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B J Ortwerth

Publications and source records attributed to B J Ortwerth.

At least 73 records · Page 4Linked to original sources
PubMed1986

Isolation and characterization of a new aminopeptidase from bovine lens.

An aminopeptidase has been purified to homogeneity from bovine lens tissue by gel filtration and DEAE-cellulose chromatography. This enzyme has a molecular weight of 96,000 under both native and denaturing conditions. The purified enzyme hydrolyzed a variety of synthetic substrates as well as di-, tri-, and higher molecular weight peptides. Significantly this enzyme is capable of hydrolyzing arginine, lysine, and proline aminoacyl bonds. The pH optimum for activity and stability was 6.0. Both a reduced sulfhydryl group and a divalent metal ion are essential for activity. The native enzyme contains 1.6 mol of zinc and 1.0 mol of copper/mol of enzyme. No activation was seen upon incubation with either magnesium or manganese; however, heavy metal ions were inhibitory. Bestatin and puromycin were effective inhibitors and no endopeptidase activity could be detected in the purified preparation. This enzyme is clearly distinct from the lens leucine aminopeptidase, but rather, is identical to a cytosolic aminopeptidase III isolated from other tissues. Evidence is presented which argues that this enzyme may be the major lens aminopeptidase under in vivo conditions.

Aminopeptidases↗
PubMed1986

Structural relationship between tRNALys2 and tRNALys4 from mouse lymphoma cells.

Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNALys2 and tRNALys4, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNALys4 has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t6A*, replacing t6A. This unidentified nucleotide is not a hypomodified form of t6A. Thus, tRNALys4 is not a simple precursor of tRNALys2. Both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNALys2 and tRNALys4 were obtained. The distinctions between tRNALys2 and tRNALys4 may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5',5'''-P1,P4-tetraphosphate, a putative signal in DNA replication.

Animals↗
PubMed1986

Solubilization of the lens water-insoluble fraction by sonication.

A method is reported whereby the solubilization of the bulk of the lens water-insoluble fraction is accomplished by a short sonication of the suspended proteins in low salt buffers. This procedure solubilized greater than 90% of a bovine lens water-insoluble fraction and 80% of the normal human lens water-insoluble fraction. Decreased protein was solubilized from cataractous lenses, but in every case sonication was at least equivalent to extraction with 6.0 M urea. Fractionation of the solubilized proteins by Agarose A-1.5 m gel filtration chromatography showed native molecular weights for bovine lens, but only partial disaggregation with human lens extracts. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) comparison of the proteins solubilized by sonication and 6.0 M urea extraction showed no major differences except that sonication solubilized more of the highly cross-linked protein which remained at the top of the gel.

Adult↗
PubMed1986

Aminopeptidase III activity in normal and cataractous lenses.

Aminopeptidase III activity was demonstrated in extracts from several different mammalian lenses by the hydrolysis of Arg-MCA at pH 6.0. No more than a two-fold difference was seen in overall specific activity. Sections of bovine lenses were removed from the periphery to the center and assayed. A sharp decline in activity was observed in the inner cortical region, and little or no activity was observed in the lens nucleus. This correlated with an increase in the presence of low molecular weight peptides as determined by SDS polyacrylamide gel electrophoresis. The properties of the aminopeptidase from human lens tissue were the same as those previously reported for the purified enzyme from bovine lens. The aminopeptidase activity of normal and cataractous lenses was compared using 4 different substrates. The cataractous lenses had significantly less total aminopeptidase activity. However, little difference in specific activity was observed based on soluble lens protein content. Similarly, electrophoretic separations of normal and cataractous soluble proteins showed little or no differences in the content of low molecular weight peptides. Therefore, this major human lens aminopeptidase remains functional in the cataractous state.

Aging↗
PubMed1984

Lysine tRNA and cell division: a G1 cell cycle mutant is temperature sensitive for the modification of tRNA5Lys to tRNA4Lys.

