Oliver Baum

High-performance membrane chromatography of serum and plasma membrane proteins

Porous discs made of poly(glycidyl methacrylate) were used for high-performance membrane chromatography (HPMC) of proteins. In model experiments, separations of standard proteins by anion-exchange HPMC using a DEAE disc were carried out. The influences of sample distribution and disc diameter and thickness on separation performance were studied. The separation disc allowed a scaling-up from analytical (diameter 10 mm) to semi-preparative (diameter 50 mm) dimensions. In an application study, separations with anion-exchange and affinity HPMC were carried out using different complex samples such as rat serum and plasma membrane proteins. In all experiments the results on poly(glycidyl methacrylate) discs were comparable to those achieved on adequate high-performance liquid chromatographic (HPLC) columns. However, the separations on HPMC discs could be carried out faster than corresponding separations on HPLC columns. The pressure drop on the discs was low even at high flow-rates. The experiments show that the poly(glycidyl methacrylate) discs used are especially suitable for the isolation of proteins and other biopolymers which occur in a diluted state in complex mixtures.

Rat dipeptidyl peptidase IV (DPP IV) exhibits endopeptidase activity with specificity for denatured fibrillar collagens

Dipeptidyl peptidase IV (DPP IV, CD 26) is an integral membrane serine protease exhibiting a well characterized exopeptidase activity. The present study shows that DPP IV also possesses a novel gelatinase activity and therefore endopeptidase activity, which was directly demonstrated by gelatin zymography. Protease inhibitor profile analysis showed that the endo‐ and exopeptidase activities of DPP IV share a common active site. Substrate specificity was detected for denatured collagen types I, II, III and V suggesting that DPP IV might contribute to collagen trimming and metabolism. On the basis of these data we propose that DPP IV and the recently sequenced gelatinolytic seprase (FAPα) represent a new subfamily of gelatinolytic integral membrane serine proteases.

Vascular endothelial growth factor is expressed in endothelial cells isolated from skeletal muscles of nitric oxide synthase knockout mice during prazosin-induced angiogenesis.

In skeletal muscles, angiogenesis can be induced by increases in wall shear stress. To identify molecules involved in the angiogenic process, a method based on the use of BS-1 lectin-coated magnetic beads was developed to isolate a cellular fraction enriched in microvascular endothelial cells which are directly exposed to wall shear stress. Using such cellular fractions from skeletal muscles of C57 mice in which angiogenesis was induced by administration with the alpha(1)-adrenergic antagonist prazosin, we found the concentration of vascular endothelial growth factor (VEGF) increased in correlation to the duration of the prazosin stimulus. In contrast, the angiopoietin-2/tie-2 system was not changed even after 4days of prazosin treatment. In neuronal nitric oxide synthase (nNOS) knockout mice, the VEGF concentration was also elevated after prazosin treatment but remained almost unchanged in endothelial nitric oxide synthase (eNOS) knockout mice. However, eNOS (and not nNOS) knockout mice expressed higher levels of VEGF under non-stimulated conditions as compared to C57 mice. These results suggest that VEGF produced in endothelial cells is involved in angiogenesis in skeletal muscles of mice responding to the administration of systemic vasodilators. NO derived from eNOS and nNOS may be an important regulator of the angiogenic response in skeletal muscles in vivo.

DISTRIBUTION AND QUANTIFICATION OF ALPHA1-INTEGRIN SUBUNIT IN RAT ORGANS

SummaryThe α1β1-integrin is known to be a receptor for collagen and laminin mediating cell-matrix interactions. A monoclonal antibody, 33.4, which specifically inhibits the α1-integrin-mediated in vitro cell-collagen binding of rat hepatocytes and hepatoma-derived A-cells (Löster et al., 1994), was used to purify by immunoaffinity chromatography the α1-integrin subunit from rat liver in large quantities for inducing a polyclonal antiserum. In immunoblot analysis on membrane extracts of several rat organs this polyclonal antiserum recognized only a 190 kDa-band, suggesting that it is highly specific for the α1-integrin subunit. A sandwich-ELISA with monoclonal antibody 33.4 and the polyclonal antiserum against the α1-integrin subunit, respectively, enabled the quantitative expression pattern of the α1-integrin subunit to be studied in different rat organs. With the exceptions of brain (not detectable) and muscle (low concentration), the α1-integrin subunit was detectable in almost all organs of the digestive, respiratory and urogenital system as well as in lymphatic organs. The highest relative concentrations of α1-integrin subunit were found in uterus, lung and spleen, whereas in seminal vesicle, stomach, parotid gland, epididymis, kidney and liver only modest concentrations were evident. The organ distribution and localization of α1-integrin subunit were studied by immunohistochemistry with monoclonal and polyclonal antibodies. Immunoreactivity was present in the plasma membranes of all smooth muscle cells, vascular endothelial cells of many organs and fibrocyte-fibroblast sheaths in the heart and kidney. Since these cells are in close contact with collagen-containing basal membranes as well as reticular fibrils, strong evidence exists that in rat tissues the α1-integrin subunit is expressed at sites where collagen is present and might be involved in vivo in cell—ollagen binding.

