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J Horwitz

Publications and source records attributed to J Horwitz.

At least 73 records · Page 4Linked to original sources
PubMed1994

Chaperone-like activity of alpha-crystallin. The effect of NADPH on its interaction with zeta-crystallin.

alpha-Crystallin, a major structural protein of the ocular lens of vertebrates, has been characterized recently as a molecular chaperone (Horwitz, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10449-10453 and Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. Biol. Chem. 268, 1517-1520). While alpha-crystallins prevent the aggregation of various proteins denatured by heat or chaotropic agents, neither the mode of interaction between target proteins and alpha-crystallin nor the specific conformational requirements, if any, of the target protein are known. Here, we demonstrate that the ability of alpha-crystallin to prevent thermally induced aggregation of zeta-crystallin/NADPH:quinone oxidoreductase, an abundant crystallin of guinea pigs and camelids, is strongly dependent on the presence of the obligate cofactor (NADPH) of the target enzyme. zeta-crystallin in the absence of NADPH is readily aggregated at 41 degrees C, and alpha-crystallin added at a 1:1 (w/w) ratio offers very little protection. In contrast, in the presence of NADPH zeta-crystallin remains stable to 45 degrees C and with the addition of alpha-crystallin (1:1 (w/w)) is protected from aggregation even at 55 degrees C. Cibacron blue 3GA, a nonmetabolized pyridine nucleotide analog, which has very high binding affinity to zeta-crystallin had similar effects. NADH and NAD+, which are not bound by zeta-crystallin, had no such effect. Complex formation between alpha-crystallin and non-native zeta-crystallin was demonstrated in the presence of either cibacron blue 3GA or NADPH. Circular dichroism spectroscopy of zeta-crystallin in the presence and absence of NADPH or cibacron blue indicated that nucleotide binding was accompanied by a change in the protein's aromatic amino acid environment but that the secondary structure was unaffected. The data suggest that subtle change in the conformation of denaturing proteins can markedly affect the ability of alpha-crystallin to protect them from aggregation.

Animals↗
PubMed1994

Catalytic subunit isoforms of mammalian lens Na,K-ATPase.

To identify the Na,K-ATPase isoforms present in the mammalian lens, seven antisera were prepared to selected peptide sequences of the catalytic (alpha) subunit. Three antisera were prepared to peptide sequences at the N-terminus of the three sequenced rat alpha isoforms. There is < 53% sequence homology among the isoforms in this region. Three antisera were prepared to peptide sequences at the ouabain binding site in the extracellular loop between membrane spanning sequences 1 and 2 of the sequenced rat alpha isoforms; sequence homology among the isoforms in this region is < 69%. An antiserum was also prepared to the carboxyl terminal region of the alpha 2 rat isoform. The sequenced isoforms (rat and human) in this region are > 94% homologous. The results from stains of Western blots of SDS-PAGE separations of lens membranes are presented. Alpha 1 is the predominant isoform of the epithelium. It is not found in cells of the central epithelium but is present in cells located more toward the equator. Alpha 3 is the catalytic subunit of the central 43% of the epithelium. The lens fiber cell membranes have a catalytic subunit that is related to the alpha 2 isoform. In the fiber cell a 98-100 kDa band stains with the antiserum to the alpha 2 N-terminus and the antiserum to the alpha 2 ouabain site. The antiserum to the alpha 2 C-terminus does not stain the 98-100 kDa band. (Preliminary reports of these results were presented at the 1992 and 1993 meetings of the Association for Research in Vision and Ophthalmology).

Amino Acid Sequence↗
PubMed1993

The substrate specificity of brain microsomal phospholipase D.