Ts-694 is a temperature sensitive mutant of hamster cells which is blocked in the G1 phase of the cell cycle at the restrictive temperature of 39 degrees. A comparison of the Lys-tRNA isoacceptors by RPC-5 chromatography showed a decrease in tRNA5Lys and an increase in tRNA4Lys at 39 degrees. This was identical to the changes seen in confluent cultures at the permissive temperature of 33 degrees. These Lys-tRNA changes were not seen in ts-694 cells blocked in G1 by isoleucine deficiency, nor in two other G1 ts mutants at the restrictive temperature. Cells trapped in S phase by a thymidine block also contained decreased levels of tRNA4Lys when raised to 39 degrees. Both tRNA4Lys levels and cell division increased when the cells were returned to the permissive temperature. An in vitro assay was established for the modification of tRNA5Lys to tRNA4Lys with tRNA6Lys and tRNA2Lys as intermediates. The first reaction is the synthesis of tRNA6Lys which involves the introduction of a modified uridine at the third position of the anticodon. Extracts of 694 cells grown at 33 degrees were able to modify rat liver [3H] tRNA5Lys to tRNA6Lys and tRNA4Lys in vitro when assayed at 25 degrees but not at 39 degrees. Extracts of Balb/c 3T3 cells, however, were more active at 39 degrees than at 25 degrees showing that the normal enzyme is not temperature sensitive. Ts-694 cell tRNA, isolated from cells grown at 33 degrees was aminoacylated at both 25 degrees and 39 degrees with rat liver synthetases. tRNA4Lys was present at both temperatures indicating that ts-694 cells do not contain a temperature sensitive tRNA4Lys.

Animals↗
PubMed1984

Lysine tRNAs from rat liver: lysine tRNA sequences are highly conserved.

The two major lysine tRNAs from rat liver, tRNA2Lys and tRNA5Lys, were sequenced by rapid gel or chromatogram readout methods. The major tRNA2Lys differs from a minor form only by a base pair in positions 29 and 41; both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Although highly related, the major tRNA2Lys and tRNA5Lys differ in four base pairs and four unpaired nucleotides, including the first position of the anticodons, but have the same base pair in positions 29 and 41. The three tRNAs maintain a m2G-U pair in the acceptor stem. Detection of this m2G is in contrast to other reports of lysine tRNAs. Sequences of lysine tRNAs are strongly conserved in higher eukaryotes.

Animals↗
PubMed1983

Competence and progression growth factors stimulate different tRNAlys modification reactions in BALB/C 3T3 cells.

Sparse cultures of Balb/C 3T3 cells were growth arrested in a medium containing 1% calf plasma. This treatment caused a decrease in tRNA4lys, a tRNA species related to cell division, and a corresponding increase in tRNA5lys. A restoration of tRNA4lys levels and an increase in cell number was obtained by the addition of either 10% calf serum or a combination of fibroblast growth factor and melanocyte stimulating activity. Neither growth factor alone evoked a proliferative response. The progression factors, insulin and multiplication stimulating activity stimulated the rapid conversion of tRNA5lys to tRNA2lys, whereas the competence factors, fibroblast growth factor and platelet-derived growth factor stimulated a slight conversion of tRNA2lys to tRNA4lys.

Animals↗
PubMed1983

The effects of growth factors on tRNALys modification.

The tRNALys population from tissue culture cells contains several isoaccepting species which are not present in the tRNALys population from tissue sources. These isoacceptors were isolated from mouse LM cells and tested for their coding properties in ribosomal binding assays and their ability to incorporate lysine into protein in a reticulocyte lysate. tRNALys5A and tRNALys6 eluted in the area of tRNALys5. All three species coded preferentially for AAA and bound with equal efficiency. tRNALys1, tRNALys3, tRNALys4, and tRNALys6 all transferred lysine into protein at a slower rate than tRNALys2 and tRNALys5, which are the native species. Several purified growth factors were tested for their ability to affect the levels of tRNALys4, a tRNA possibly associated with cell division. When Balb/c 3T3 cells were grown in medium containing plasma instead of serum, there was a decrease in tRNALys2, tRNALys3, tRNALys4 and an increase in tRNALys5 and tRNALys6. The addition of either FGF or PDGF returned the tRNALys profile to normal. The extent of the tRNALys changes depended upon the concentration of growth factor added. FGF was able to cause a 35% decrease in the tRNALys5 peak with a corresponding increase in tRNALys2 within 1 h of the addition of the factor. These data suggest that competence factors have the ability to stimulate the modification of specific tRNALys isoacceptors.