Nitric oxide synthase-1 is enriched in fast-twitch oxidative myofibers

Abstract. Nitric oxide synthase-1 (NOS-1) is found in high concentrations in skeletal muscles, where its synthesis product nitric oxide (NO) is reported to be involved in a number of processes, including the modulation of the oxidative metabolism of myofibers. Performing immunoblot analysis and quantification of formazan produced by its specific NADPH diaphorase activity, we found NOS-1 to be enriched in rat skeletal muscles with a high proportion of fast-twitch myofibers. Since these myofibers represent a metabolically heterogeneous subpopulation, we extended our investigation to the level of individual myofibers. Using serial sections we combined myosin heavy chain-based fiber-typing with quantitative succinate dehydrogenase histochemistry to determine three groups of fiber-types, comprising fast-oxidative, fast-glycolytic and slow-oxidative myofibers. Image analysis showed that NOS-1 diaphorase activity is significantly enriched in fast-oxidative myofibers compared with fast-glycolytic and slow-oxidative ones. In order to characterize potential biological effects of the fiber-type-specific enrichment of NOS-1, we performed cytochrome oxidase histochemistry in the presence of the NO donors NOC-9 and SNAP. Both NO donors reduced cytochrome oxidase activity in all myofibers investigated with almost identical semi-maximal inhibition rates, although fast-oxidative and slow-oxidative myofibers contained twice as much basal catalytic activity than fast-glycolytic ones. In summary, we suggest that the NOS-1/NO system of skeletal muscles exerts its biological role especially in fast-oxidative myofibers, since these myofibers express more NOS-1 than fast-glycolytic or slow-oxidative ones and also contain the highest concentrations of cytochrome oxidases as potential target molecules of NO.

The specificity of the histochemical NADPH diaphorase reaction for nitric oxide synthase-1 in skeletal muscles is increased in the presence of urea.

Nitric oxide synthase-1 (NOS-1) can be demonstrated in the sarcolemma region of myofibers in rodent skeletal muscles with the use of NADPH diaphorase histochemistry. Since other, especially intrafibrar enzymes also exhibit NADPH diaphorase activity, we tried to increase the specificity of the histochemical reaction for NOS-1. A qualitative and quantitative analysis was performed on cryostat sections of fast-twitch oxidative myofiber-rich tongue and fast-twitch glycolytic myofibers-rich tibialis anterior muscle derived from C57 mice and NOS-1 deficient knockout mice. All myofibers of both C57 mice and NOS-1 knockout mice contained significant intrafibrar NADPH diaphorase activity which was inhibited to almost background levels when 2 M urea was added to the incubation medium. On the other hand, myofibers of C57 mice but not of NOS-1-deficient knockout mice exhibited NADPH diaphorase activity in their sarcolemma region which was only weakly reduced in the presence of 2 M urea as was demonstrated by image analysis. Quantitative data on the activity of NADPH diaphorase(s) were obtained in situ by photometric analysis of formazan extracted from cryostat sections. The catalytic activity in tongue and tibialis anterior muscle was reduced in presence of 2 M urea to approximately 27% in C57 mice and to 7-17% in NOS-1 knockout mice, respectively. An in vitro NADPH diaphorase assay performed on homogenates of skeletal muscles also revealed an inhibitory effect of 2 M urea in both mouse strains and, additionally, indicated an upregulation of NADPH diaphorase activity in NOS-1 knockout mice. Finally, an immunodepletion analysis demonstrated that NOS-1 comprises 38% of the total NADPH diaphorase activity in tongue and approximately 59% in tibialis anterior muscle in C57 mice. In conclusion, we recommend the addition of 2 M urea to the incubation medium to increase the specificity of the NADPH diaphorase reaction to localise NOS-1 with the use of catalytic histochemistry.

Association of soluble guanylate cyclase with the sarcolemma of mammalian skeletal muscle fibers.

Previous investigations have shown that NO-producing nitric oxide synthase (NOS)-1 and CO-generating heme oxygenase (HO-2) are associated with the sarcolemma of skeletal muscle fibers in many mammalian species. Despite numerous roles ascribed to NO and possibly also CO in skeletal muscle, a specific receptor for both gases has hitherto not been found in myofibers. Therefore, in the present work the appearance of the alpha1, beta1 and beta2 subunits of soluble guanylate cyclase (sGC), the most commonly known receptor for NO and potentially also CO, was analysed in mammalian skeletal muscles using immunoblotting and immunohistochemistry. Immunoblotting with an antibody against the beta1 subunit of sGC revealed a band of 70 kDa corresponding to the molecular weight of this protein. Immunohistochemistry with antibodies against the alpha1, beta1 and beta2 sGC subunits showed that the larger part of positivity was present in the sarcolemma region of skeletal muscle fibers and colocalized with NOS-1 mainly in type II myofibers and with HO-2 in type I and type II myofibers. For the first time, sarcolemmal association of sGC and its colocalization with NOS-1 generating the sGC-activator NO and with HO-2 producing the potential sGC upregulator CO have been demonstrated in the present study. These results enable a better understanding of the role of NO and CO in myofibers and suggest a so far unknown molecular mechanism for the interaction of sGC with the sarcolemma.