Neurotransmitters activate a phospholipase D that is though to specifically hydrolyse phosphatidylcholine. This enzyme has a unique property known as transphosphatidylation: in the presence of an appropriate nucleophilic receptor such as an alcohol, phospholipase D will catalyse the production of phosphatidyl-alcohol. We have studied phospholipase D using an in vitro assay that uses [3H]butanol of high specific radioactivity (15 Ci/mmol) as an acceptor. In the presence of [3H]butanol and phosphatidylcholine, a microsomal membrane fraction from rat brain catalysed the production of phosphatidyl[3H]butanol. Phospholipase D activity was dependent upon the presence of a detergent; the optimal sodium oleate concentration was between 4 and 6 mM. The RF of the phosphatidyl[3H]butanol on t.l.c. was identical to the RF of the phosphatidylbutanol formed when [3H]phosphatidylcholine was incubated with 100 mM butanol. These data confirm the identity of phosphatidyl[3H]butanol. One important advantage of this assay is that the substrate does not need to be labelled. We have used this advantage to examine the substrate specificity of phospholipase D. Microsomal phospholipase D appears to hydrolyse phosphatidylcholine most efficiently. There is a relatively small but significant activity against phosphatidylethanolamine and phosphatidylserine, and there is no significant activity against phosphatidylinositol. As the head-group becomes more like choline, the phospholipid becomes a better substrate for phospholipase D. The addition of one methyl group leads to a large increase in activity. Fatty acid composition does not play a role in determining the substrate specificity. This assay should be useful in furthering our understanding of this important enzyme.

Animals↗
PubMed1993

The C-terminal region of alpha-crystallin: involvement in protection against heat-induced denaturation.

Recent studies have demonstrated that the alpha-crystallins can protect other proteins against heat-induced denaturation and aggregation. To determine the possible involvement of the C-terminal region in this activity, the alpha-crystallins were subjected to limited tryptic digestion, and the amount of cleavage from the N-terminal and C-terminal regions of the alpha-A and alpha-B crystallin chains was assessed using antisera specific for these regions. Limited tryptic digestion resulted in cleavage only from the C-terminal region of alpha-A crystallin. This trypsin-treated alpha-A crystallin preparation showed a decreased ability to protect proteins from heat-induced aggregation using an in vitro assay. Together, these results demonstrate that the C-terminal region of alpha-A crystallin is important for its ability to protect against heat-induced aggregation, which is consistent with the hypothesis that post-translational changes that are known to occur at the C-terminal region may have significant effects on the ability of alpha-A crystallin to protect against protein denaturation in vivo.

Alcohol Dehydrogenase↗
PubMed1993

J1-crystallins of the cubomedusan jellyfish lens constitute a novel family encoded in at least three intronless genes.

The transparent cellular eye lens of the jellyfish (Tripedalia cystophora) contains three major proteins called J1-, J2-, and J3-crystallins. Here we have isolated cDNAs encoding three novel 37-kDa J1-crystallin polypeptides (J1A, J1B, and J1C) sharing 84-98% identity in amino acid sequence among themselves. Each polypeptide is encoded in a separate gene lacking introns. In contrast to the striking similarity of the coding regions, the 5'- and 3'-untranslated sequences of the three J1-crystallin mRNAs are completely different, consistent with an ancient duplication of their genes. Thermostability experiments showed that J1-crystallins remain soluble at 50 degrees C, but precipitate at 60 degrees C, suggesting that these major lens proteins are neither heat shock proteins nor unusually heat-resistant as are many vertebrate crystallins. Although J1 mRNAs appear polyadenylated, no typical polyadenylation signal was detected in the cDNAs. Surprisingly, the only obvious similarities among the 5'-flanking regions of the three J1-crystallin genes are putative TATA boxes and several CAAT sequences, consistent with fewer evolutionary constraints on the regulatory sequences than on the coding sequences of these crystallin genes.

Amino Acid Sequence↗
PubMed1993

Alpha-crystallin, a molecular chaperone, forms a stable complex with carbonic anhydrase upon heat denaturation.

