A HISTOCHEMICAL METHOD FOR DISTINGUISHING BETWEEN SIDE-CHAIN AND TERMINAL (α-ACYLAMIDO) CARBOXYL GROUPS OF PROTEINS

The specificity of the Barrnett-Seligman method for the histochemical demonstration of α-acylamido carboxyl groups (C terminal) of proteins is dependent on the conversion of such groups to ketones by the action of acetic anhydride and absolute pyridine. Studies on model compounds show that the side-chain carboxyl groups also react in the method and that most of the final color developed can be attributed to these carboxyls, rather than to the C terminal carboxyl groups. It is postulated that the side-chain carboxyls react by formation of mixed anhydrides in the presence of acetic anhydride and pyridine. This mixed anhydride then could link with a hydrazide to form a dihydrazide, which is capable of coupling with a diazo dye. Acetic anhydride treatment alone, without pyridine, also yields mixed anhydride. The mixed anhydride derived from the side-chain carboxyls can be destroyed by base, whereas the methyl ketone derived from the C terminal carboxyl is unaffected, and this treatment makes the method specific for C terminal carboxyl groups. Tissues treated in such a fashion demonstrate that all the color reaction obtained in the method is due to side-chain carboxyls, and that C terminal groups yield little or no staining as would be expected for "average" molecular weight proteins.     

The effect of chondromucoprotein on the solubility and peroxidatic properties of haemoglobin and methaemalbumin in aqueous solution and blood

1. Insoluble complexes, formed by electrostatic interaction between chondromucoprotein and chromoproteins (haemoglobin, methaemalbumin), were studied by measurement of precipitated pigment and by decrease in peroxidatic activity, maximum formation from aqueous solution occurring at pH 4·0–4·6. 2. Chondromucoprotein did not form complexes with plasma haptoglobins and haptohaemoglobins under these conditions, and high concentrations had no significant effect on colorimetric estimates of serum haptoglobin, although the peroxidatic activity of haemoglobinaemic serum was depressed owing to formation of chondromucoprotein–methaemalbumin complex. 3. The complexes formed by interaction between chondromucoprotein and plasma proteins contain two protein-bound biologically active components (plasminogen, haematin), as a result of co-precipitation after interaction between their carriers and chondromucoprotein. The possible presence of other biologically active trace components is discussed. 4. The results are related to complex-formation between other plasma proteins and chondromucoprotein, and possible implications arising from the complex-forming properties of tissue and urine chondromucoprotein are referred to. It is concluded that the inability of chondromucoprotein to form complexes with normal urine proteins is due to a deficiency of fibrinogen, β-lipoproteins and chromoproteins, which, in plasma, form a large proportion of the proteins involved in complex-formation.     

Some evidence confirming the specificity of Barrnett and Seligman's technique for demonstrating side-chain carboxyl groups in proteins

Summary There is some confusion in the literature as to whether the protein carboxyl groups demonstrable with Barrnett and Seligman's histochemical technique are C-terminal or side-chain. It seems that in the first step of this technique, hot mixtures of acetic anhydride and pyridine convert the commonly-occurring side-chain carboxyls into mixed acid anhydrides. That they do has now been confirmed; proteins in tissue sections treated with such acetic anhydride mixtures do not react with hydroxynaphthoic acid hydrazide after being left for a long time in either cold methanol or a cold solution of aniline in xylene. In this they resemble the acid anhydrides used for synthezising peptides in vitro. Thus all the lilac-reddish colours observable in tissue proteins on which the Barrnett and Seligman technique proper has been carried out can be ascribed to side-chain carboxyl groups.     

Collagen-induced platelet aggregation and release. I effects of side-chain modifications and role of arginyl residues

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen.These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.     

Collagen-induced platelet aggregation and release. II critical size and structural requirements of collagen

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen.These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.     

Reversed phase chromatographic analysis of Nostoc and Euglena isoleucyl-tRNAs aminoacylated in vitro in homologous and heterologous systems.

