of the bovine, porcine and rat peptides, although exhibiting a striking homology, clearly shows that 40% of the substitutions have led to a charge change.     

The primary structure of the gamma-subunit of the ATPase from Synechocystis 6803

The nucleotide sequence of the gene coding for the F0F1-ATPase gamma-subunit (atpC) from the transformable cyanobacterium Synchocystis 6083 has been determined. The deduced translation product consists of 314 amino acid residues and is highly homologous (72% identical residues) to the sequences of other cyanobacterial gamma-subunits. The Synechocystis 6803 sequence is also homologous to the chloroplast gamma-sequence. Like in the other cyanobacterial subunits, only the first of the 3 cysteine residues, which are involved in energy-linked functions of the gamma-subunit in spinach chloroplasts, is conserved in Synechocystis 6803.