Alpha-crystallin, a major eye lens protein of vertebrates has been characterized as a molecular chaperone based on its ability to inhibit the aggregation of proteins undergoing thermal denaturation (Horwitz, J., Proc. Natl. Acad. Sci. USA 1992, 89, 10449-10453). To understand the mechanisms underlying this chaperone-like activity, the present study addressed molecular interactions between alpha-crystallin and its target proteins. Using carbonic anhydrase as a model target protein, we demonstrate complex formation between the 2 proteins upon heating, as assessed by the criteria of agarose gel electrophoresis, immunoprecipitation, ultrafiltration and gel filtration chromatography. The complex of alpha-crystallin and carbonic anhydrase is stable, at room temperature and at 4 degrees C, for over 18 hours, and is non-covalent in nature. The results also indicate that alpha-crystallin binds the early non-native form of the target protein.

Animals↗
PubMed1993

Structural and functional similarities of bovine alpha-crystallin and mouse small heat-shock protein. A family of chaperones.

alpha-Crystallin, composed of the subunits alpha A and alpha B, is a major vertebrate eye lens protein, accomplishing a structural role in maintaining lens stability and transparency. Both subunits also occur in low amounts outside the lens, where their precise function is unknown. They are structurally related to the small heat-shock proteins (HSPs), and increasing evidence indicates that they have also functional similarities with the small HSPs. To extend our insight into these structural and functional relationships, the mouse small HSP (HSP25) was compared with bovine alpha-crystallin, with respect to several known properties of the latter. We show that alpha-crystallin and HSP25 resemble each other in secondary structure and have similar stability toward urea dissociation at pH 7.0. Mixed polymers can be formed from any combination of alpha A-crystallin, alpha B-crystallin, and HSP25 subunits. Furthermore, we demonstrate that HSP25, like alpha-crystallin, can function as a molecular chaperone, by suppressing heat-induced aggregation of other proteins, and is an efficient inhibitor of elastase. Finally, HSP25 is found to be a substrate for protein cross-linking by tissue-type transglutaminase, like alpha B-crystallin. Our results thus corroborate that alpha-crystallin and the small HSPs have comparable functions, probably being involved in the protection of other proteins under conditions of stress.

Animals↗
PubMed1993

Enzyme activities and crystallin profiles of clear and cataractous lenses of the RCS rat.

Royal College of Surgeons (RCS) rats have hereditary retinal degeneration in association with posterior subcapsular opacities. Cataract formation is thought to be correlated with an increase in lipid peroxidation products in the vitreous (Zigler and Hess, 1985). In order to examine the possibility that parallel changes in enzyme activity are occurring within the lens, we analysed the activity of four key enzymes and the crystallin protein profile. We compared RCS rat lenses at three different stages of cataract formation to clear lenses of the nonpigmented RCS rat, lenses from pigmented RCS rat and to normal (Fisher) rat. Our data shows that concomitant with the appearance of the RCS cataract, the ratio of the crystallins beta 1, beta H and gamma to the total lens protein was reduced. The crystallin profile of a clear RCS lens was similar to that of a normal (Fisher) lens. No significant difference in the activity of the enzymes hexokinase and glucose-6-phosphate dehydrogenase (G6PD) was found among the lenses, however the activity of glutathione reductase and aldolase was reduced in the cataractous lenses.

Animals↗
PubMed1993

DL-propranolol inhibits lens hexokinase activity and affects lens optics.

A clinico-biochemical study indicated that the beta-blocker DL-propranolol may affect human lens epithelial hexokinase (HK) activity. In that study five key enzymes were analysed in 192 freshly excised human lens epithelia obtained during cataract surgery. In a large number of patients the epithelial HK was found to be inactive. Medical records of these patients showed widespread use of the drug DL-propranolol. In vitro experiments demonstrated a direct inhibitory effect of the drug on human lens HK activity. Lens refractive function was monitored during long term bovine lens culture experiments in which the potential cataractogenic agent was added to the culture media. DL-propranolol in a concentration of 0.1 mM reduced HK activity in bovine lens epithelium after 72 hr in organ culture and disrupted lens light focusing ability after 250 hr of incubation. Kinetic studies of HK inhibition suggested a competitive inhibitory effect of the drug on the enzyme.

