D. C. Watts

Phosphagen Kinases and Evolution in the Echinodermata

Molecular weights and distribution of kinases in echinoderms suggest that creatine kinase arose in the gamete of a common ancestral species and emerged in contemporary adult forms by parallel evolution.

Biological features of the new A2G--adr mouse mutant with abnormal muscle function.

A new mouse mutant (A2G-adr) with abnormal muscle function is described and has been compared with the 129 Re dystrophic mouse. The mutation, which is due to an autosomal gene defect, results in myotonic-like spasms, progressive muscle weakness and a reduced lifespan. Affected animals were consistently lighter than normal littermates; comparison of organ weights and organ-to-bodyweight ratios indicated a slower growth rate in the mutants.

Hybridization of matrix-bound MM-creatine kinase with BB-creatine kinase and arginine kinase

Dimeric rabbit muscle creatine kinase (MM-CK) was bound to CNBr-activated Sepharose 4B by one of its subunits (MM-CKA). Treatment of MM-CKA with guanidine hydrochloride released the unbound subunit to yield the matrix-bound monomer (M-CKB). M-CKB recombined with dissociated MM-CK soluble subunits to reconstitute a matrix-bound dimer (MM-CKC). M-CKB also associated with dissociated subunits of BB-CK from crude extracts of rabbit brain and of arginine kinase from sea cucumber muscle (MM-AK) to form the matrix-bound heterohybrids MB-CKC and M-CK/M-AKC, respectively. Guanidine hydrochloride gradient elution studies showed that MM-CKA, MM-CKC and MB-CKC were all dissociated at the same concentration of the denaturant (0.96 M), while the M-CK/M-AKC heterohybrid was less stable, dissociating at 0.5 M. The specific interaction between subunits of echinoderm and mammalian phosphagen kinases to form a hybrid enzyme of dual substrate specificity supports the view that these enzymes had a common evolutionary origin.

Osmotic control and urea biosynthesis in selachians

1. 1. Dogfish (Scyliorhinus canicula) gained weight and soon died when kept in a mixture of modified sea water and tap water (60% v/v) of freezing-point (f.p.) −1·20°C: when the f.p. of this mixture was restored to that (about) −1·96°C) of the sea water by adding urea (2·6 g to 100 ml) or sucrose (12·0 g to 100 ml), the fish survived and maintained weight. 2. 2. Carbamoyl phosphate synthetase (ATP: carbamate phosphotransferase (dephosphorylating), E.C. 2.7.2.5), activated by addition of manganese ions, was increased in liver homogenates from dogfish kept in dilute sea water, but decreased in those from fish kept in urea-dilute sea water; homogenates from fish in sucrose-dilute sea water showed “normal” carbamoyl phosphate synthetase activity. 3. 3. Carbamoyl phosphate synthetase activity in liver homogenates from four species of elasmobranchs, including S. canicula, was stimulated more effectively by Mn2+ than by Mg2+. 4. 4. The results confirm previously expressed views that whilst urea has an osmotic function, control of its concentration is not part of a mechanism by which marine elasmobranchs decrease osmotic stress. The apparent urea loss by dogfish in diluted sea water can be entirely explained by dilution of the body fluids as water is imbibed.

Glycolytic, Pentose-Phosphate Shunt and Transaminase Enzymes in Gastrocnemius Muscle, Liver, Heart, and Brain of Two Mouse Mutants, 129 J-dy and A2G-adr, with Abnormal Muscle Function

Abstract: Aldolase and phosphoglycerate kinase activity were markedly reduced in muscle from two mouse mutants, 129 J‐dy and A2G‐adr, with abnormal muscle development. The pentose‐phosphate shunt enzymes, glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase, were both greatly increased in the gastrocnemius of 129 J‐dy mice, but only the former was slightly increased in A2G‐adr muscle. Alanine and aspartate aminotransferase activities were normal or low in 129 J‐dy muscle but increased to approximately 200% in A2G‐adr muscle. Liver from 129 J‐dy mice showed increased activity of glucose‐6‐phosphate dehydrogenase. These findings are compatible with the well‐recognised lipid involvement in the 129 J‐dy mutant but indicate that an abnormality of amino acid metabolism in relation to energy supply is probably more important in the A2G‐adr mutant.

Inhibition of creatinine kinase by iodoalkanes: Further appraisal of the essential nature of the reactive thiol group

Abstract Creatine kinase (ATP: creatine N- phosphotransferase ) is completely inhibited by low molecular weigh iodoalkanes in a pseudo first order reaction. Analysis of this and other data suggests that covalent modification per se is not a sufficient criterion to establish whether or not an enzyme group is essential for catalysis.

