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The substrate specificity of porcine pepsin has been altered by site-directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro, Yaa is Glu. Leu, Gln or Lys, Nph is p-nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two- to fourfold higher kcat/Km, compared with wild-type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild-type pepsin cleaved a peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen alpha1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met cleaved site Ser-Phe at a rate 20-fold higher than the wild-type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu-Ser (similar to cathepsin D) and at a rate 23-fold higher than the wild-type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.

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Collagenous peptides containing the Ehrlich chromogen (EC), a trifunctional cross-link of proposed pyrrolic structure, were selectively isolated from a tryptic digest of bovine perimysial collagen by coupling to a diazotised support. Peptides containing pyridinoline (Pyr), another trifunctional cross-link but based on a 3-hydroxypyridinium ring, were isolated from the uncoupled material. The isolated cross-linked peptides were purified by chromatographic procedures and subsequently characterised by amino acid and sequence analyses. EC occurred in stoichiometric amounts in three-chained peptides derived from type I collagen cross-link regions. In contrast, Pyr was found in non-stoichiometric amounts in three-chained peptides where two of the chains were identified as the 76 amino-terminal residues of the α1 (III) collagen chain. The third chain in these Pyr cross-linked peptides was derived from the C-terminal helical cross-link region of either type III collagen or the corresponding region of type I collagen, with the former region predominating. These findings suggest that EC and Pyr cross-links of perimysial collagen are associated mainly with type I and type III collagen respectively.

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Collagenous peptides containing the Ehrlich chromogen (EC) were selectively isolated from a tryptic digest of bovine tendon by coupling to a diazotized polyacrylamide support. The isolated p-phenol-azo-EC peptides were purified and characterized by amino acid and sequence analyses. EC occurred in stoichiometric amounts in trimeric cross-linked chains originating from the known cross-link regions of type-I collagen. The major locus of the EC was alpha 2(I)Hyl-933 x alpha 1(I)Lys(Hyl)-9N x alpha 2(I)Lys(Hyl)-5N but it was also shown to occur at the loci alpha 1(I)Hyl-87 x alpha 1(I)Lys(Hyl)-16C x alpha 1(I)Lys(Hyl)-16C and alpha 1(I)Hyl-930 x alpha 1(I)Lys(Hyl)-9N x alpha 2(I)Lys(Hyl)-5N. After sequence analyses of the C-terminal helical cross-link region alpha 2(I)928-963, corrections are presented for residues 927, 930, 932 and 933 of the bovine alpha 2(I) chain. The collagen-associated EC is postulated to be a trisubstituted pyrrole formed by the reaction of the aldehyde form of a telopeptidyl lysine residue with a bifunctional keto amino cross-link. It is also proposed that when the telopeptidyl lysine residue is hydroxylated the above reaction will result in pyridinoline formation.

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The thermal stability of intramuscular collagen, as determined using differential scanning calorimetry, was measured in five muscles from 75 goats with known birth dates ranging in age from one day to 13 years. The collagen cross-link pyridinoline, and the collagen-associated, and putative cross-link, Ehrlich Chromogen were also measured. Five different muscles were examined and the effects of age compared to those found in the tendon of the longissimus dorsi muscle. The differences between intramuscular collagen and tendon collagen were found to be much greater than those between the intramuscular collagens of different muscles. Intramuscular collagen is more thermally stable than tendon collagen due to higher levels of heat-stable cross-links. However the increase in thermal stability of intramuscular collagen with age could not be explained simply in terms of the cross-links measured.

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A newborn child with neonatal neutropenia as a result of the presence of maternal IgG isoantibodies against neutrophil granulocyte blood group antigens is reported. Mechanism, diagnostics and therapy of the disease are discussed. The diagnosis not only has consequences for the child, but also for the mother and following pregnancies. A review of the most important causes of neonatal neutropenia is given.

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The hydrothermal isometric tension and thermal transition temperature of collagen were determined in tendons from three different calf muscles. The levels of the nonreducible collagen crosslink, pyridinoline, and the collagen-associated Ehrlich chromogen were also measured in the three tendons. The reducible collagen crosslinks, hydroxylysinonorleucine, dihydroxylysinonorleucine, and histidinohydroxymerodesmosine were measured in two tendons. The thermal properties and levels of crosslinks were found to vary considerably between the different tendons, and also at different sites in two of the tendons. A strong correlation was observed between the thermal transition temperatures and the hydrothermal isometric tensions of the nine tendon sites examined. Both thermal properties correlated with the concentration of both pyridinoline and Ehrlich chromogen. The analogous behavior of the collagen-associated Ehrlich chromogen and the pyridinoline crosslink supports the role of the Ehrlich chromogen as a nonreducible crosslink.