Animals↗
PubMed1993

A biochemical characterization of the photophore lenses of the midshipman fish, Porichthys notatus Girard.

The present study is a biochemical characterization of the photophore lenses of the midshipman fish, Porichthys notatus, a species that bears 800 photophores distributed over the body surface. The biochemical properties of the photophore lenses were compared with those of the eye lens with which they share a similar developmental origin and analogous function. To achieve a high refractive index, the vertebrate eye lens has a relatively high concentration of structural proteins (20-50%, depending on species) and a simple protein composition, that is, relatively few proteins are synthesized in comparison to other tissues. Similarly, the photophore lenses of P. notatus had a relatively high protein concentration (average = 29%, n = 5) and approximately 60% of the total soluble protein was represented by two subunit species of 33 kD and 35 kD on denaturing polyacrylamide gels. The structural proteins of the eye lens are of two principle types: 1) beta and gamma polypeptides which belong to vertebrate lens-specific crystallin families, and, 2) enzymes recruited into the lens which take on the function of structural proteins. Here, we report that the two major photophore lens subunits of 33 kD and 35 kD are biochemically similar to each other, but are clearly distinct from any of the previously characterized crystallins. Therefore, we propose that photophore lenses appear to recruit a novel protein.

Amino Acid Sequence↗
PubMed1993

Comparison of the homologous carboxy-terminal domain and tail of alpha-crystallin and small heat shock protein.

The C-terminal domain and tail, which is the most conserved region of the alpha-crystallin/small heat shock protein (HSP) family, was obtained from rat alpha A-crystallin, bovine alpha B-crystallin and mouse HSP25. All three domains have primarily beta-sheet conformation and less than 10% of alpha-helix, like the proteins from which they are derived. Whereas the C-terminal part of alpha A-crystallin forms dimers or tetramers, the corresponding regions of alpha B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the alpha-crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.

Amino Acid Sequence↗
PubMed1993

Phagocytosis of outer segments by retinal pigment epithelium: phagosome-lysosome interaction.

We studied phagocytosis of rod outer segments (ROS) by the retinal pigmented epithelium (RPE) using rapid freezing, freeze-drying, and electron microscopic immunocytochemistry. Phagocytosis of photoreceptor outer segment tips by the RPE occurs daily, and in rats the shedding of these tips is light-entrained to a circadian rhythm. We studied the phagocytic process 5, 30, 90, and 150 min after light onset or after subjective light onset in rats entrained to a 12-hr dark-12-hr light cycle. Lysosomes were labeled with antibodies to cathepsin D, a major lysosomal enzyme responsible for opsin degradation. Phagosomes and phagolysosomes were recognized because of the lamellar structure of their photoreceptor-derived contents. We found a population of lysosomes that fuse with one another before they interact with phagosomes. This fusion can be triggered either by light or by endogenous circadian mechanisms. We also found that lysosome-phagosome interaction occurs after the ingestion stage is completed and that this interaction occurs in two steps. First, smaller lysosomes fuse with phagosomes. Subsequently, larger lysosomes appear to interact with phagosomes via pore-like or bridge-like structures. It is proposed that interchange of contents takes place through these structures.

Animals↗
PubMed1993

Distribution and activity of glutathione-S-transferase in normal human lenses and in cataractous human epithelia.

The distribution of glutathione-S-transferase (GST) activity was determined in frozen normal human lenses. The highest activity of GST was found in the peripheral and equatorial regions, whereas the lowest activity was found in the nucleus. Western blot showed that both mu and pi isoenzymes of GST were present in human lenses. This result is similar to that found in rat lenses. In addition, GST activity was analyzed in 50 lens epithelia which were obtained during cataract surgery. Twenty-seven lens epithelia showed no activity. Statistically significant association was found between cortical and mixed cortical--nuclear cataract and loss of GST activity. No association was found between pure nuclear cataract and loss of epithelial GST activity.

Adult↗