To investigate the mechanisms governing collagen interaction with blood platelets, the effects of side-chain modifications on collagen-induced platelet aggregation and release of serotonin were studied. Since many chemical modifications alter the ability of collagen to form fibers that, according to current theory, may complicate interpretation of data, we eliminated this possibility by using collagen stabilized in a native-type fibrillar structure by treatment with either glutaraldehyde or ultraviolet irradiation. Acetylation, methylation, succinylation, treatment with 2,4-dinitrofluorobenzene, 2,4,6-trinitrobenzene sulfonic acid or 1,2-cyclohexanedione, and deguanidination with hypobromite were used to modify collagen side-chain reactive groups: amino, carboxyl, hydroxyl and guanidino. Both unmodified monomeric dispersed and fibrillar collagen preparations initiated platelet aggregation and release, although the kinetics and magnitude of the response were different. Monomeric collagen which had been modified by deguanidination, methylation or succinylation, failed to polymerize in physiological conditions and did not induce platelet aggregation and release. However, none of the chemical modifications of stabilized native-type collagen fibers, except treatment with hypobromite or cyclohexanedione, had an effect on collagen-induced platelet aggregation and release. Both hypobromite and cyclohexanedione modified guanidino groups of arginyl residues. Results showed that the ability of a collagen sample to induce platelet aggregation and release of serotonin is dependent on the arginine content of fibrillar collagen. These data demonstrate that manipulation of amino, carboxyl and hydroxyl groups is unimportant as long as the native-type fibrillar structure is maintained, and that arginyl residues are directly involved in collagen-platelet interaction. Moreover, the data suggest that only the arginyl residues in the Y position of the tripeptide unit Gly-X-Y of collagen are responsible.     

Environmental enhancement of in vitro chondrogenesis. IV. Stimulation of somite chondrogenesis by exogenous chondromucoprotein

Proteoglycan complex extracted from embryonic cartilage (chondromucoprotein) with 4.0 M guanidinium chloride greatly stimulates in vitro somite chondrogenesis. In the presence of exogenous chondromucoprotein (CMP) which consists predominantly of proteochondroitin sulfate, there is a large increase in the amount of differentiating cartilage which can be detected visually in somite explants. There is a 2–3-fold increase in the amount of sulfated glycosaminoglycans (including chondroitin 4- and 6-sulfate) accumulated by somite explants supplied with exogenous CMP complex. These results are of potential significance, since during the period of interaction between the notochord or spinal cord and somitic mesoderm, the notochord and spinal cord synthesize and secrete proteoglycan.     

New histochemical techniques for the demonstration of carboxyl groups in mucosubstances

Synopsis The histochemical demonstration of the carboxyl groups of mucosubstances was studied by two methods at the light and electron microscopic levels. Conditions for activating carbohydrate carboxyl groups were elucidated from which a modified carbodiimide reaction procedure was worked out. With this procedure several acid mucosubstances could be demonstrated; some were characterized as sialomucoproteins. The mechanism of the carbodiimide reaction is discussed.A method is also discussed for increasing the electron opacity of Alcian Blue-stained mucosubstances with a sulphide-silver reaction.     

A fluorescent water soluable carbodiimide-2-hydroxy-3-naphthoic acid hydrozide reaction for the demonstration of carboxyl groups in proteins and mucosubstances.

The carbodiimide-2-hydroxy-3-haphthoic acid hydrazide reaction as developed by Geyer (1964) was used without subsequent diazonium coupling as a fluorescent method for the demonstration of carboxyl groups in both proteins and mucosubstances. The topological distribution of the fluorophore was similar to that reported by Geyer. Quantitative microfluorometric studies on cartilage sections revealed differences in detail between emissions in cartilage matrix mucoprotein as compared to the dense connective tissue associated with the perichondrium which consists principally of protein. It would also appear that the primary fluorescent emission of unstained preparations at 450 mm should be useful in microfluorometric determinations of proteins.     