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Transverse tubules (t-tubules) were prepared from muscle by dissociation of intact triads during centrifugation in ion-free sucrose gradients. They were further purified by the removal of contaminating sarcoplasmic reticulum after loading with calcium phosphate. Purification was accompanied by enrichment in markers specific for t-tubules, e.g., nitrendipine binding sites. According to gel electrophoresis the purified t-tubules contained three major protein bands of 104, 70, and 30 kDa. When solubilized with detergents there was a two- to threefold increase in Mg2+-ATPase activity, and a corresponding increase in the 30-kDa protein band. The 104-kDa protein was shown to be a (Na+ + K+)-ATPase because of its phosphorylation by [gamma-32P]ATP in the presence of sodium ions. The orientation of the t-tubule membrane was predominantly inside-out.

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High pressure treatment (150 MPa for 10 min at 35°C) of rabbit longissimus dorsi muscles reduced the yield of a heavy microsomal preparation (8000-28 000 g) by approximately 90%. This was shown to be due to the loss of the sarcoplasmic reticulum, the major component of microsomal preparations, from untreated muscles. While microsomal preparations from pressure-treated muscles were shown to have lost all of their (Ca(2+), Mg(2+))-ATPase activity they retained all their Mg(2+)-ATPase (basal) activity. Sucrose density gradient centrifugation of these preparations yielded two fractions. By reason of its low density, high cholesterol content, high Mg(2+)-ATPase activity, SDS-gel protein profile and solubility in lysolecithin, the lightest fraction appeared to be derived from the transvers tubules. The heavier fraction, present in greater quantity but with a lower Mg(2+)-ATPase activity than the light fraction, was probably derived from the sarcolemma.

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A fluorometric assay for the K+-dependent phosphatase activity of the (Na+ + K+)-ATPase in both purified and membrane-bound forms is described. The assay utilizes 3-O-methylfluorescein phosphate as substrate and measures the fluorescence of the 3-O-methylfluorescein produced by hydrolysis of the substrate. The assay described is an order of magnitude more sensitive than the assay employing p-nitrophenylphosphate, the substrate most commonly used to measure this activity. The assay is also suitable for the specific measurement of (Na+ + K+)-ATPase activities in membranes which contain high levels of other ATPase activities.

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A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.

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The effects of both high voltage and low voltage electrical stimulation were studied in rabbit Longissimus dorsi muscles. The rate of fall of pH as well as the activities of phosphorylase a, phosphorylase kinase, and phosphorylase phosphatase were measured. The effects on the yield, ATPase activities, and calcium permeability of the sarcoplasmic reticulum (SR) were also measured. Although only high voltage stimulation increased the post-stimulation rate of pH fall, both types of electrical stimulation increased the phosphorylase a activity, apparently by increasing the activity of phosphorylase kinase and destroying a large part of the phosphorylase phosphatase activity. Electrical stimulation reduced the yield of SR and increased its basal ATPase activity. It also increased the ability of the SR to retain accumulated calcium. We conclude that the different rates of pH fall observed following the two types of stimulation are due to the differential effects of these treatments on one of the ATPase activities, probably the myofibrillar ATPase.

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The effects of high pressure (150 MPa) on the regulation of phosphorylase activity in pre-rigor rabbit muscles have been studied at 35° and 0°C. At 35°C muscle contracts, phosphorylase is activated and the muscle pH falls to 5·8 in 2 min. Coinciding with these changes, phosphorylase phosphatase activity falls rapidly, while phosphorylase kinase, although active for longer, loses its activity as the pH falls. Both of these enzymes are completely inactivated after 5 min under pressure, while phosphorylase still retains 80% of its activity under these conditions. The effects of pressure on the activities of these enzymes in white and red muscles of rabbits were compared, with a greater effect being observed in white muscles. At 0°C, muscles subjected to high pressure did not contract, but at this temperature the three enzyme activities (phosphorylase, phosphorylase kinase and phosphorylase phosphatase) were all lost at a greater rate than at 35°C, although the pH of the muscle did not fall below 6·5. The effects of high pressure treatment on isolated phosphorylase a and b and phosphorylase kinase were also studied at both 0° and 35°C and the results obtained closely paralleled those observed in whole muscle.

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