Characterization of molecular aggregates of α1β1-integrin and other rat liver membrane proteins by combination of size-exclusion chromatography and chemical cross-linking

Abstract Many membrane proteins display their biological activity in molecular aggregates of interacting counterparts. The analysis of these aggregates remains difficult; especially intermolecular complexes of membrane proteins tend to dissociate or artificially aggregate during detergent extraction out of membranes. Thus, the existence of protein aggregates was investigated by two approaches. First, after modest detergent extraction, the presence of three well characterized rat liver membrane proteins, α 1 β 1 -integrin, dipeptidyl aminopeptidase IV (DPP IV) and cell-CAM 105 (CAM = cell adhesion molecule), in aggregates could be demonstrated when investigated by size-exclusion chromatography (SEC) under non-denaturating conditions. However, the applied detergents partially influenced the resolution of the separation reducing the ability to discriminate between native and artificial protein aggregates. To circumvent these problems, a second approach based on covalent cross-linking of native protein complexes by dithiobis(succinimidylpropionate) was combined with the performance of denaturating SEC. Under such optimized conditions the expression of α 1 β 1 -integrin as heterodimer and DPP IV as homodimer was confirmed. In addition, some high-molecular-mass complexes of all model proteins consisting of unknown components could also be detected. Taken together, non-denaturating SEC and chemical cross-linking in combination with denaturating SEC represent methodological approaches for the characterization of protein aggregates.

Skeletal Muscle Fibres Show NADPH Diaphorase Activity Associated with Mitochondria, the Sarcoplasmic Reticulum and the NOS-1-containing Sarcolemma

The subcellular appearance of NADPH diaphorase activity in different rat skeletal muscles has been analyzed. Both a sarcolemma-associated as well as a non-sarcolemma-associated NADPH diaphorase-dependent generation of formazan was observed. The sarcolemma-associated NADPH diaphorase staining appeared regularly in two manifestations: one observed in longitudinal sections as dotted costameres at the cell surface which accordingly appeared in transversal sections as rings surrounding the myofibre surface. At this site, nitric oxide synthase (NOS)-1 was located. The second sarcolemma-associated site of NADPH diaphorase staining was found as bundles of longitudinal-orientated stripes of hitherto unidentified origin. The non-sarcolemma-associated production of formazan was likewise manifested at two sites: the first was found regularly in longitudinal sections as intense sarcomere-like striations occurring parallel to the I-bands and indicating mitochondria. The second non-sarcolemma-associated NADPH diaphorase staining was realized as fine longitudinal filaments of variable occurrence connecting the mitochondria and presumably belonging to the sarcoplasmic reticulum. Attempts to identify single NADPH diaphorase(s) existing in skeletal muscles by incubation with specific inhibitors failed but showed the presence of two different subpopulations of NADPH diaphorases in myofibres: a urea-resistant fraction in the sarcolemma region containing NOS-1 and a non-sarcolemma-associated, urea-sensitive fraction depleted of NOS-1.

Chemical cross‐linking leads to two high molecular mass aggregates of rat α 1 β 1 integrin differing in their conformation but not in their composition

In order to detect protein interactions of the collagen/laminin receptor α 1 β 1 integrin, covalent chemical cross‐linking was performed with the homo‐bifunctional, amine reactive reagents DSS (disuccinimidylsuberate) and DSP (dithiobis(succinimidylpropionate)). After cross‐linking of the 190 kDa rat α 1 integrin subunit, immunoblotting revealed two additional, immunoreactive, high molecular mass complexes (M r 240/290 k). Generation of the 240/290 kDa aggregates depended on the presence of the intact tertiary protein structure. As shown with immunoaffinity purified proteins, the 240/290 kDa aggregates consist exclusively of α 1 and β 1 integrin subunits. No other cross‐linked proteins associated with the α 1 or β 1 subunit were detected. In contrast to the non‐cross‐linkable α 1 β 1 integrin, the 240/290 kDa aggregates presumably represent active forms of the adhesion receptor, because both bound in vitro to collagen I and IV. This ability of α 1 β 1 integrin to cross‐link and produce two additional high molecular mass forms is shared by rat α 9 β 1 integrin. Thus, the cross‐linking approach directly indicates that β 1 integrins occur in different conformations caused by variations in the folding and/or spatial arrangement of their subunits.