Some fish muscle esterases and their variation in stocks of the herring (Clupea harengus L.). The nature of esterase variation

Abstract 1. 1. Electrophoretograms of α-naphthyl acetate and α-naphthyl butyrate esterases from white muscle of one-hundred Blackwater, ninety-nine Dunmore and fifty Ballantrae herring contained from two to ten bands of activity, but no single esterase band occurred in every fish. 2. 2. There was no evidence for allelic variation or multiple forms produced by subunit interaction but the relative intensities of bands 1 and 10 indicated that these could be inherited as full dose, half-dose or no dose. 3. 3. Esterase distribution was markedly different in the Dunmore and Blackwater stocks. The Ballantrae stock was intermediate in character. 4. 4. Different properties of the esterases were revealed by selective inhibition with N-ethylmaleimide, parachlormercuribenzoate, phenylmethylsulphonyl fluoride and physostigmine salicylate. Neuraminidase altered the electrophoretic mobility of bands 1, 3, 4 and 7. 5. 5. Esterase variation is suggested to result, in part, from the binding of sialic acid residues.

Gastrocnemius Muscle Lipids in Relation to Diet in Two Mouse Mutants, 129Re-dy and A2G-adr, with Abnormal Muscle Function

Abstract: The lipids of gastrocnemius muscle from normal and dystrophic (dy) mice of the Bar Harbor, 129Re strain were studied. Animals were fed diets containing either 3.1% or 1.1% of total calories as linoleic acid. Lipid analyses were also done on muscle from a new mouse mutant, A2G‐adr, which has abnormal muscle function, characterised by an arrested development of the righting response. These animals were fed the “high” linoleic acid diet only. Total lipid, triacylglycerol, and cholesterol were elevated in the 129Re‐dy irrespective of the diet, whereas A2G‐adr possessed significantly higher levels of cholesterol. Total phosphorus (μg P/g muscle) and cholesterol/phospholipid ratios were elevated in the dy strains only. Cardiolipin was raised in the dy (“low” linoleic diet) and adr muscle, whereas phosphatidylcholine was lower in the adr strain only. Linoleic acid esterified to phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine was elevated whereas arachidonic acid in phosphatidylserine was decreased in both mutants. Docosahexanoic acid (22:6) in all three dy phospholipids was decreased, independent of dietary treatment. The adr strain possessed normal levels of this fatty acid. The results specifically point to an abnormality in long‐chain polyunsaturated fatty acid metabolism in gastrocnemius muscle in the 129Re‐dy mutant; in the adr mutant they could reflect an abnormal increase in the number of muscle mitochondria.

The diagnostic value of plasma myoglobin levels in the adult and fetus at-risk for Duchenne muscular dystrophy.

In boys with Duchenne muscular dystrophy (DMD) plasma myoglobin levels remained approximately constant with age while creatine kinase (CK) activity progressively decreased. For carrier detection, plasma myoglobin level was found to be less reliable than CK activity. The myoglobin level was raised only in some of those subjects who also showed a raised CK activity and was normal in those with a normal CK activity. The myoglobin level in fetal muscle at 18-22 weeks gestational age was found to be low compared with adult skeletal muscle levels and, consequently, the myoglobin level in fetal plasma and in amniotic fluid was found to be negligible. It is concluded that measurement of myoglobin offers no advantage over CK for the investigation of any aspect of DMD.

Distribution, specificity and function of some proteases, general esterases and cholinesterases from several species of starfish

1. 1. The distribution and pattern of proteases, lipases and esterases in extracts from the pyloric caeca and skin of several species of starfish have been determined by starch gel electrophoresis coupled with specific staining. In Asterias rubens the cardiac stomach, intestine and rectal sacs were also investigated. 2. 2. Most protease activity originates in the caecae. The skin of A. rubens contained insignificant activity towards artificial protease substrates, but while that of Solaster papposus was about 10 per cent of the caecal level, it would not digest gelatin. The significance of these findings for external digestion is discussed. 3. 3. The proteases are first formed as inactive zymogens and become spontaneously active after extraction. The number of bands resolved by electrophoresis increases in the first day of extraction and then remains constant. 4. 4. Using artificial substrates and specific inhibitors, distinct trypsin and chymotrypsin-like activities could be demonstrated in tissue extracts. Their distribution in various species is reported. Two bands of trypsin, two bands of chymotrypsin and one band of p-nitrophenylacetate esterase could be resolved in extracts of A. rubens by electrophoresis on Sephadex blocks. Michaelis constants and other properties of the partially purified enzymes and those in crude extracts are reported. 5. 5. In caecal extracts tributyrin esterase showed two bands of activity on starch gel and these corresponded to the major bands of p-nitrophenyl acetate esterase activity. Triolein esterase activity was weaker and corresponded with the most electropositive of the other esterase bands. These esterase activities were distinct from the protease activity. A. rubens skin esterase was inactive with p-nitrophenyl acetate. 6. 6. Extracts were also investigated for the presence of enzymes similar to human acetylcholine esterase and serum cholinesterase. Evidence was obtained for an acetylcholinesterase which is tentatively suggested to be involved in nervous function.