Animals↗
PubMed1983

Perturbation of the mitochondrial lysine tRNA population by virus-induced transformation or stress of mammalian cells: functional properties and nucleotide sequence of a mitochondrially associated lysine tRNA.

Eleven isoaccepting lysine tRNAs from mammalian sources are demonstrable by RPC-5 chromatography and polyacrylamide gel electrophoresis. The appearance and amounts of these isoacceptors varies with the source and growth state of cells. One isoacceptor, tRNALys6, observed in preparations of tRNA from some virus-transformed cells in culture, has been characterized by determining functional properties, cellular location, and its nucleotide sequence. tRNALys6 responds primarily to the lysine codon AAA, but it is not used efficiently in a wheat germ translational system in vitro. Compared with lysine isoacceptors 1, 2, 4, 5a, and 5, [3H]lysine appears in vivo in tRNALys6 with a delay of about 3 h. This delay may in part be a result of a less functional tRNA, but a compartmented state of tRNALys6 also appears to be important. tRNALys6 is associated with mitoplasts prepared from KA31 fibroblasts. The nucleotide sequence of tRNALys6 was determined by rapid postlabeling procedures involving limited hydrolysis in formamide, 32P-labeling of 5' ends of fragments with polynucleotide kinase, separation of the nested set of fragments in polyacrylamide denaturing gels, release of 5'-labeled nucleotides with RNase T2, and identification of the released nucleotides by chromatography on PEI cellulose. Confirmation of the positions of major nucleotides was done by using limited digestions by RNases of tRNALys6 labeled with 32P on the 3' terminus in a gel readout procedure. The nucleotide sequence of tRNALys6 differs from that of cytoplasmic lysine tRNAs and mammalian mitochondrial lysine tRNAs. It contains U*, an unidentified modified uridine occurring in the anticodon of some mitochondrial tRNAs. tRNALys6 appears to occur in very limited amounts, or not at all, in most cells unless stressed, but when present it is associated with mitochondria, although it is probably coded in the nucleus.

Animals↗
PubMed1983

Age-related and distributional changes in the trypsin inhibitor activity of bovine lens.

Age-related changes in the bovine lens trypsin inhibitor activity were measured by assaying water soluble extracts of 10 concentric slices from the periphery to the center of the lens. Inhibitor assays were carried out at pH 7.0 and 7.9 using prenatal, calf and mature lenses. The inhibitor at pH 7.0 remained constant throughout the lens but the pH 7.9 activity decreased sharply in the lens nucleus. This was particularly true for the prenatal and calf lenses. Agarose A-15 m gel filtration of the water soluble inhibitor activity showed a decrease in inhibitor in the alpha-crystallin region and a corresponding increase in inhibitor activity in the HMW protein peak. Inhibitor assays were carried out on the water insoluble fractions following solubilization in 6.0 M-urea. Little or no inhibitor activity was seen in the outer cortical fractions but the inner cortex and nucleus contained high levels of inhibitor activity in the water insoluble fraction with specific activities 7- to 10-fold higher than the comparable crude lens extracts. These data suggest that the lens inhibitor activity at pH 7.0 and 7.9 aggregate into a HMW complex and with time preferentially enter the water insoluble fraction. The distribution of a purified 5.5 K inhibitor protein between the water soluble and the water insoluble fraction was measured. In the periphery all of this inhibitor was in the water soluble fraction, but toward the center of the lens this inhibitor began shifting to the water insoluble fraction. Slices taken from the lens nuclear region showed that all the inhibitor was in the water insoluble fraction as detected by both activity measurements and SDS-PAGE.

Aging↗
PubMed1983

Purification and properties of a protein from bovine lens which inhibits trypsin and two endogenous lens proteinases.