Synthesis of beta- and gamma-fluorenylmethyl esters of respectively N alpha-Boc-L-aspartic acid and N alpha-Boc-L-glutamic acid

The orthogonal synthesis of N alpha-Boc-L-aspartic acid-gamma-fluorenylmethyl ester and N alpha-Boc-L-glutamic acid-delta-fluorenylmethyl ester is reported. This is a four-step synthesis that relies on the selective esterification of the side-chain carboxyl groups on N alpha-CBZ-L-aspartic acid and N alpha-CBZ-L-glutamic acid. Such selectivity is accomplished by initially protecting the alpha-carboxyl group through the formation of the corresponding 5-oxo-4-oxazolidinone ring. Following side-chain esterification, the alpha-carboxyl and alpha-amino groups are deprotected with acidolysis. Finally, the alpha-amino group is reprotected with the t-butyl-oxycarbonyl (Boc) group. Thus aspartic acid and glutamic acid have their side-chain carboxyl groups protected with the base-labile fluorenylmethyl ester (OFm) and their alpha-amino groups protected with the acid-labile Boc group. These residues, when used in conjunction with N alpha-Boc-N epsilon-Fmoc-L-lysine, are important in the formation of side-chain to side-chain cyclizations, via an amide bridge, during solid-phase peptide synthesis.     

A histochemical study of chondroid tissue in Limulus and Octopus

Summary In a comparative histochemical study of Octopus and Limulus chondroid tissues and mouse tracheal cartilage, it was demonstrated that both invertebrate chondroids behave as less acid mucopolysaccharides than those in mouse cartilage. Octopus chondroid was less reactive than Limulus chondroid. Cetyl pyridinium chloride blockage of toluidin blue metachramasia could be unblocked in mouse cartilage by an hour's treatment in 0.5 M KCl, but even extended periods in 2.0 M KCl failed to accomplish this in the invertebrate chondroids. With a number of methods for demonstrating proteins, Octopus chondroid was less reactive than Limulus chondroid. Limulus chondroid matrix was intensely stained by methods for demonstrating protein thiol groups, but essentially no staining was observed in Octopus chondroid matrix. These studies indicated that the composition and/or structure of matrix material in the invertebrate chondroids differ from one another, and in turn differ from conditions in vertebrate hyaline cartilage.This work was supported by a grant (HD-1499-04) and a Career Development Award (5-K3-6176-04) from the National Institute of Child Health and Human Development of the U.S. Public Health Service.A contribution of the Sea Horse Key Marine Laboratory of the University of Florida.     

Chondroclasts in osteoneogenesis

Allogenic, demineralized bone powder (DBP) was implanted into rat rectus abdominis muscle to induce osteoneogenesis. The main induction steps are invasion of the implant by host mesenchyme cells, differentiation of cartilage, invasion by blood capillaries or angiogenesis, differentiation of osteoblasts and bone marrow. The result is the formation of a cancellous ossicle. Giant polykarions appear in the implant after calcification of the cartilage matrix. As the DBP particles are not resorbed in the implant, these polykarions could either be foreign body giant cells brought about in reaction to foreign matrix or chondroclasts which resorb the cartilage. The results obtained by histological and histochemical methods (McNeals-von Kossa stain, tartrate resistant acid phosphatase reaction), as well as ultrastructural studies, lead to the conclusion that these large polynucleated cells are chondroclasts.     

Dynamic changes in acid mucopolysaccharides during mineralization of the mandibular condylar cartilage

Summary Sequential histochemical changes related to acid mucopolysaccharides (AMPS) were studied in the calcifying cartilage of the mandibular condyle. Non-decalcified, 1 Eponembedded sections were subjected to a variety of histochemical procedures. The results indicate that AMPS are synthesized and secreted mainly by hypertrophic chondrocytes in the premineralizing zone. Within the matrix at the mineralization front the AMPS complexes are apparently degraded by lysosomal enzymes to yield a highly anionic fraction which is maintained in the matrix. This fraction could function as the site for mineralization and cationic dye reaction which allows for histochemical visualization.Supported in part by Grant DE 00163 from the National Institute of Dental Research, U.S.P.H.S.     

