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S Zaremba

Publications and source records attributed to S Zaremba.

35 records · Page 2Linked to original sources
PubMed1990

Neuronal subsets express multiple high-molecular-weight cell-surface glycoconjugates defined by monoclonal antibodies Cat-301 and VC1.1.

Cat-301 and VC1.1 are monoclonal antibodies that recognize surface-associated molecules on subsets of mammalian CNS neurons. Earlier work demonstrated that Cat-301 recognizes a 680-kDa chondroitin sulfate proteoglycan (PG). VC1.1 has been shown to recognize 3 polypeptide bands on Western blot analysis; a major band at 95-105 kDa and additional bands at 145 kDa and 170 kDa. In the present report, we show that VC1.1 also reacts with a high-molecular-weight glycoconjugate. Immunoprecipitation experiments and biochemical characterizations indicate that Cat-301 and VC1.1 define at least 3 distinct high-molecular-weight antigens. The VC1.1 antigens react with antikeratan sulfate antibodies, while the Cat-301 antigens do not. By immunodepletion, we show that some VC1.1 antigens are Cat-301 positive, while others are Cat-301 negative. In addition, Cat-301-reactive proteoglycans are heterogeneous with respect to the presence or absence of VC1.1 epitopes. Double-label immunofluorescence studies with these 2 antibodies are consistent with the biochemical results and show that there are 3 classes of immunoreactive neurons in the cat CNS:Cat-301+/VC1.1+, Cat-301-/VC1.1+, and Cat-301+/VC1.1-. These results indicate that structural microheterogeneity exists among Cat-301 and VC1.1 high-molecular-weight glycoconjugates. This heterogeneity may be a reflection of the diverse neuronal phenotypes that are recognized by Cat-301 and VC1.1 in the mammalian CNS.

Animals↗
PubMed1989

Characterization of an activity-dependent, neuronal surface proteoglycan identified with monoclonal antibody Cat-301.

Monoclonal antibody Cat-301 was previously shown to recognize a surface-associated antigen on subsets of mammalian CNS neurons whose expression is regulated by neuronal activity early in an animal's postnatal life. We now present the partial purification and characterization of the Cat-301 antigen and demonstrate that it is a chondroitin sulfate proteoglycan. Extracellular localization of the Cat-301 epitope is demonstrated by staining live, intact neurons in situ. Extraction of the antigen from membranes in the absence of detergent indicates that it is either a peripheral membrane protein or a component of an extracellular matrix. The Cat-301 antigen migrates on Western blots of SDS gels with a molecular weight of integral of 680,000 dalton and is purified by DEAE chromatography and Sepharose gel filtration in 8 M urea (pH 4.9) buffer. The antigen is sensitive to chondroitinase ABC, indicating that it is a chondroitin sulfate proteoglycan. Furthermore, we provide strong evidence that the biochemically characterized antigen is indeed the histologically detected species by using a second antibody, Cat-304, that produces immunohistological staining patterns identical to those of Cat-301 and reacts with the purified antigen, but at a distinct epitope. Our earlier developmental findings and the present localization and biochemical results suggest that the antigen may play a role in the maturation of functional connections between neurons, perhaps through stabilization of axosomatic and axodendritic synapses.

Animals↗
PubMed1985

Limited proteolytic digestion of coated vesicle assembly polypeptides abolishes reassembly activity.

We have previously identified a fraction containing several assembly polypeptides (AP) that promotes reassembly of clathrin into vesicle-free coat structures [Zaremba S, Keen JH: J Cell Biol 97:1339, 1983]. The AP are prepared from purified bovine brain-coated vesicles by extraction with 0.5 M TRIS-HCl followed by Sepharose CL-4B column chromatography. Centrifugation in sucrose gradients under nonassembly conditions supports earlier observations suggesting that four active polypeptides in the AP preparation, of Mr approximately 110,000, 100,000, 50,000, and 16,500 are present in a discrete complex that is incorporated as a unit into reassembled coats. The 16,500-dalton polypeptide does not coelectrophorese with authentic bovine brain calmodulin and does not exhibit calmodulin's Ca2+-induced shift in electrophoretic mobility. When the partially purified AP fraction was digested with elastase, the Mr approximately 110,000 and 100,000 polypeptides were rapidly degraded with little or no effect on the Mr approximately 50,000 and 16,500 bands. This treatment abolished the in vitro coat-forming ability of the AP fraction and the loss of activity closely parallels the loss of the Mr approximately 100,000 band. Disappearance of the Mr approximately 110,000 and 100,000 bands is accompanied by the generation of new bands at Mr approximately 76,000 and 65,000. When the elastase-treated AP is examined by sucrose gradient sedimentation in nonassembly buffers, the new bands continue to cosediment with the Mr approximately 50,000 and 16,500 polypeptides. This indicates that the elastase digestion has cleaved off a fragment of the Mr approximately 110,000 and 100,000 bands, leaving behind a truncated, inactive AP complex. A protein kinase activity has been detected in coated vesicle preparations that utilizes the 50,000-dalton AP as its preferred substrate [Keen JH, Zaremba S: J Cell Biol 97:174a, 1983]. Elastase treatment does not abolish this activity, indicating that the kinase by itself is not sufficient for maintaining reassembly activity.

