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Biomedical subjects

C M Kay

Publications and source records attributed to C M Kay.

At least 217 records · Page 12Linked to original sources
PubMed1975

Purification and characterization of Aspergillus niger exo-1,4-glucosidase.

A specific exo-1,4-glucosidase (1,4-alpha-D-glucan glucohydrooase, EC 3.2.1.3) from Aspergillus niger has been partially purified and subsequently characterized by biochemical, physico-chemical and optical methods. Molecular sieve chromatography yields an enzyme with maximal activity at pH 4.2-4.5 close to its isoelectric point. Reduction and carboxymethylation leads to complete loss of activity and O-acetylation of 3 of the 13 tyrosine residues results in loss of 20 % of the activity. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that the native enzyme consists of two major components of molecular weights 63 000 and 57 500, respectively. Small amounts of dissociated material of molecular weight 28 000 and 16 000 as well as aggregates of the order of 100 000 are also present to the extent of 2-5% of the total potein. Following reduction and carboxymethylation under forcing conditions, the bands around 60 000 diminish and the 28 000-30 000, 16 000 and aggregate bands are dominant...

Amino Acid Sequence↗
PubMed1975

Molecular and biological studies on cardiac muscle calcium-binding protein (TN-C).

TN-C was purified from bovine cardiac muscle. In the absence of Ca-2+, cardiac TN-C has an intrinsic sedimentation coefficient of 1.93 S and a molecular weight of 18 000 daltons. Cardiac TN-C reverses the inhibitory effect of skeletal TN-I on the Mg-2+-activated ATPase of a skeletal synthetic actomyosin preparation in the presence of skeletal tropomyoson. Circular dichroism (CD) studies indicate that cardiac TN-C undergoes a major conformational change upon binding Ca-2+. A similar response is elicited by Sr-2+, whereas Mg-2+ has a much less pronounced effect. The presence of Mg-2+ does not alter the net effects of either Ca-2+ or Sr-2+. Cardiac TN-C is rich in acidic amino acid residues. UV absorption, near UV CD, and fluorimetric studies show that the protein lacks tryptophan and has a relatively high phenylalanine to tyrosine ratio. The results of this study invite direct comparisons with results reported for the skeletal muscle analogue of cardiac TN-C.

Adenosine Triphosphatases↗
PubMed1975

Rabbit cardiac myosin. I. Physical and chemical characterization of the native molecule.

Rabbit cardiac myosin, isolated from frozen tissue, was effectively purified by batchwise treatment with DEAE-cellulose in addition to suing cilution-precipitation techniques. An extensive experimental program was subsequently carried out with respect to the enzymic amino acid, optical and physicochemical properties of native cardiac myosin. This program has included the following: examination of the effects of pH and varying concentrations of ATP, CaCl2, MgCl2, and PCMB on its ATPase activity; measurement of its circular dichroic spectrum in solvent buffers, at different pH or containing ATP in the absence or presence of Ca-2+ or Mg-2+ ions; study of the concentration dependence of its viscosity and sedimentation velocity at low temperatures; and investigation of its molecular weight by the Archibald method and low- and high-speed sedimentation equilibrium. The results of these studies were consistent with the interpretation that cardiac myosin is comprised of highly asymmetric, semi-rigid molecules with a molecular weight in the order of 4.7 times 10-5, which display non-ideality even in solvent buffers of high ionic strength at neurtal pH. In addition, computer analysis of the high-speed sedimentation equilibrium data has provided evidence for the presence of a self-association reaction at low protein concentration. Even though the specif ATPase activity of cardiac myosin was found to be approximately one-third that reported for skeletal myosin in all cases, it was concluded, on the the basis of the essentially analogous physical and chemical properties of rabbit cardiac and skeletal myosin, that the two proteins are very similar in terms of molecular size, shape, and secondary structure.

Adenosine Triphosphatases↗
PubMed1975

Rabbit cardiac myosin. II. Proteolytic fragmentation with insolubilized papain.

The substructure of the cardiac myosin molecule was examined by the limited proteolytic digestion of the parent molecule with (dialdehyde starch)-methylenedianiline-mercuripapain, S-MDA-mercuripapain, at low temperatures and neutral pH, using moderate enzyme to myosin rations. Pertinent properties of the insoluble enzyme complex were also examined. Kinetic, ultracentrifugal, and chromatographic observations of the fragmentation process revealed that a single type of lytic reaction occurs during the early stages, predominately releasing heavy meromyosin subfragment 1 (HMM-S1) and myosin rods. With further time digestion, the rods are additionally cleaved yielding light meromyosin and HMM-S2, and HMM-S1 is found to be partially degraded. The major proteolytic subfragments were isolated, purified, and characterized with respect to their enzymatic, optical, amino acid, and physicochemical properties. Only HMM-S1 exhibited Ca-2+-activated ATPase activity, and at a level three- to fourfold higher than that of native myosin. Moreover, its hydrohynamic properties suggest that it is globular in structure. On the other hand, light meromyosin-A (LMM-A) (which consists mainly of rods), and HMM-S2 appear to be highly asymmetric, rigid, alpha-helical molecules devoid of the amino acid proline. Strong similarities were evident in all aspects upon comparison of these results with documented information concerning the skeletal system. On the basis of the physical and chemical properties of the proteolytic subfragments relative to that of native myosin, it was further concluded that the cardiac myosin molecule is a double-stranded, alpha-helical rod ending in tow subfragment 1 globules, of which only one may be enzymatically active at a time.

Adenosine Triphosphatases↗
PubMed1975

The isolation and characterization of the ATPase inhibitory protein (TN-I) from bovine cardiac muscle.

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated APTase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 +/- 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 +/- 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.

Adenosine Triphosphatases↗