Fluorescence of 2-hydroxy-3-naphthoic acid hydrazide derivatives of side-chain carboxyl groups of proteins

Summary Sections processed through the first part of the Barrnett and Seligman (1958) procedure, which is considered to be selective for side-chain carboxyl groups of proteins, are fluorescent when exposed to incident blue light. The results of a number of tests suggest that the fluorescence can be ascribed principally to binding of 2-hydroxy-3-naphthoic acid hydrazide (HNAH) by mixed acid anhydride derivatives of side-chain carboxyl groups of proteins. Thus, the procedure appears to be a fluorescent counterpart of the bright-field method of Barrnett and Seligman.     

Evidence that endogenous cyclic AMP does not modulate serum sulfation factor action on embryonic chicken cartilage

The role of cartilage cyclic AMP as a mediator or modulator of serum sulfation factor (SSF) action on embryonic chicken cartilage was assessed. Media with concentrations of rat serum (7.5%) sufficient to maximally stimulate chondromucoprotein synthesis as measured by ³⁵SO₄ incorporation did not change cartilage cyclic AMP levels. Theophylline (2.5mM) doubled cyclic AMP in cartilage incubated in media but had no effect on ³⁵SO₄ incorporation. In media containing 5% rat serum, theophylline at 0.5, 1.5 and 2.5mM caused a similar and significant rise in tissue cyclic AMP but only 2.5mM inhibited SSF stimulated ³⁵SO₄ incorporation. The data indicate that cartilage cyclic AMP neither mediates nor modulates SSF action on cartilage chondromucoprotein synthesis.     

Covalent immobilization of proteins and peptides for solid-phase sequencing using prepacked capillary columns

The use of prepacked capillary columns for immobilizing proteins and peptides for solid-phase Edman degradation is described. Capillary tubes with an internal volume of about 30 microliters are filled with glass beads bearing isothiocyanato groups (DITC-glass), aminophenyl groups (AP-glass), or aminoethylaminopropyl groups (AEAP-glass) and are sealed with porous plugs. Proteins or peptides in appropriate buffers are introduced into the columns by capillary action and are covalently coupled to the glass beads, either by reaction of lysine side-chain amino groups with DITC-glass, by carbodi-imide-mediated reaction of carboxyl groups with AP-glass, or by reaction of homoserine lactone groups with AEAP-glass. Optimization of attachment conditions is described. The capillary columns are loaded into the sequencer and, when sequencing has been completed, are discarded. This technique greatly simplifies polypeptide immobilization and is suitable for microsequencing (less than 50-1000 pmol) or macrosequencing (1-50 nmol).     

Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein

A glycoprotein that exhibits alkaline phosphatase activity and binds Ca2+ with high affinity has been extracted and purified from cartilage matrix vesicles by fast protein liquid chromatography. Antibodies against this glycoprotein were used to analyze its distribution in chondrocytes and in the matrix of calcifying cartilage. Under the light microscope, using immunoperoxidase or immunofluorescence techniques, the glycoprotein is localized in chondrocytes of the resting zone. At this level, the extracellular matrix does not show any reaction. In the cartilage plate, between the proliferating and the hypertrophic region, a weak immune reactivity is seen in the cytoplasm, whereas in the intercolumnar matrix the collagen fibers appear clearly stained. Stained granular structures, distributed with a pattern similar to that of matrix vesicles, are also visible. Calcified matrix is the most stained area. These results were confirmed under the electron microscope using both immunoperoxidase and protein A-gold techniques. In parallel studies, enzyme activity was also analyzed by histochemical methods. Whereas resting cartilage, the intercellular matrix of the resting zone, and calcified matrix do not exhibit any enzyme activity, the zones of maturing and hypertrophic chondrocytes are highly reactive. Some weak reactivity is also shown by chondrocytes of the resting zone. The observation that this glycoprotein (which binds Ca2+ and has alkaline phosphatase activity) is synthesized in chondrocytes and is exported to the extracellular matrix at the time when calcification begins, suggests that it plays a specific role in the process of calcification.     