Animals↗
PubMed1985

A reliable method for assessing topographical arrangement of proteins in the chromaffin granule membrane.

An efficient and reliable method for preparing chromaffin granules and membranes for topographical analysis has been designed to meet the following criteria. Only labeling of truly intact organelles is analyzed for cytoplasmic surface probes. No resealing of open membranes occurs under specified reaction conditions. Saturation of available sites must be demonstrated. Problems in meeting these conditions are discussed, in particular the interference of granule lysate with labeling reactions.

Adrenal Medulla↗
PubMed1983

Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats.

A protein activity has been identified in extracts of coated vesicles that enables purified clathrin triskelions to reassemble in vitro into coat structures of uniform size. Coats formed in the presence of this preparation, regardless of the buffer system employed, are uniform in size with a mean diameter of 78 nm (+/- 5 nm SD) and a sedimentation coefficient (S20,w) of approximately 250S. Analysis of the reassembled coats on dodecyl sulfate acrylamide gels reveals that they have specifically incorporated three polypeptides from the preparation: those of Mr congruent to 52,000, 100,000, and 110,000. The 52,000-, 100,000-, and 110,000-mol-wt polypeptides are incorporated in molar ratios of 0.85, 1.11, and 0.26, respectively, per three clathrin monomers (equivalent to one triskelion). We therefore designate these as assembly polypeptides (AP). In contrast, coats formed from clathrin alone, under permissive buffer conditions, are larger (400S), more heterogeneous in size (101 nm +/- 15 nm SD), and are composed only of clathrin and its associated light chains. These biochemical and biophysical characteristics distinguish AP-reassembled coats from coats formed by triskelions alone. AP-reassembled coats can be isolated, dissociated, then reassembled in the absence of any other factors. This recycling indicates that all the information needed for reassembly is present in the coat-incorporated polypeptides themselves. Reassembly is stoichiometric and saturable with respect to both clathrin and AP concentration. In the presence of AP, significant coat reassembly occurs at clathrin concentrations as low as 0.06 mg/ml. AP-mediated reassembly proceeds at 4 degrees, 22 degrees, and 37 degrees C. Coat formation also proceeds efficiently at intracellular pH values (7.2-7.5) in the presence of AP. In its absence, reassembly does not occur at all above pH 6.7. In summary, AP promotes clathrin reassembly into coat structures of uniform size and distinctive composition under physiologically relevant salt, temperature, and pH conditions. In addition, the close similarity in size between AP-reassembled coats in vitro and coated membranes in the Golgi region in vivo raises the possibility that AP in the cell may be associated with this subpopulation of coat structures.

Animals↗
PubMed1983

Activity of nonspecific esterases in the marrow of mice following intraperitoneal administration of concanavalin A and PHA.

The activity of nonspecific esterases was studied in the bone marrow cells of mice after single intraperitoneal injections of Con A and PHA-M in the dose of 30 mg per 1 kg of body weight. Samples were collected 24, 72 and 120 hours after the injection of lectins. A cellular reaction to the activity of nonspecific esterases was tested after the method of Heckner using naphthol chloroacetate AS-D as an esterase substrate. In the control group the cells belonging to the granulocytic system except these being in mature forms, showed an apparent activity. A negative esterase reaction was revealed also in megakaryocytes, monocytes and lymphocytes. The administration of Con A intensified the esterase reaction in the cells belonging to the granulocytic series, except the mature forms. The esterase activity appeared in megakaryocytes, monocytes and lymphocytes. Most numerous changes in the activity were noted 72 hours following the injection. PHA-M was found to exert a reverse effect, weakening the esterase reaction in comparison to both the controls and the Con A--group.

Animals↗
PubMed1981

Transmembrane nature of chromaffin granule dopamine beta-monooxygenase.

The topographical arrangement of membrane-bound dopamine beta-monooxygenase in the chromaffin granule was investigated by proteolytic treatment and radiolabeling of intact granules or the isolated granule membranes. Topographical analysis was furthered by precipitating detergent extracts of treated membranes with specific antibodies to dopamine beta-monooxygenase, followed by electrophoresis of the immunoprecipitate in acrylamide-dodecyl sulfate. Trypsin or pronase treatment of intact granules cleaved small portions of membrane-bound dopamine beta-monooxygenase, resulting in proteins with slightly lower molecular weights. Membranes bearing these smaller forms of membrane-bound dopamine beta-monooxygenase were fully active enzymatically. Pronase treatment of isolated membranes cleaved all of the membrane-bound dopamine beta-monooxygenase antigenic sites from the membrane and also removed the enzymatic activity. Lactoperoxidase-catalyzed iodination revealed that membrane-bound dopamine beta-monooxygenase can be labeled in intact granules, but to a greater extent in isolated membranes. Quantitative peptide mapping showed that the distribution of label among tryptic peptides of membrane-bound dopamine beta-monooxygenase differed, depending on whether intact granules or isolated membranes had been labeled. The results of these experiments provide evidence that membrane-bound dopamine beta-monooxygenase is a transmembrane protein which has its active site exclusively on the intragranular face, plus a unique portion of its polypeptide chain exposed on the cytoplasmic face of the intact granule.

Adrenal Medulla↗