An inhibitor of trypsin-like proteinases was isolated from the water-soluble proteins of bovine lens cortex. The inhibitor was purified by four simple procedures: the separation of the inhibitor fraction by Agarose A-1.5 m gel filtration, extraction with 2.5% TCA at 70 degrees C, ammonium sulfate precipitation of the TCA-soluble proteins and a final separation by gel filtration chromatography. This preparation was found to be homogeneous by SDS-PAGE with an approximate subunit molecular weight of 5500 daltons. Gel filtration separated the ammonium sulfate precipitate into an inactive high-molecular-weight peak which eluted in the void volume, and two peaks of approximately 40 000 daltons and 10 000 daltons. Both low-molecular-weight peaks gave a single 5500 dalton band on SDS-PAGE, but only the 40 000 dalton peak was active when concentrated and assayed with bovine trypsin. These data suggest that the inhibitor is present in multimeric forms in solution, but only the octamer appears to be active. Antibodies prepared against the purified inhibitor showed a single precipitin line, while no reaction was seen with an alpha-crystallin antiserum. Upon storage in solution all of the inhibitor became converted into a high-molecular-weight form which was completely inactive. SDS-PAGE dissociated the inhibitor aggregate into a major 44 000 dalton band along with several minor bands. Amino acid analysis showed that the purified inhibitor contains a very high content of hydrophobic residues. The lens inhibitor was effective in reducing the activity of trypsin, but complete inhibition was not seen even at high inhibitor levels. A rapid and complete inhibition was observed, however, with two endogenous trypsin-like proteinases isolated from the alpha-crystallin region.

Amino Acids↗
PubMed1983

Isolation and characterization of a 25K serine proteinase from bovine lens cortex.

A lens serine proteinase with trypsin-like specificity has been purified to homogeneity. This is one of two serine proteinases associated with the alpha-crystallin fraction from bovine lens. The purification was accomplished by a combination of isoelectric precipitation, activation to release the proteinase, gel-filtration and affinity chromatography. The purified proteinase showed a single protein band of 25 000 daltons on SDS-PAGE. A single protein band was also seen on non-denaturing gels which correlated with the location of the proteinase activity. The proteinase had a pH optimum between 7.2 and 8.2, was stable between pH 5.8 and 8.6 but was unstable above 40 degrees C upon heating. The enzyme lacked any requirement for metal ions and hydrolyzed arginine, lysine and asparagine substrates. alpha-Crystallin, and especially the B-chain of alpha-crystalline, was rapidly hydrolyzed by the proteinase compared to other lens crystallins. Metallo- and cysteine-proteinase inhibitors had no effect upon the enzyme activity whereas three different serine-proteinase inhibitors completely abolished all activity. A number of protein and peptide trypsin inhibitors also completely inhibited the lens 25K serine proteinase.

Animals↗
PubMed1980

The nucleotide sequence of two bovine lens phenylalanine tRNAs. Possible activation of a new phenylalanine tRNA gene during differentiation of lens cells.

Bovine lens fiber cells contain two major isoacceptors of phenylalanine tRNA, tRNA1Phe and tRNA2Phe whereas lens epithelial cells contain mainly tRNA2Phe (Ortwerth, B.J,, Yonuschot, G.R., Heidlege, J.F., Chu-Der, O.M.Y., Juarez, D., and Hedgcoth, C. (1975) Exp. Eye Res. 20, 417-426). Thus, the induction of a new species of tRNAPhe accompanies the differentiation of lens cells in vivo. These two species of tRNAPhe have been purified and their sequences have been analyzed. The results of the analyses show that the sequence of tRNA2Phe is identical with beef liver tRNAPhe while tRNA1Phe differs from tRNA2Phe by a guanosine to adenosine substitution at position 57. Therefore, the T psi CG sequence in the ribosylthymine loop of tRNA2Phe is replaced by T psi CA in tRNA1Phe, This observation demonstrates a case in which a change in sequence of tRNA isoacceptors accompanies cell development rather than changes in the level of post-transcriptional modification as noted in other systems (reviewed by Osterman L.A. (1979) Biochimie (Paris) 61, 323-342). The conversion of guanosine to adenosine in the ribosylthymine loop of lens tRNA1Phe suggests a possible activation of a new tRNAPhe gene during the differentiation of lens cells, although the possibility that this replacement may be catalyzed by a transglycosylase type enzyme has not been ruled out.

Animals↗