Modifications of connective tissue matrices by an enzyme extracted from cartilage

Summary The degradative properties of an enzyme extracted from bovine costal cartilage were studied histochemically, autoradiographically, and electronmicroscopically. Previous work had indicated that this enzyme catalyzes the degradation of proteinpolysaccharide, light fraction (PP-L) from bovine costal cartilage to a proteinpolysaccharide with a lower protein content than the original substrate. Attempts were therefore made to ascertain if this protease was species specific and if proteinpolysaccharides other than those present in cartilage were susceptible to enzyme digestion. To this end, the effect of the enzyme on human fetal cartilage and jaws and the epiphyseal plates of neonatal and postnatal rats was studied. Furthermore, ³⁵S-H₂SO₄ was injected into pregnant rats, the fetuses were removed and sections of them were digested with the enzyme and then autoradiographed. In the histochemical experiments Alcian Blue with MgCl₂ was used for the staining of tissue polyanions and Bromphenol Blue for the detection of free basic groups. Finally, the limbs of 20 day-old rats were utilized in electronmicroscopic studies.Within the limitations of the techniques utilized the results obtained elucidated the following characteristics of the action of the enzyme on the tissues: 1. the products resulting from the action of this protease are more soluble than the proteinpolysaccharides originally present in the tissues, 2. the extent to which the enzyme affects the tissue depends directly on the state of maturation of the tissue and, therefore, on the state of aggregation of the matrix, and 3. the enzyme is not species or tissue specific.Autoradiograms of sections incubated with the enzyme also indicate an enhanced solubility of proteinpolysaccharides.The effects of the enzyme on ultrathin sections were manifested in an increased affinity for phosphotungstic acid staining of the extrafibrillar matrix and of particles in the lacunae of degenerating chondrocytes. The latter showed a distribution similar to that of particles seen in semithin sections stained with Azure A.Supported by Grant DE-02110-03-04 of the National Institutes of Public Health, Bethesda, Md.     

A method for histochemical demonstration of protein-bound amino groups by light microscopy

We have established an efficient method for the histochemical demonstration of protein-bound amino groups by light microscopy, using a ninhydrin or alloxan-thiocarbohydrazide-silver proteinate (NHY or ALX-TCH-SP) sequence followed by a physical development (PD) procedure. As a result of the present experimental studies on Carnoy's solution-fixed paraffin sections of a series of rat tissues, including three types of major salivary glands, liver, pancreas, stomach, duodenum, jejunum, colon, kidney, prostate, and spleen, the sensitivity and specificity of the new method were found to be sufficient. In the tissues tested, protein-bound amino groups were visualized by distinct brownish or blackish reaction products. Comparisons of the particular method with the NHY or ALX-Schiff methods employed hitherto have substantiated the fact that the former method leads to apparently higher visibility of reaction products than the latter.     

Sites of sulfatation in the chondrocytes of the articular cartilage of the rabbit

35S sulfate uptake by the articular cartilage chondrocytes, from biopsies of rabbit, have been studied by high resolution autoradiography. The Golgi apparatus, rough endoplasmic reticulum, cytosol, cytoplasmic membrane and extracellular space were considered as cell compartments in the quantitative analysis of the autoradiograms. The results obtained show: 1) a high activity of radiosotope incorporation in the Golgi apparatus; 2) a fast rhythm of transfer of the substances labelled in the Golgi apparatus to the cell membrane; 3) significant labelling of the rough endoplasmic reticulum, throughout the experiment. It is concluded: 1) The grains observed in the rough endoplasmic reticulum show a significant radioisotope uptake on this level, and this evidence some sulfotransferase activity. 2) The high 35S sulfate uptake level which is observed in the Golgi apparatus demonstrates that the highest sulfotransferase enzyme activity is located in this cell area, thus showing that the "early" sulfation that began in the rough endoplasmic reticulum was completed by a "late" sulfation in the Golgi apparatus. It is here that complete chondromucoprotein building takes place before being excreted. 3) The high transfer level of the labelled substances from the Golgi apparatus shows that the sulfated product secretion for building the cartilage matrix takes place rapidly since a great label increase can be already observed at the beginning of the chase period in the outer surrounding area of the chondrocyte membrane.