George Kalnitsky

THE RELATION OF STRUCTURE TO ENZYMATIC ACTIVITY IN RIBONUCLEASE

Before proceeding with our experiments relating structure to function in the enzyme bovine pancreatic ribonuclease (RNase) and before attempting to isolate an active fragment of KNase containing an active center (if it exists), we felt it necessary to improve the methods of assaying for RNase. The first part of this presentation, therefore, will deal with the assay procedures employed and with the effects of salts and urea on these procedures and on the substrate, yeast sodium ribonucleate (YRNA). The second part will attempt to provide answers to the following questions relating the structure of the ribonuclease molecule to its enzymatic activity: (1) Are the 4 disulfide bonds necessary for enzyme activity? (2) Is the tightly coiled form of the molecule essential for its activity? ( 3 ) Is the whole, intact protein essential for its catalytic activity?

The catalysis of the oxidation of some dihydroxybenzene derivatives by various metallic ions

Mn++, Co++, Cu++ and to a certain extent Fe++ catalyzed the oxidation of epinine, 3-hydroxytyramine, N-methyl DOPA, DOPA and catechol, in approximately decreasing order of activity. Tyrosine was not oxidized. In general, Mn++ was the most effective catalyst. During the oxidation of each substrate, CO2 was evolved and hydrogen peroxide was formed. The metallic ion-catalyzed reaction differs from the the tyrosinase-catalyzed oxidation of these compounds in many ways, chief of which are the optimum pH, the rate of oxidation, the specificity toward substrates, the amount of oxygen absorbed per molecule of substrate oxidized and the formation of hydrogen peroxide.

The activity of ribonuclease after digestion with carboxypeptidase

Abstract On incubating ribonuclease (RNase) with a crystalline carboxypeptides preparation, a number of amino acids were liberated. Of the 17 amino acids in the RNase molecule, all except cystine and proline have thus far been quantitatively determined as products of this hydrolytic reaction with no loss in the original activity of the RNase. During the early phases of the digestion, the activity of the RNase was increased. A separation of the digested from the undigested RNase molecules has been achieved by chromatography of the digests in secondary-butyl alcohol: water (3:1) at 3° C for 15 hours. The native RNase molecules did not migrate under these conditions and were quite stable, whereas the digested RNase molecules migrated and were less stable. The data obtained with this procedure correlates reasonably well with the amino acid determinations as a measure of the per cent of the RNase molecules digested. The extent of digestion thus far achieved approximates 80% of the RNase molecules present, with no loss in enzymic activity. It is calculated that approximately 15% of the amino acid residues which constitute RNase are not essential for the enzymic activity, but appear to be necessary for the stability of the molecule.

Partially hydrolized ribonuclease with enzymic activity

Abstract Crystalline ribonuclease (RNase) was digested with crystalline carboxypeptidase untreated with di iso propylfluorophosphate. The elution patterns of the digested RNase from Amberlite IRC-50 (XE-64) were found to be constant unless inactivation occurred during the digestion period. The quantitative differences between the elution patterns of fully-active and partially-inactivated RNase (measured by protein determination) correlated with the per cent inactivation of the enzyme (measured by RNase assay). With approximately 70% of the enzymic activity remaining, RNase may be divested of 3 of its 9 moles of valine, 3 to 4 of the 12 moles of alanine, at least 1 of the 15 moles of aspartic acid 1.3 of the 3 moles of phenylalanine, 1.5 of the 2 moles of leucine, 2 of the 3 moles of isoleucine and 2 to 4 of the 4 moles of histidine. The liberation of both leucine residues in the RNase molecule is not clearly related to enzyme inactivation, whereas the liberation of 1 of the 4 histidine residues and 1 or 2 of the 3 isoleucine residues appears to be associated with inactivation of the enzyme. From these data, and from the partial structural formula of the RNase molecule made available by the work of Hirs, Moore and Stein , it is estimated that the major portion of the RNase molecule is not essential for its catalytic activity, under the conditions of these experiments, and that the “active center” of this enzyme is located relatively closer to the C-terminal end of the molecule.

Identification of Sterols on Filter Paper and Their Separation by Paper Partition Chromatography.

Summary Methods for identification and separation of several non-keto steroids in 10 μg quantities on paper chromatograms are described.

