Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein

Dynein was extracted from outer arm-less axonemes of the mutant oda1 and fractionated by high-pressure liquid chromatography on a MonoQ column into seven distinct subspecies (named a-g). Each subspecies contained one or two heavy chains and several medium-sized and light chains; by vanadate/UV-induced photocleavage and SDS- polyacrylamide gel electrophoresis, eight distinct heavy chains were identified. Analysis of the mutant axonemes indicated that the subspecies f (containing two heavy chains) is missing in the inner-arm mutant ida1 and the subspecies a, c and d are missing in the mutant ida4. Six subspecies (all but f) supported microtubule translocation with the maximal rate ranging from 2 to 12 micrometre s-1 and the apparent Km for ATP ranging from about 10 to 100 micromolar. All the subspecies translocated microtubules with the plus end leading, indicating that all the inner-arm dyneins are minus end-directed motors. Five subspecies (all but b and f) displayed microtubule rotation during translocation at rates of up to about 10 Hz. Unexpectedly, the Km values for ATP for translocation and rotation did not always agree; because of this, the pitch of the movement was variable with some subspecies. These observations indicate that axonemes are equipped with several inner-arm subspecies and that torque generation is a feature common to many of them.

An isozyme of hexokinase specific for the human red blood cell (HKR) [see comments]

The hexokinase (HK) of the human red blood cell (RBC) was separated into two distinct major isozymes by fast protein liquid chromatography using a linear salt gradient on a MonoQ column. The first isozyme (HKI) eluted as a sharp peak at the same position as HKI of human liver. The second isozyme eluted between HKI and HKII of human white blood cells, and it appeared to be unique to the RBC (it was designated HKR). From a gel filtration column, HKR eluted before HKI, suggesting that it was larger than HKI by several kilodaltons. In a mitochondria-enriched fraction from human reticulocytes, no HKR was found; thus, HKR was not a mitochondrial enzyme. Despite these differences in chromatographic behavior, size, and mitochondrial binding, both forms behaved kinetically as HKI. RBC from normal blood contained HKI and HKR at an equal activity, but in reticulocyte-rich RBC, HKR dominated. When RBC of increasing age was separated by buoyant density ultracentrifugation, the total HK activity decayed in a biphasic manner, with half-lives respectively of approximately 15 and approximately 51 days. When isolated by MonoQ column from each age-separated fraction, HKR was the major form in the youngest RBC, and decreased rapidly with cell age, with a t 1/2 of approximately 10 days, representing a negligible activity in the oldest RBC. Instead, HKI was relatively stable through the entire life span of the RBC, with a t 1/2 of approximately 66 days. Thus, HKR appears to be an RBC-specific isozyme that is predominant in the reticulocyte and is then rapidly degraded. During maturation of the RBC, the fast decay of HKR contributes to the early sharp decline of HK activity and the slow decay of HKI to the later gradual decline.

An isozyme of hexokinase specific for the human red blood cell (HKR) [see comments]

The hexokinase (HK) of the human red blood cell (RBC) was separated into two distinct major isozymes by fast protein liquid chromatography using a linear salt gradient on a MonoQ column. The first isozyme (HKI) eluted as a sharp peak at the same position as HKI of human liver. The second isozyme eluted between HKI and HKII of human white blood cells, and it appeared to be unique to the RBC (it was designated HKR). From a gel filtration column, HKR eluted before HKI, suggesting that it was larger than HKI by several kilodaltons. In a mitochondria-enriched fraction from human reticulocytes, no HKR was found; thus, HKR was not a mitochondrial enzyme. Despite these differences in chromatographic behavior, size, and mitochondrial binding, both forms behaved kinetically as HKI. RBC from normal blood contained HKI and HKR at an equal activity, but in reticulocyte-rich RBC, HKR dominated. When RBC of increasing age was separated by buoyant density ultracentrifugation, the total HK activity decayed in a biphasic manner, with half-lives respectively of approximately 15 and approximately 51 days. When isolated by MonoQ column from each age-separated fraction, HKR was the major form in the youngest RBC, and decreased rapidly with cell age, with a t 1/2 of approximately 10 days, representing a negligible activity in the oldest RBC. Instead, HKI was relatively stable through the entire life span of the RBC, with a t 1/2 of approximately 66 days. Thus, HKR appears to be an RBC-specific isozyme that is predominant in the reticulocyte and is then rapidly degraded. During maturation of the RBC, the fast decay of HKR contributes to the early sharp decline of HK activity and the slow decay of HKI to the later gradual decline.

Mitochondrial aspartate aminotransferase determined by "Fast Protein Liquid Chromatography"

We describe an improved separation of the isoenzymes of aspartate aminotransferase (EC 2.6.1.1), based on ion-exchange chromatography. Involving the "Fast Protein Liquid Chromatography" system (Pharmacia) with a MonoQ column, this rapid, reproducible method for quantifying the mitochondrial enzyme shows good resolution and sensitivity, and results correlate well with those by an established immunochemical method.

Lysine Decarboxylase from Hafnia alvei: Purification, Molecular Data and Preparation of Polyclonal Antibodies

The purification and molecular properties of lysine decarboxylase from Hafnia alvei and the preparation of polyvalent antibodies specific for this enzyme are described. The enzyme was purified within two HPLC steps on a TSK G 4000 SW and a MonoQ column to homogeneity. The subunit of the enzyme has a molecular weight of approximately 80.000 d. Under “native” conditions it seems to form aggregates up to ten subunits. Lysine decarboxylase from H. alvei contains one mol pyridoxal phosphate per mol subunit. Antibodies against the lysine decarboxylase were purified by affinity chromatography.