Some properties of the manganese and copper catalyzed oxidation of catechol and some other ortho-dihydroxybenzene derivatives☆

Abstract The type of buffer used had a profound effect on the Mn ++ catalyzed oxidation of the ortho dihydroxybenzene compounds used. Veronal or tris (hydroxymethyl) amino methane were found to be the most suitable buffers for the study of these reactions. Glycine, phosphate, citrate and borate buffers inhibited the reaction. The optimum Mn ++ concentration for the 3-hydroxytyramine and DOPA oxidation was 2.5ṡ10 −4 M . Higher Mn ++ concentrations increased the rate of oxidation only slightly. The optimum Cu ++ concentration was found to be 2.5ṡ10 −3 M . Under the conditions of these experiments chelates were formed between Cu ++ and catechol, DOPA, N-methyl DOPA and 3-hydroxytyramine. No chelation was detected between Mn ++ and these compounds. Catechol was oxidized by MnO 2 and KMnO 4 .

Differential Centrifugation Studies of Guinea Pig Lung Proteases

Summary Five subcellular fractions were isolated from guinea pig lung homogenates by differential centrifugation. These fractions were defined biochemically by the analysis of 18 enzymes representing different subcellular compartments. Succinate dehydrogenase and cytochrome oxidase distributed with the heavy mitochondrial fraction, while N-acetyl-β-glucosaminidase, acid p-nitrophenylphos-phatase, cathepsins A, Bl, and D, dipeptidyl-peptidases I and II, and elastolytic esterase distributed with the light mitochondrial fraction. Alkaline p-nitrophenylphosphatase, glucose 6-phosphatase, dipeptidylpeptidase IV, neutral protease, and alkaline protease all demonstrated a “microsomal” enrichment. In the cytosol were found lactate dehydrogenase, dipeptidylpeptidase III, and a dipeptidase. The lung subcellular fractions were heterogeneous with cross-contamination between the heavy mitochondrial, light mitochondrial, and “microsomal” fractions. The enzyme distributions noted were similar to those found in other tissues.

Highly restricted specificity of the serine proteinase aspergillopeptidase B.

Abstract The oxidized A- and B-chains of bovine insulin were hydrolyzed with a homogeneous preparation of Aspergillopeptidase B and the resulting peptides were determined by amino acid analysis after separation by column chromatography. The enzyme showed a remarkably narrower specificity than previously reported. At 0°C the enzyme cleaved essentially only the bond Glu-Asn (position 17–18) in the A-chain and the bond Leu-Tyr (position 15–16) in the B-chain. The enzyme, therefore, would appear useful in producing limited proteolysis for structure-function studies.

PROTEOLYTIC ACTIVITY IN BOVINE DENTAL PULP.

Abstract The autolysis of bovine dental pulp by endogenous proteolytic enzymes has been demonstrated by means of a colorimetric ninhydrin method. The activity is heat-labile and acts optimally at pH 3.0. Acid-denaturated hemoglobin proved to be a useful exogenous substrate for the cathepsins of bovine dental pulp. Optimal hemoglobin hydrolysis was shown to occur at pH 4.0. The activity was concentrated fivefold by heat fractionation under closely controlled conditions (pH 5.0, 56 ° C, 60 seconds). Further concentration of the activity was obtained by Sephadex G-75 filtration and hydroxyapatite column chromatography. The total hemoglobin-splitting activity present in bovine dental pulp was separated into four active fractions on the hydroxyapatite column. Two of the fractions differed considerably in their pH optima, which were observed to be at pH 3 and pH 5, respectively. The relative purification of the pH 3 enzyme was 80 when compared with the starting material.

Muscle ribosome detachment factor. Does it have a role in the pathogenesis of Duchenne muscular dystrophy

A protein that detaches ribosomes from rough microsomal membranes (“detachment factor”) (DF) was isolated from the cytosol fraction of rat and human muscle. The procedure of isolation included differential centrifugation, precipitation with ammonium sulfate and column chromatography with Sephadex G‐100. The protein which is not completely homogenous, has a molecular weight of 50,000–60,000 daltons, is heat labile and has an optimum pH at 7.4–7.6. The DF activity of the protein is inhibited by soybean trypsin inhibitor (73%), pepstatin (67%), and leupeptin (42%), although no proteolysis could be measured. The DF activity was tested on muscle samples (rough microsomal membranes) obtained from Duchenne muscular dystrophy (DMD) patients (7 cases) and normal controls (16 cases). The yield of membrane bound ribosomes (MBR) obtained from muscle samples of DMD patients by extraction with DF is 5‐fold higher than from muscle samples of normal controls. The difference in MBR yield is not related to the type of DF. Similar values for MBR were obtained with rat and human (normal and dystrophic) DF. Ribosomal protein synthesis (RPS) with ribosomes extracted by DF showed values similar to the RPS of detergent extracted ribosomes. Our findings suggest the observed increase in membrane bound ribosomes in DMD probably results from increased levels of mRNAs coding for membrane and secretory proteins such as collagen.